Este trabalho foi financiado pela Fundação Marshall JB.

1. Prepare Animal e Equipamentos para Procedimento <ol><li> Antes de iniciar a cirurgia, preparar o local cirúrgico e do animal. Fazer o cateter da carótida, inserindo uma agulha de calibre 23 em uma extremidade romba, de 12 &quot;de tubagem PE50. Cortar um chanfro de aproximadamente 45 ° na extremidade oposta do cateter. Esterilizar equipamento antes do procedimento. Usar uma capota de cabelo, máscara cirúrgica e luvas estéreis. </li><li> Coloque uma almofada de aquecimento sobre a superfície onde a cirurgia será realizada. Ative-a e deixe-o aquecer. Cobrir a área com um pano absorvente banco. </li><li> Verifique se você tem manitol 25% em solução sem cristais. Se estiverem presentes cristais, dissolvê-los, colocando o frasco num banho de água quente a 80 ° C e agitando periodicamente. </li><li> Registar a massa do animal. </li><li> Prepare uma diluição de propofol para entrega a 0,8 mg / kg / min, a uma taxa de 0,1 ml / min. </li><li> Colocar o rato na caixa de indução durante 3 min. Definições: 5% de isoflurano, 2 L / mintaxa de fluxo no ventilador. </li><li> Em cima de uma toalha descartável, fazer a barba do pescoço e duas coxas. </li></ol> 2. Entubar the Rat <ol><li> Colocar a parte de trás dos animais na caixa de indução durante 3 min. </li><li> Prepare o seu tubo endotraqueal com cateter IV. Para este tamanho e linhagem de rato, usamos um cateter 18 ga. O tubo deve ser grande o suficiente para permitir a ventilação adequada, mas pequena o suficiente para caber na traquéia sem causar danos. Puxe a agulha para fora do cateter de plástico e cuidadosamente quebrar a cerca de agulha. 1 cm da sua ponta afiada. A agulha bunted será a sua caneta. Colocar a parte de trás da agulha no cateter. </li><li> Desligar o rato pelos seus dentes sobre uma superfície inclinada. </li><li> Brilhar uma luz diretamente contra o pescoço do animal e alinhados entre suas orelhas. </li><li> Inserir uma cerca pequena espátula. 3 centímetros para a orofaringe. Levante para cima, enquanto puxando a língua para fora da boca. </li><li> Visualize a abertura da traqueia. Você vai seeA pequeno buraco aberto e próximo com cada respiração. </li><li> Insira cuidadosamente cateter no orifício. Às vezes você pode sentir o plástico entrar na cartilagem da traqueia. A traquéia é sensível. Não force o cateter. </li><li> Definir o ventilador a 1 L / min e 60 bpm e o isoflurano a 2%. </li><li> Conectar o cateter ao ventilador e o tubo de fita com a superfície de operação. </li><li> Verificar que o animal é anestesiado, realizando um aperto do dedo do pé. </li></ol> 3. Estabelecer Propofol Anesthesia IV <ol><li> Fixe o rato gravando todos os quatro membros ea cauda à superfície operacional. </li><li> Esfregue o pescoço raspada e sites virilha com clorexidina sabão três vezes. Em seguida, esfregue com clorexidina lavar três vezes. </li><li> Anexar uma seringa de propofol com um conjunto de extensão IV. Montar a seringa numa bomba de infusão. </li><li> Utilizando um bisturi, cortar através da pele da coxa rapada. Evitar o corte do músculo. </li><li> Abrir a incisão com uma forçaps para visualizar a veia femoral. </li><li> Puxar o tecido ao longo da artéria para expor o recipiente. </li><li> Insira um 26 ga Monoject veterinária cateter IV na veia. Quando a agulha perfura a parede da veia você vai sentir-se mais fácil de empurrar. Retrair a agulha e empurra o cateter dentro do vaso. </li><li> O início da infusão de propofol a 0,1 ml / min. </li><li> 3-5 min após o início da perfusão de propofol, mudar o gás de anestesia a uma mistura de 50% de O 2 e 50% de N 2 O e desligar o isoflurano. </li></ol> 4. Expor a bifurcação da artéria carótida comum <ol><li> Faça uma incisão na linha no pescoço com lâmina de bisturi. </li><li> Cortar entre os dois grandes glândulas salivares com tesoura ou bisturi. </li><li> Dissecar o tecido lateralmente à linha média para expor a artéria carótida. Use uma pinça para abrir as camadas de tecido. Você pode usar uma tesoura para retirar peças que você expõem com a pinça e determinar a ser minimally vascular. Controlar a hemorragia com gaze, cotonetes, e cauterização, se necessário. </li><li> Injectar 3 IP ml de soro fisiológico se houve moderada a forte sangramento. </li><li> Use afastadores para puxar tecido para longe da carótida. Encontrar a bifurcação. </li><li> Exponha a artéria occipital apenas distal à bifurcação. Cauterizar o entre fórceps e separá-lo com uma electrocauterização. </li><li> Use uma pinça para expor totalmente a bifurcação da carótida e tanto quanto do ECA possível. </li><li> Expor a artéria tireóide superior ramificando-se o ECA distal à artéria occipital. Cauterizar o entre fórceps e separá-lo com uma electrocauterização. </li></ol> 5. Cateterização da artéria carótida externa <ol><li> Coloque 2 comprimentos de 4/0 de seda sob o ECA. </li><li> Pegue um comprimento de sutura com sua pinça hemostática e recolher o ECA volta caudalmente (em direção aos pés do rato). </li><li> Tome a sutura outros e amarrar até cranialmente (para a boca do rato) como w possívelnó um cirurgião om o. Isso forma uma ligadura permanente para sacrificar o ECA. </li><li> Remova a pinça hemostática primeiro (o que foi aplicar tensão caudal) e pegar a sutura outro (o que é amarrado ao redor do ECA) e aplicar tensão suave cranialmente com a pinça hemostática. </li><li> Amarre outro nó cirurgião livremente (não proteger - deixar mal amarrado) distal para proximal da bifurcação e à ligadura permanente. </li><li> Irrigar com lidocaína a 1% para relaxar a artéria. </li><li> Preencha o seu cateter com solução salina heparinizada. </li><li> Colocar um clipe recipiente temporário tão perto quanto possível da bifurcação. Clipe lugar vaso em um ângulo para maximizar o espaço para a cateterização. Deslize o nó frouxo na direção do clipe. </li><li> Faça um pequeno arteriotomia (corte) no ECA apenas proximal à ligadura usando microtesoura. Não deve haver nenhum sangramento, se o clipe e ligadura são seguras. </li><li> Usando fórceps para controlar as paredes da ECA, passe a extremidade chanfrada do cateter para dentro do vaso. </li&gt; <li> Fixe o cateter com o nó de sutura frouxa. Ligar a artéria ao redor do cateter. </li><li> Remova o clipe e limpar a área com um cotonete. </li><li> Avançar o cateter, para cerca de 1 mm distal à bifurcação. Segurar a artéria com uma pinça proximal ao nó de assegurar a embarcação está imobilizada. </li></ol> 6. Administrar Manitol <ol><li> Encher um 8 &quot;conjunto de extensão IV e uma seringa de 6 ml com manitol sem a criação de quaisquer bolhas de ar na Conserva-se a incubadora a 37 ° C. Nota: trabalho rapidamente com manitol - será prontamente precipitar para fora da solução, à temperatura ambiente.. </li><li> A fim de visualizar BBBD, pode administrar corante azul de Evans e vê-lo introduzir as áreas do cérebro em que a barreira tenha sido interrompida. Para fazer isso, dar ao animal 2 ml / kg de azul de Evans a 2% através de um local novo IV na perna contralateral ao propofol. </li><li> Coloque a extensão definida para um mM 5 filtrar e preenchê-lo com o manitol para remover o ar bubbles. Depois, fixe esta atinja a torneira sem criar bolhas. </li><li> Administrar manitol a 0,09 ml / seg a 25 seg. Preste atenção para o manitol para alcançar o equilíbrio com o fluxo na carótida comum em um ponto imediatamente proximal à ACI. Ele deve aparecer a pulsar com a taxa de rato coração. Se o manitol simplesmente flui para o coração que você superar a pressão do rato sangue. Isto pode causar complicações potencialmente fatais e BBBD pobres. </li></ol>

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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=Improving+drug+delivery+to+intracerebral+tumor+and+surrounding+brain+in+a+rodent+model:+a+comparison+of+osmotic+versus+bradykinin+modification+of+the+blood-brain+and/or+blood-tumor+barriers.">Improving drug delivery to intracerebral tumor and surrounding brain in a rodent model: a comparison of osmotic versus bradykinin modification of the blood-brain and/or blood-tumor barriers.</a> Neurosurgery. 43, 879 (1998).</li><li>Kinoshita, M., McDannold, N., Jolesz, F. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+administered+in+conjunction+with+osmotic+blood-brain+barrier+modification+in+patients+with+brain+metastases.">Chemotherapy administered in conjunction with osmotic blood-brain barrier modification in patients with brain metastases.</a> Journal of neuro-oncology. 4, 195-207 (1987).</li><li>Doolittle, N. D., 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=Safety+and+efficacy+of+a+multicenter+study+using+intraarterial+chemotherapy+in+conjunction+with+osmotic+opening+of+the+blood-brain+barrier+for+the+treatment+of+patients+with+malignant+brain+tumors.">Safety and efficacy of a multicenter study using intraarterial chemotherapy in conjunction with osmotic opening of the blood-brain barrier for the treatment of patients with malignant brain tumors.</a> Cancer. 88, 637-647 (2000).</li><li>Guillaume, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-arterial+chemotherapy+with+osmotic+blood-brain+barrier+disruption+for+aggressive+oligodendroglial+tumors:+results+of+a+phase+I+study.">Intra-arterial chemotherapy with osmotic blood-brain barrier disruption for aggressive oligodendroglial tumors: results of a phase I study.</a> Neurosurgery. 66, 48 (2010).</li><li>Remsen, L. G., 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+influence+of+anesthetic+choice,+PaCO2,+and+other+factors+on+osmotic+blood-brain+barrier+disruption+in+rats+with+brain+tumor+xenografts.">The influence of anesthetic choice, PaCO2, and other factors on osmotic blood-brain barrier disruption in rats with brain tumor xenografts.</a> Anesthesia &amp; Analgesia. 88, 559-559 (1999).</li><li>Nilaver, G., 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=Delivery+of+herpesvirus+and+adenovirus+to+nude+rat+intracerebral+tumors+after+osmotic+blood-brain+barrier+disruption.">Delivery of herpesvirus and adenovirus to nude rat intracerebral tumors after osmotic blood-brain barrier disruption.</a> Proceedings of the National Academy of Sciences. 92, 9829 (1995).</li><li>Fortin, D., McCormick, C. I., Remsen, L. G., Nixon, R., Neuwelt, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+neurotoxicity+of+etoposide+phosphate+administered+in+combination+with+other+chemotherapeutic+agents+after+blood-brain+barrier+modification+to+enhance+delivery,+using+propofol+for+general+anesthesia,+in+a+rat+model.">Unexpected neurotoxicity of etoposide phosphate administered in combination with other chemotherapeutic agents after blood-brain barrier modification to enhance delivery, using propofol for general anesthesia, in a rat model.</a> Neurosurgery. 47, 199 (2000).</li><li>Neuwelt, E. 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=Primary+CNS+lymphoma+treated+with+osmotic+blood-brain+barrier+disruption:+prolonged+survival+and+preservation+of+cognitive+function.">Primary CNS lymphoma treated with osmotic blood-brain barrier disruption: prolonged survival and preservation of cognitive function.</a> Journal of clinical oncology. 9, 1580 (1991).</li><li>Remsen, L. G., Trail, P. A., Hellstr&#246;m, I., Hellstr&#246;m, K. E., Neuwelt, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+delivery+improves+the+efficacy+of+a+tumor-specific+doxorubicin+immunoconjugate+in+a+human+brain+tumor+xenograft+model.">Enhanced delivery improves the efficacy of a tumor-specific doxorubicin immunoconjugate in a human brain tumor xenograft model.</a> Neurosurgery. 46, 704 (2000).</li><li>Neuwelt, E. A., Pagel, M. A., Kraemer, D. F., Peterson, D. R., Muldoon, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+chemoprotection+without+compromise+of+chemotherapy+efficacy+in+a+rat+brain+tumor+model.">Bone marrow chemoprotection without compromise of chemotherapy efficacy in a rat brain tumor model.</a> Journal of Pharmacology and Experimental Therapeutics. 309, 594-599 (2004).</li></ol>

Não há conflitos de interesse declarados.

Existem alguns meios de maximizar a eficácia de BBBD. É importante minimizar a hemorragia durante a fase de corte para baixo. A pressão arterial ea freqüência cardíaca pode ser afetada por sangramento substancial e esses fatores são conhecidos por afetar o grau de BBBD 13. Sangramento pode ser reduzido pelo uso de suturas para ligar grandes vasos, tais como o da tiróide superior e as artérias occipitais, as quais devem ser divididas. Além disso, a electrocauterização pode ser usado para dividir vasos e dissecar áreas que têm um fornecimento de sangue rico. É também importante para manter o ar e sólidos de todas as linhas colocadas no animal - particularmente o catéter da carótida. Trabalhar rapidamente com o manitol ajuda a assegurar que os cristais não se formam no interior da seringa ou tubo. É importante a utilização de uma taxa de fluxo de manitol, que é eficaz para o tamanho e estirpe de rato utilizado nesta experiência. A taxa de fluxo da infusão de manitol devem coincidir com a pressão no Caroti comum do ratod artéria tal que uma elevada concentração de fluxos de manitol na ICA sem que flui para baixo a carótida comum para o coração e os pulmões, o que pode causar danos. Pesquisadores a escolha de um tamanho diferente e tensão de rato do que o utilizado neste estudo deve realizar experimentos para encontrar a taxa de fluxo correto manitol. Uma das limitações deste procedimento é que ele requer o sacrifício da ECA. Uma vez que o ECA é cateterizada, torna-se difícil e pouco confiável a cateterização novamente. Assim, este processo não pode ser efectuada duas vezes no mesmo hemisfério. Adicionalmente, os compostos apresentados na sequência BBBD devem ser cuidadosamente testadas para a neurotoxicidade. Algumas drogas que são normalmente seguros tornar-se perigoso, uma vez que atravessa a barreira sangue-cérebro. Apesar destas limitações, BBBD tem sido amplamente utilizado para estudar a entrega melhorada de quimioterapia para tumores cerebrais 8,15-18. Também pode ser útil para outras aplicações terapêuticas em which uma droga eficaz não atravessa a barreira sangue-cérebro e é conhecida a administração de manitol para ser seguro.

<table border="1"><tbody><tr><td> Nome de material </td><td> Companhia </td><td> Número de catálogo </td><td> Comentário </td></tr><tr><td> Ratos Long Evans </td><td> Harlan Laboratories </td><td></td><td> 210-250 g, do sexo masculino </td></tr><tr><td> PE 50 Tubulação </td><td> Beckton-Dickinson </td><td></td><td></td></tr><tr><td> Calibre 18 x 2,5 &quot;cateter IV </td><td> Terumo </td><td></td><td> Para tubo ET </td></tr><tr><td> 30 &quot;extensão IV define </td><td> Abbott </td><td></td><td></td></tr><tr><td> 26 medidor de cateter IV veterinária </td><td> Monoject </td><td></td><td></td></tr><tr><td> O corante azul de Evans </td><td> Sigma </td><td> E2129 </td><td></td></tr><tr><td> Bipolar </td><td> Codman </td><td></td><td></td></tr><tr><td> Filtro, 5 micrómetros </td><td> Braun </td><td></td><td></td></tr></tbody></table>

rat model of blood-brain barrier disruption to allow targeted neurovascular therapeutics

Células endoteliais com junções apertadas, juntamente com a membrana de base e os pés de extremidade de astrócitos rodeiam os vasos sanguíneos cerebrais, para formar a barreira sangue-cérebro 1. A barreira selectivamente exclui moléculas de cruzamento entre o sangue e o cérebro com base no seu tamanho e carga. Esta função pode impedir a entrega de terapêuticas para doenças neurológicas. Um certo número de fármacos quimioterapêuticos, por exemplo, não irá efectivamente atravessar a barreira sangue-cérebro para atingir as células tumorais 2. Assim, melhorando a libertação de fármacos através da barreira sangue-cérebro, é uma área de interesse. Os métodos mais comuns para melhorar a administração de fármacos ao cérebro, são perfusão cerebral directa e barreira hemato-encefálica ruptura 3. Directos garantias de infusão intracerebrais terapias que chegar ao cérebro, no entanto, este método tem uma capacidade limitada para dispersar a droga 4. Hemato-encefálica ruptura da barreira (BBBD) albaixos de drogas para fluir directamente a partir do sistema circulatório no cérebro e, assim, atingir mais eficazmente dispersas células tumorais. Três métodos de interrupção barreira incluem rompimento barreira osmótica, a interrupção barreira farmacológico e ultra-som focalizado com microbolhas. Ruptura osmótica, iniciada por Neuwelt, utiliza uma solução hipertónica de 25% de manitol que desidrata as células da barreira sangue-cérebro, causando-lhes a encolher e perturbam as suas junções apertadas. Ruptura da barreira também pode ser realizada com compostos farmacologicamente vasoactivos, tais como a histamina 5 e 6 de bradicinina. Este método, no entanto, é selectiva em primeiro lugar para a barreira cérebro-tumoral 7. Além disso, RMP-7, um análogo do péptido bradiquinina, verificou-se ser inferior quando comparado cabeça-a-cabeça com BBBD osmótico com manitol 25% 8. Outro método, de ultra-som focado (FUS) em conjunto com agentes de contraste de microbolhas de ultra-som, também tem sido demonstradoa reversivelmente abrir a barreira sangue-cérebro 9. Em comparação com o FUS, porém, manitol 25% tem um longo histórico de segurança em pacientes humanos que o torna uma ferramenta comprovada para pesquisa translacional 10-12. A fim de realizar BBBD, manitol devem ser entregues a uma taxa elevada directamente na circulação arterial cerebral. Em seres humanos, um cateter endovascular é guiado para o cérebro, onde o fluxo rápido e directo pode ser realizado. Este protocolo de modelos humanos BBBD tão perto quanto possível. Na sequência de um corte para baixo em relação à bifurcação da artéria carótida comum, um cateter é inserido dentro da ECA retrógrado e usados ​​para entregar o manitol directamente para dentro da artéria carótida interna (ICA) a circulação. Propofol e N 2 O anestesia são usados ​​para a sua capacidade para aumentar a eficácia de ruptura da barreira 13. Se for executado adequadamente, este procedimento tem a capacidade de segurança, de forma eficaz, e reversivelmente abrir a barreira sangue-cérebro e improve a entrega de medicamentos que normalmente não chegam ao cérebro 8,13,14.

<img alt="Figura 1" src="/files/ftp_upload/50019/50019fig1.jpg" /> Figura 1. Visualizando hematoencefálica ruptura da barreira através de extravasamento de corante azul de Evans. Corante azul de Evans é um pigmento que se liga a albumina e não é extravasado para o cérebro, em condições fisiológicas. O rompimento da barreira sangue-cérebro de um lado do cérebro permite azul de Evans para introduzir e manchar o azul hemisfério interrompido enquanto o hemisfério não-interrompido permanece inalterado. Deste modo, é útil como marcador de sangue-cérebro ruptura da barreira. Figura 1A é um exemplo de um cérebro sem sangue-cérebro ruptura da barreira. A Figura 1B é um exemplo de um azul manchado cérebro após BBBD osmótica usando 25% de manitol. Note-se que um hemisfério está tingido de azul, enquanto a outra permanece imaculada. Alguns azul entrou no hemisfério esquerdo, perto da fenda longitudinal medial, como resultado da mistura do fluxo de sangue no instante tele Círculo de Willis.

Watch this Scientific Journal Video about Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics at JoVE.com

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Hemato-encefálica ruptura da barreira auxilia a distribuição de certas drogas para o cérebro. Manitol entregue intra-arterialmente células vizinhas encolhe vasos sanguíneos, a fim de interromper a barreira física.

Modelo de ratos de sangue-cérebro Rompimento de barreira para permitir alvejado Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

rat-model-blood-brain-barrier-disruption-to-allow-targeted

Nanyang Technological University

Research

JoVE Journal

Medicine

19.3K visualizações.  Vanderbilt University School of Medicine. Hemato-encefálica ruptura da barreira auxilia a distribuição de certas drogas para o cérebro. Manitol entregue intra-arterialmente células vizinhas encolhe vasos sanguíneos, a fim de interromper a barreira física.

Vídeo: Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Guiada por MRI rompimento da barreira hemato-encefálica utilizando Ultrassom Focalizado transcraniana em um modelo de rato

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Medicina

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

A administração intranasal de Terapêutica do SNC para Ratos Awake

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Estique em células cerebrais endoteliais microvasculares (cEnd) como um<em&gt; In Vitro</em&gt; Modelo de lesão cerebral traumática do cérebro Barreira Hemato

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

Criação de um<em&gt; In Vitro</em&gt; Modelo de Rato barreira hemato-encefálica (BBB): A Focus on BBB impermeabilidade e Transportes Receptor mediada

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Oxigênio-Glucose Privação e reoxigenação como um<em&gt; In Vitro</em&gt; Isquemia e reperfusão Lesão modelo para estudar Disfunção Barreira Hematoencefálica

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

A Rat carótida Balão Injury modelo para testar Therapeutics remodela Anti-vasculares

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Retroductal submandibular Glândula instilação e localizada fracionado irradiação em um modelo de rato de salivar Hipofunção

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Ensaios In Vitro para Avaliar Formação e Perturbação de Embarque de Barreira de Sangue-Cérebro

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Melhor método para o estabelecimento de uma In Vitro barreira hemato - encefálica modelo baseado em células endoteliais do cérebro de suínos

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...