Os autores gostariam de agradecer Verlene Henry e Lindsay Holmes para a assistência no desenvolvimento deste modelo. Este trabalho foi parcialmente financiado pela Fundação S. James McDonnell (# 220020173).

1. Linhagens de células <ol><li> U87MG células glioma são cultivadas em frascos grandes de tecido cultura com DMEM-F12 suplementado com 5% de soro fetal bovino (FBS). </li><li> As células são colhidas por lavagem frascos duas vezes com fosfato quente Buffered Saline (PBS) e incubando-os a 37 ° C por 5 minutos com 10 ml de PBS contendo 0,25% de tripsina e EDTA 0,05%. Uma vez que as células são levantadas, eles são colocados em um tubo de 50 ml contendo 10 ml de meios de cultura e centrifugadas (300 g X para 4 min). </li><li> Após a lavagem, as células são ressuspendidas a uma concentração de 10 x 10 6 / ml em meios de cultura, o que permitiu uma inoculação de 50.000 células / 5 mL. </li><li> As células são mantidas em gelo até injeção intracraniana. </li></ol> 2. Guia parafuso intracraniana bolting Este procedimento pode ser realizado alguns dias antes da injeção das células. Todos os procedimentos aqui descritos foram realizados sob estritas condições estéreis. <ol><li> Camundongos (BALB / c nu / nu feminino; 5-6 semanas, aproximadamente 18g) são numerados para fins de identificação, pesados ​​e anestesiados com uma injeção intraperitoneal (ip) de uma mistura de cetamina (100 mg / kg) e xilazina (5 mg / kg). </li><li> A pele é varrida para baixo com uma solução de iodo e de uma pequena incisão (2-3 mm) é feita no lado direito da linha média e anterior à linha interaural. Isso expõe as suturas coronal e sagital do crânio. O bregma é posicionado na junção destas duas suturas. </li><li> O guia de ponto de entrada do parafuso é então marcada a um ponto 2.5 mm lateral e 1 mm anterior ao bregma. Este ponto está localizado diretamente acima do núcleo caudado 1. </li><li> A 1 x 1 mm de profundidade buraco é perfurado com uma mão estéril realizada broca através do crânio para a dura. </li><li> Um parafuso guia esterilizado que se estenderá 1,6 milímetros abaixo da superfície crânios na dura é, então, fugiu para o buraco com uma chave de fenda até que esteja alinhado com o crânio (Figura 1A). </li><li> Um estilete estéril ou dummy parafuso é então colocada no orifício central do parafuso guia para fechar a abertura (Figura 1B). </li><li> A ferida é fechada com adesivo de tecido Vetbond (n-butil cianoacrilato) e os ratos recebem uma injeção intraperitoneal de reversine (pequenos animais) (0,1 ml / kg) e carprofeno (5 mg/kg/100 mL) para analgesia. Os ratos são, então, permitiu recuperar em uma esteira de aquecimento (36 ° C), que pode levar até 20 minutos. Ratos são freqüentemente monitoradas e observadas durante este tempo de recuperação até que sejam plenamente consciente. </li></ol> 3. Enxerto celular intracraniana <ol><li> A seringa estéril Hamilton algemado é preparado pela aplicação de um pequeno anel de plástico para a ponta da agulha permitindo apenas dois milímetros da agulha para estender abaixo da saída do parafuso guia (Figura 1B). Do ponto de inoculação final é, portanto, 3,5 milímetros abaixo da superfície do crânio. </li><li> Quatro dias após a cirurgia parafuso guia, os ratos são novamente anestesiados como descrito acima (2.1) e uma pequena incisão é feita sobre o parafuso guia para retirar o estilete. </li><li> A seringa Hamilton algemado é então preenchido com 5 mL de bem comum, tendo as células precaução para não criar ou elaborar as bolhas de ar. </li><li> A seringa é fixada à bomba de perfusão ea agulha é inserida no parafuso guia (Figura 1C). As células são então infundido a uma taxa de 30 mL por hora. </li><li> O aparelho automatizado (Figura 1D) permitiu-nos a injetar até 10 animais de cada vez, a um fluxo constante. As células também podem ser injetados manualmente usando a seringa hamilton algemado ou, alternativamente, a seringa pode ser algemado por uma ponteira 200 mL que foi cortado por 3 mm para permitir que a ponta da agulha se estender além do parafuso guia por 2 mm. A injeção deve ser realizada em um ritmo constante estacionário. </li><li> Quando a infusão estiver concluída, a seringa é cuidadosamente removido eo estilete é substituído no parafuso guia. A ferida é fechada colada e os ratos são dadas medicamentos de recuperação como antes (2,7) e tempo para se recuperar em uma esteira de aquecimento. </li></ol> 4. Desafio terapêutico <ol><li> Quatro dias após a inoculação de células U87MG, os ratos são pesados ​​e divididos aleatoriamente em grupos controle e tratamento. </li><li> O grupo controle é dada uma injeção intra-peritoneal (IP) da PBS (100 l), enquanto o grupo de tratamento é dada uma injeção IP de AMG102 (100 mg em 100 ml de PBS). </li><li> As injeções são repetidas a cada segundo dia por 14 dias, um total de 6 injeções (Figura 2). </li><li> Após a última injeção, os ratos são monitoradas diariamente e pesados ​​a cada segundo dia. </li><li> Quando os ratos começaram a mostrar significativas disfunções neurológicas (saldo perturbação, paralisia), desidratação, ou perda de peso mais de 10% ou moribundo, eles foram abatidos de forma humana. Estes humano ponto final dia foram, então, registada na curva de sobrevida de Kaplan-Meier. </li></ol> 5. Avaliação de eficácia terapêutica </p&gt; <ol><li> A curva de sobrevida de Kaplan-Meier foi gerada usando GraphPad Prism 4,03 para Windows; GraphPad Software, San Diego, Califórnia, EUA, <a href="http://www.graphpad.com">www.graphpad.com</a> 3. Mortes ou euthanizations foram marcados como ponto final de eventos e classificada como 1. Animais que permaneceram vivos até o final do experimento foram registradas como não-acontecimentos e pontuada como 0. </li></ol> 6. Resultados representativos: Animais de controle começou a mostrar sinais de perturbação neurológica e perda de peso 23 dias após a inoculação inicial de células. Este foi um atraso significativo no grupo tratado AMG102 a 35 dias. Na verdade o dia 35, 77% (n = 09/07) do grupo controle foram sacrificados. A curva de sobrevida de Kaplan-Meier demonstra claramente o aumento significativo da sobrevida em resposta a AMG102 tratamento (Figura 3). Por 70 dias, 88% dos ratos controle foram sacrificados (n = 09/08) em comparação com 37% (n = 08/03) de AMG102 grupo tratado. A análise histológica do cérebro confirmou que todos os nove animais do grupo controle desenvolveram tumores em comparação com apenas 3 dos 8 AMG102 ratos tratados (Figura 4). <img alt="Figura 1" src="/files/ftp_upload/3157/3157fig1.jpg" /> Figura 1: Imagem (A) mostra parafuso guia intracraniana posicionados em uma área localizada diretamente acima do núcleo caudado. O algemado hamilton seringa (pré-carregado com células) é então colocado dentro do parafuso guia de forma que 2 mm de agulha se estende além do guia e as células são injetadas 3,5 milímetros no interior do núcleo caudado. A seringa é posteriormente removido e substituído por um estilete (B). O dispositivo automático de infusão (C) pode injetar até 10 animais de cada vez. Aqui mostrados são cinco injeções simultâneas (D). <img alt="Figura 2" src="/files/ftp_upload/3157/3157fig2.jpg" /> Figura 2: Esquema de protocolo do estudo. Células foram injetadas no dia 0 com o tratamento começa no dia 4. Animais controle receberam uma injeção de PBS (100 l) IP enquanto o grupo de tratamento recebeu uma injeção de AMG102 (100 μg/100 mL) IP. Estas injecções foram dadas a cada segundo dia por 10 dias. Um total de 6 injeções foram dadas. Animais foram monitorados por 70 dias para sinais de distúrbios neurológicos e físicos. <img alt="Figura 3" src="/files/ftp_upload/3157/3157fig3.jpg" /> Figura 3: Kaplan-Meier comparando a sobrevivência e controle de AMG102 camundongos tratados. Significante de sobrevivência foi obtida após o tratamento com AMG102 (p = 0,005). <img alt="Figura 4" src="/files/ftp_upload/3157/3157fig4.jpg" /> Figura 4: Microfotografias de seções cérebro coronal demonstrando o desenvolvimento de tumores nos animais controle (A) e não o desenvolvimento do cérebro tumor no AMG102 animais tratados (B). Barra de escala é de 1 mm.

<ol>
	<li>Lal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+implantable+guide-screw+system+for+brain+tumor+studies+in+small+animals.">An implantable guide-screw system for brain tumor studies in small animals.</a> J Neurosurg. 92, 326-333 (2000).</li><li>Jun, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMG+102,+a+fully+human+anti-hepatocyte+growth+factor/scatter+factor+neutralizing+antibody,+enhances+the+efficacy+of+temozolomide+or+docetaxel+in+U-87+MG+cells+and+xenografts.">AMG 102, a fully human anti-hepatocyte growth factor/scatter factor neutralizing antibody, enhances the efficacy of temozolomide or docetaxel in U-87 MG cells and xenografts.</a> Clin Cancer Res. 13, 6735-6742 (2007).</li><li>Pillay, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasticity+of+oncogene+addiction:+implications+for+targeted+therapies+directed+to+receptor+tyrosine+kinases.">The plasticity of oncogene addiction: implications for targeted therapies directed to receptor tyrosine kinases.</a> Neoplasia. 11, 448-458 (2009).</li><li>Buchanan, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosensitization+of+glioma+cells+by+modulation+of+Met+signaling+with+the+hepatocyte+growth+factor+neutralizing+antibody.+AMG102.">Radiosensitization of glioma cells by modulation of Met signaling with the hepatocyte growth factor neutralizing antibody. AMG102.</a> J Cell Mol Med. 15, 1999-2006 (2011).</li><li>Wen, P. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+II+study+evaluating+the+efficacy+and+safety+of+AMG+102+(rilotumumab)+in+patients+with+recurrent+glioblastoma.">A phase II study evaluating the efficacy and safety of AMG 102 (rilotumumab) in patients with recurrent glioblastoma.</a> Neuro Oncol. 13, 437-446 (2011).</li><li>Caretti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+Tumor+Growth+and+Post-Irradiation+Recurrence+in+a+Diffuse+Intrinsic+Pontine+Glioma+Mouse+Model.">Monitoring of Tumor Growth and Post-Irradiation Recurrence in a Diffuse Intrinsic Pontine Glioma Mouse Model.</a> Brain Pathol. 4, 441-451 (2011).</li><li>Jallo, G. I., Volkov, A., Wong, C., Carson, B. S., Penno, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+brainstem+tumor+model:+functional+and+histopathological+characterization.">A novel brainstem tumor model: functional and histopathological characterization.</a> Childs Nerv. Syst. 22, 1519-1525 (2006).</li><li>Johns, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficacy+of+epidermal+growth+factor+receptor-specific+antibodies+against+gliomaxenografts+is+influenced+by+receptor+levels,+activation+status,+and+heterodimerization.">The efficacy of epidermal growth factor receptor-specific antibodies against gliomaxenografts is influenced by receptor levels, activation status, and heterodimerization.</a> Clin Cancer Res. 13, 1911-1925 (2007).</li><li>Scott, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+clinical+trial+with+monoclonal+antibody+ch806+targeting+transitional+state+and+mutant+epidermal+growth+factor+receptors.">A phase I clinical trial with monoclonal antibody ch806 targeting transitional state and mutant epidermal growth factor receptors.</a> Proc. Natl. Acad. Sci. U. S. A. 104, 4071-4076 (2007).</li><li>Niyazi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+options+for+recurrent+malignant+glioma.">Therapeutic options for recurrent malignant glioma.</a> Radiother. Oncol. 98, 1-14 (2011).</li></ol>

Este trabalho foi financiado por uma bolsa de pesquisa da Amgen Inc.

O método parafuso intracraniana guia aqui apresentado permite a criação rápida e reprodutível de xenoenxertos intracraniana e nas mãos de um técnico de animais treinados, este procedimento é muito facilmente realizada sem a necessidade de um dispositivo estereotáxico. Nosso método combina o uso de um parafuso guia, para localizar com precisão a região do cérebro mais adequado para o desenvolvimento do tumor in vivo, e uma bomba de infusão automatizado que permite a inoculação simultânea de até 10 animais de cada vez. O guia procedimento de aumento do parafuso leva menos de 5 minutos para ser realizado. A infusão de células automatizadas de 30 mL / hora leva apenas 10 minutos para um volume de 5 mL. Isso garante uma dosagem exacta a uma taxa constante e reduz o risco de expulsão de células para a área extra-craniana devido ao aumento da pressão intracraniana. Temos também realizado este procedimento com 10 volumes mL e não observou qualquer crescimento tumoral extra-craniano, devido ao refluxo celular. Como a bomba de infusão automática também permite que vários animais a ser inoculado simultaneamente que pode injetar até 60 animais por hora, em comparação a um procedimento de estereotaxia, que é limitada a aproximadamente 15 animais por hora. O passo mais crítico neste procedimento é a localização do parafuso guia no crânio como sua posição determina a localização final da maior parte do tumor. Se o parafuso guia é muito central, as células serão injetadas em uma região entre os dois hemisférios do cérebro enquanto que se a guia é posicionada para a frente, as células não vai ser injetado no cérebro. Portanto, é essencial para ser o mais preciso possível para localizar o guia 2,5 milímetros laterais e 1 mm anterior ao bregma 1. Neste estudo foi utilizado um parafuso guia de aço, embora parafusos-guia de plástico também estão disponíveis para aqueles que necessitam de estudos de imagens cerebrais, tais como ressonância magnética ou PET 6. Mesmo se a bomba de infusão automatizado é o custo proibitivo para alguns grupos, as células ainda podem ser injetadas manualmente mais rapidamente do que as abordagens estereotáxica. No geral, este método rápido, preciso e reprodutível pode ser usado para estudar meduloblastoma glioma e os tumores do tronco cerebral 7. Este procedimento tem sido realizado em mais de 500 ratos entre os nossos dois laboratórios com mortes devido a reações inesperadas anestésico (&lt;1%). Ratos que foram aparafusadas com o parafuso guia e, posteriormente, infundido com células não morrem ou sofrem qualquer complicações neurológicas, como resultado do procedimento. Animais tenham sobrevivido nos últimos 100 dias, sem quaisquer complicações. Embora nós preferimos usar um mouse sem pêlos, um mouse imuno-comprometidos com o cabelo como o SCID seria tão apropriado, desde que o cabelo crânio foi removido antes da cirurgia para manter o local limpo e para evitar que o cabelo interferir com parafuso guia. Existem diversas novas terapêuticas actualmente a ser desenvolvido para o tratamento de glioma 8-10. Recentemente, nós mostramos que xenoenxertos subcutânea U87MG glioma pode ser inibida pelo tratamento com AMG102 3; um anticorpo humanizado direcionado para HGF actualmente em fase de avaliação clínica em glioma 5. Foi utilizado o método descrito aqui para estabelecer tumores intracranianos U87MG no cérebro de ratos nude. Nosso estudo mostra claramente a utilidade do método e demonstra que AMG102 significativamente inibe o crescimento de xenoenxertos U87MG ortotópico. Vamos continuar a usar este modelo para determinar o que outras terapêuticas podem ser usadas em combinação com AMG102 para obter a inibição mais eficaz de crescimento do tumor.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> DMEM/F12 </td><td> Invitrogen </td><td> 12634010 </td><td></td></tr><tr><td> FCS </td><td> Bovogen </td><td> SFBs </td><td></td></tr><tr><td> Tripsina </td><td> Invitrogen </td><td> 15050065 </td><td></td></tr><tr><td> EDTA </td><td> Sigma </td><td> E6758 </td><td></td></tr><tr><td> Balb / c nu / nu ratos </td><td> ARC Perth </td><td></td><td></td></tr><tr><td> Titular do parafuso </td><td> Um plásticos </td><td> SD-1 </td><td></td></tr><tr><td> Titular da broca </td><td> Um plásticos </td><td> DH-1 </td><td></td></tr><tr><td> Broca </td><td> Um plásticos </td><td> D # 60 </td><td></td></tr><tr><td> Parafuso guia (metal) </td><td> Um plásticos </td><td> C212SG </td><td></td></tr><tr><td> Manequim parafuso (stylus) </td><td> Um plásticos </td><td> C212SD </td><td></td></tr><tr><td> Hamilton seringa (10 mL 26g agulha cimentada) </td><td> Divisão de graça Discovery Science </td><td> 80075 </td><td></td></tr><tr><td> PHD bomba de infusão 22/2000 </td><td> Harvard Apparatus </td><td> 70-2003 </td><td> Opcional </td></tr><tr><td> Vetbond </td><td> 3M </td><td> 1469SB </td><td></td></tr><tr><td> Thermo pad </td><td> Harvard Apparatus </td><td> 340390 </td><td></td></tr></tbody></table>

a simple guide screw method for intracranial xenograft studies in mice

A enxertia de células tumorais humanas no cérebro de camundongos imunossuprimidos é um método estabelecido para o estudo dos cânceres de cérebro, incluindo glioblastoma (glioma) e meduloblastoma. A abordagem estereotáxica amplamente utilizado só permite a injeção de um único animal de cada vez, é trabalhoso e requer equipamentos altamente especializados. O método parafuso guia, inicialmente desenvolvido por Lal et al., 1 foi desenvolvido para eliminar procedimentos estereotáxica pesado. Vamos agora descrever uma abordagem modificada parafuso guia que é rápida e excepcionalmente segura; sendo que ambos são críticos considerações éticas. Nomeadamente, o nosso procedimento agora incorpora uma bomba de infusão que permite que até 10 animais a ser simultaneamente injetados com células tumorais. Para demonstrar a utilidade deste procedimento, nós estabelecemos células glioma U87MG como xenoenxertos intracraniana em camundongos, que foram então tratados com AMG102; um anticorpo totalmente humano direcionado para HGF / fator de dispersão actualmente em fase de avaliação clínica 2-5. Injeção sistêmica de AMG102 prolongou significativamente a sobrevivência de todos os camundongos com xenotransplantes U87MG intracraniana e resultou em uma série de curas completas. Este estudo demonstra que o método parafuso guia é uma abordagem, de baixo custo altamente reprodutível para o estabelecimento de xenoenxertos intracraniana. Além disso, fornece um modelo fisiológico relevantes para a validação de novas estratégias terapêuticas para o tratamento de câncer no cérebro.

Watch this Scientific Journal Video about A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice at JoVE.com

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

A fim de avaliar romance paradigmas terapêuticos para o tratamento de glioma, fisiológicos modelos relevantes são essenciais. Nós utilizamos um procedimento de parafuso implantáveis ​​guia para criação de modelos de xenoenxerto intracraniana que é mais rápido e mais seguro do que as abordagens estereotáxica.

Um método simples parafuso Guia para intracraniana Estudos Xenoenxerto em Ratos

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

a-simple-guide-screw-method-for-intracranial-xenograft-studies-in-mice

Research

JoVE Journal

Medicine

23.0K visualizações.  Monash Institute of Medical Research. A fim de avaliar romance paradigmas terapêuticos para o tratamento de glioma, fisiológicos modelos relevantes são essenciais. Nós utilizamos um procedimento de parafuso implantáveis ​​guia para criação de modelos de xenoenxerto intracraniana que é mais rápido e mais seguro do que as abordagens estereotáxica.

Vídeo: A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections1. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications2,3. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections4. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins5. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. 
Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy6. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.

Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins&#x2019; circulations. 
Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.

Guia de Cateterismo Fio Assistida e injeção de corante colorido para o Mapeamento de Vascular monocoriônicas placentas gêmeas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC ...

Medicina

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

Medida da Pressão Intracraniana peridural em ratos, utilizando um transdutor de pressão de fibra óptica

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Um método simples de intubação Lung Rato

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

A Novel de alta resolução<em&gt; In vivo</em&gt; Técnica de Imagem para estudar a resposta dinâmica de estruturas intracranianas ao crescimento do tumor e Terapêutica

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

O<em&gt; Em ovo</em&gt; CAM-ensaio como um Xenograftmodel para o Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

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 ...

Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is required. In most cases, the preferred route of administration is via oral delivery. At the present time, oral delivery is mostly accomplished using an oral gavage procedure, in part, because it can achieve consistent and precise dosing in the animal model. Although this method is well established it does have complications that can result in a high rate of animal attrition. To this end, the procedure introduced here describes an alternative to the oral gavage method in which the desired drug is incorporated into a tastant, orally dissolving strip (ODS) that can simply be presented to the test animal where it is then rapidly taken up with minimal manipulation of the test subject. Herein, we demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

The following protocol describes a novel method for chronic oral drug administration using an orally dissolving strip (ODS) in lieu of the more commonly used oral gavage method. We demonstrate that preclinical, oral drug delivery using the ODS method represents a safe, convenient, and humane alternative to oral gavage.

Utilizando um Dissolver Faixa por via oral para estudos farmacológicos e toxicológicos: uma alternativa simples e Humane a sonda oral for Animals

Prior to testing novel therapeutics in humans, short and long term preclinical (i.e., animal), repetitive pharmacological and toxicological testing is ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Um método melhorado para rápida de intubação da traqueia em Ratos

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

Um método para quantificar Desempenho Membro Superior na Vida Diária utilizem acelerómetros

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...