Os autores gostariam de agradecer Shawna Rideout-Gros e Garces Alexandra para a sua ajuda com o cuidado de animais, e Wu Ping para sua assistência técnica. O suporte para este trabalho foi fornecido pelo Instituto Nacional de Saúde sob concessão não. EB003268, eo Programa de Pesquisa do Canadá Presidentes.

1. Ultra-sonografia e ressonância magnética de configuração Uma ressonância magnética compatível com três eixos do sistema de ultra-som foi utilizado neste estudo (FUS Instruments, Inc., Toronto, Ontario, Canadá). Dois transdutores de ultra-som foram utilizados diferentes: um in-house construído 551,5 kHz transdutor esfericamente focada (raio de curvatura = 60 mm, diâmetro externo = 75 mm, diâmetro interno = 20 mm), e uma MHz 1,503, matriz 8-sector com integrado PZT hidrofone (Imasonic Inc., Voray-sur-L&#39;Orgnon, França) dirigido como um único elemento transdutor esfericamente focado (FN = 0,8, abertura = 10 cm). Uma ressonância magnética compatível com PVDF receptor 8 foi utilizado para gravar emissões acústicas quando o transdutor kHz 551,5 foi usado. Se o equipamento for utilizado outro, são sugeridas as seguintes: <ol><li> Seleccionar um adequado, transdutor de ultra-som calibrado com uma frequência central na gama MHz MHz 0,25 -1,5 e um número f-(raio de curvatura / abertura) de 1 ou menos. </li><li> Coloque um fi banho de águapreencheram com água morna, desgaseificado, água deionizada na cama de um T 1,5 ou 3 T MRI. O banho de água deve ter uma placa de topo, que pode segurar os animais. Montar o transdutor de ultra-som no tanque em uma fase de posicionamento MRI compatível de três eixos ou sistema de 9, de frente para a superfície da água. </li><li> Executar o cabo do transdutor para o equipamento de ultra-som de condução através de um painel de penetração. </li><li> Gerar o sinal de condução do transdutor usando um gerador de função e amplificador de potência. Use um circuito externo de correspondência para minimizar a energia reflectida eléctrica. </li><li> Sabendo o comprimento focal transdutor, coloque o foco transdutor na superfície da água e sonicar da superfície da água para gerar uma fonte de água visível. Passos 1,6 e 1,7 descrevem como registrar o foco usando a fonte de água gerado. Um método mais preciso para localizar o foco, o qual envolve sonicando um calor de absorção de gel e de imagem a elevação da temperatura resultante no foco com RM, podem ser encontrados em 9 &lt;/ Sup&gt;. </li><li> Marcar a posição do foco usando um marcador que será visível nas imagens de RM. Isto pode ser feito usando uma placa com um orifício central que vai encher com água quando colocado sobre o foco. O buraco cheio de água será visível, na ressonância magnética e proporcionará coordenadas focais em dois planos, enquanto que a coordenada na direcção axial do transdutor pode ser determinada a partir da superfície da água. </li><li> Usando uma seqüência localizador 3-plano, a imagem do marcador de foco e registrar a localização do foco no sistema de coordenadas de ressonância magnética. </li><li> Para monitorizar as emissões acústicas, montar um hidrofone 8 MR-compatível que é usado como um detector de cavitação passiva (PCD) no banho de água dirigido para o foco do transdutor, ou utilizar um transdutor com hidrofone integrado. </li><li> Executar os cabos de PCD para um cartão de escopo que é suficientemente rápida para capturar rajadas de até 10 ms a PRF até 2 Hz (por exemplo, ATS460, AlazarTech, Pointe-Claire, Quebec, Canadá). Os cabos devem ser aterrado para a penetração painel ou RF escudo para minimizar o ruído. </li></ol> 2. Preparação dos animais <ol><li> Anestesiar os animais utilizam o gás isoflurano. Uma vez que isofluorano tem um efeito sobre BBB ruptura 10, os animais devem ser tomadas fora do gás de 10 minutos antes do início da experiência. O local de pomada lubrificante ocular em cada olho, no início da anestesia para proteger a córnea de dessecação ou outras lesões. </li><li> Usando um barbeador elétrico raspar a pele da parte superior da cabeça do animal e do pescoço, em seguida, remova a pele restante usando um creme depilatório (por exemplo, Nair, Church &amp; Dwight Co., Princeton, NJ, EUA) e lavar o couro cabeludo com sabão neutro e água. </li><li> Preparar a cauda com um esfrega pele betadine seguido por uma lavagem bridine. Executar um wipedown final com álcool, a fim de visualizar a veia da cauda. </li><li> Inserir um cateter de calibre 22 com 3-forma torneira na veia da cauda e lavar com uma mistura de heparina / solução salina (33 U / mL) para evitar a formação de coágulosno cateter. </li><li> Entregar anestésicos injectáveis ​​(40-50 mg de cetamina / kg, 10 mg / kg de xilazina), através de injecção intramuscular, e remover o animal a partir do isofluorano. </li><li> Coloque a supina animal anestesiado sobre o sistema de posicionamento de ultra-som com a parte superior da cabeça contactando o banho de água através de um furo no topo da placa-(Fig.1). Uma pequena quantidade de ultra-som gel talvez aplicado ao topo da cabeça do animal para minimizar a possibilidade de airbubbles aprisionados. </li><li> Tape as pernas para o sistema de posicionamento. A cabeça pode ser mantida no lugar usando uma barra de mordida, se disponível, ou fita colocada firmemente em todo o queixo. </li><li> Cobrir o animal com uma toalha e cobertor circulação de água, a fim de mantê-lo aquecido. </li></ol> 3. Target Selection <ol><li> Adquirir base axial T 2-T imagens ponderadas e 1-RM ponderada do cérebro. Se estiver usando ressonância magnética de 1,5 T e dedicado a cabeça do tamanho de bobina de superfície RF-receber, adequado scum parâmetros podem ser: T 2 ponderada: FSE, TE = 60 ms, TR = 2000 ms T 1 ponderada: FSE, TE = 10 ms, TR = 500 ms </li><li> Selecione o destino a partir do T 2-ponderadas varreduras, evitando os ventrículos ea linha média do cérebro, e selecionando uma profundidade média do cérebro. </li><li> Mova o foco do transdutor para o local de destino. </li></ol> 4. Preparação de microbolhas Microbolhas Definity (Lantheus Medical Imaging, MA, EUA) são utilizados por vários grupos de FUS mediadas microbolhas induzidas BBBD 2,5,7. Dosagens adequadas para outros tipos de microbolhas pode ser encontrada na literatura 11,12. <ol><li> Activar as microbolhas Definity e lentamente elaborar um pequeno volume para uma seringa de 1 mL utilizando uma agulha de 18 gauge. </li><li> Remover ar preso da seringa delicadamente movendo o êmbolo para trás e para frente. Não toque na seringa, pois isso pode romper as bolhas. </li><li> Diluir o Definity em condições normais<html> solução salina numa proporção de 10:01 de solução salina para Definty injetando-se lentamente o volume necessário de Definity para uma seringa de soro fisiológico. Se uma entrega de infusão é usado, o Definity pode ser ainda mais diluída, a 50:1 ou 100:1. </li><li> Inverta suavemente a seringa para misturar completamente o Definity e solução salina até uma aparência mesmo seja alcançado. Inversão suave da seringa imediatamente antes da injecção pode também ser necessário se as bolhas começaram a flutuar para fora da suspensão. </li><li> Calcular o volume da dose requerida com base em 0,02 mL / kg de Definity, ou 0,2 ml / kg da solução (menos 10:1 de diluição). </li></ol> 5. Entrega de ultra-som <ol><li> Defina os parâmetros ultra-som, usando rajadas de direito baixos do ciclo, não sonicações onda contínua. Parâmetros de sonicação adequadas a 0,5 MHz são 0,23 MPa de pressão situ, 10 rajadas ms a 1 Hz PRF, 2 min. tempo de sonicação total. Em 1,5 MHz adequado das pressões in situ cair em torno de 0,45-0,5 MPa. </li></ol>_content &quot;&gt; pressões adequadas em diferentes freqüências pode ser estimada utilizando um índice de mecânica de 0,46 13. <ol start="2"><li> Verificar que a cabeça do animal é ainda acoplado à água. </li><li> Injectar as microbolhas lentamente o cateter da veia da cauda e lave com 0,5 mL de solução salina normal. Começar a sonicação em simultâneo com o início da injecção. </li></ol> 6. Avaliação da ressonância magnética Resultado de Tratamento <ol><li> Após a sonicação, injectar um agente de contraste para IRM (por exemplo, 0,2 ml / kg Omniscan, a GE Healthcare) através do cateter da veia da cauda seguido por uma lavagem de 0,5 ml de solução salina. </li><li> Realizar T de imagem 1-ponderada até que o pico de contraste tenha passado. Sítios sonicação que tenham sido rompidos com sucesso vai mostrar maior reforço do que o tecido circundante. </li><li> Execute T imagens 2-ponderada para verificar se há edema. Sinal de alta nos locais de sonicação é indicativo de edema. </li></ol> 7. RepresentanteResultados Agentes de contraste para IRM pode ser entregue com êxito através do BBB com ultra-som focalizado e circulando microbolhas. A Figura 2 mostra pré e pós-típico FUS T 1-w imagens. A Figura 2B mostra um contraste maior (CE) T 1-w imagem com distintas aberturas focais em quatro locais sonicados. Sonicação locais 1 e 2 mostram uma intensificação particularmente brilhante. Na Fig.3 locais 1 e 2 também pode ser visto para corresponder com T 2-w sinal elevada, indicando edema. A extensão de T 2-w edema pode por vezes ser mais facilmente visualizada em fatias sagital. A Figura 4 mostra CE-T 1-w e T 2-w fatias sagital através de sonicação locais 1 e 3. Edema é visível na posição 1, mas não a localização 3. A análise espectral dos capturados dados acústicos de emissões (Fig. 5) pode mostrar emissões harmônicas e / ou sub / ultra emissões harmônicas quando cavitação estável está ocorrendo. Harmonics também pode surgir a partir de tecido não-linearidades, enquanto que as emissões sub e ultraharmonic só pode ocorrer como um resultado da actividade de bolha 14. A pressões mais elevadas emissões de banda larga, indicando cavitação inercial pode também ser detectado. No entanto, estes têm sido associados com maiores quantidades de extravasamento dos glóbulos vermelhos e microdanos do que sonicações sem cavitação inercial 11. A utilização de frequências mais elevadas sonicação resulta em aberturas mais localizadas, devido ao tamanho mais pequeno ponto focal. A Figura 6 mostra que as frequências mais altas podem ser utilizadas para criar regiões mais pequenas de abertura. Isso permite que a investigação de efeitos meados do cérebro com menos perto do crânio efeitos. <img alt="Figura 1." src="/files/ftp_upload/3555/3555fig1.jpg" /> Figura 1. Configuração experimental. <spuma class = &quot;pdflinebreak&quot;&gt; <img alt="Figura 2." src="/files/ftp_upload/3555/3555fig2.jpg" /> Figura 2. Pre (à esquerda) e pós (à direita) tratamento T 1-w imagens de um cérebro de rato mostrando aumento em quatro locais de sonicação. <img alt="Figura 3." src="/files/ftp_upload/3555/3555fig3.jpg" /> Figura 3. Pré (à esquerda) e pós (à direita) tratamento T 2-w imagens de um cérebro de rato (mesmo animal como Fig.2) mostrando T 2-w edema em locais de sonicação 1 e 2. <img alt="Figura 4." src="/files/ftp_upload/3555/3555fig4.jpg" /> Figura 4. Pós-tratamento sagital T 1-w (esquerda) e T 2-w (direita) a partir de imagens do cérebro de rato mesma Figs. 2 e 3. A abertura no local 1 (esquerda) correlaciona-se com T 2-w edema (direita). Localização 3 mostra edema abertura (à esquerda), mas não T 2-w. </p&gt; <img alt="Figura 5." src="/files/ftp_upload/3555/3555fig5.jpg" /> Figura 5. Espectro de freqüência a partir de dados capturados durante uma única explosão de 10 ms a 551,5 kHz. A frequência fundamental (f 0), bem como harmónicas (2 f 0) e sub / ultraharmonics (0,5 f 0, 1,5 f 0) são visíveis. <img alt="Figura 6." src="/files/ftp_upload/3555/3555fig6.jpg" /> Figura 6. Pós-tratamento CE-T 1-w axial (à esquerda) e sagital (direita) imagens de um cérebro de rato sonicado em quatro locais em 1,503 MHz. Aberturas BBB neste frequência são vistos como sendo mais localizada.

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K. e R. Hynynen Chopra são co-fundadores da FUS Instruments, Inc. R. Chopra, A. Waspe e K. Hynynen são acionistas em Instrumentos FUS, Inc. K. Hynynen recebe suporte de pesquisa de instrumentos FUS, Inc.

Preparação dos animais e microbolhas são os aspectos mais críticos deste procedimento. O cabelo na cabeça do animal deve ser completamente removido para evitar atenuante do feixe de ultra-som. O BBB pode ser interrompido sob isofluorano anestésico, no entanto, torna-se mais difícil de obter uma abertura consistente. As microbolhas devem sempre ser manuseados com cuidado e uma bitola de pequeno, as agulhas de grande diâmetro deve ser usado quando a elaboração, a fim de evitar a queb...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Pequenos Animais Focada sistema de ultra- </td><td> FUS Instruments, Inc. </td><td> RK-100 </td></tr><tr><td> Definity </td><td> Lantheus Medical Imaging </td><td></td></tr><tr><td> Omniscan </td><td> GE Healthcare </td><td></td></tr></tbody></table>

mri-guided disruption of the blood-brain barrier using transcranial focused ultrasound in a rat model

Ultra-som concentrado ruptura (FUS) da barreira sangue-cérebro (BBB) ​​é uma técnica cada vez mais investigada para contornar a 1-5 BBB. O BBB é um obstáculo significativo para tratamentos farmacêuticas de distúrbios cerebrais em que limita a passagem de moléculas da vasculatura dentro do tecido cerebral a moléculas inferior a cerca de 500 Da em tamanho 6. FUS induzida perturbação BBB (BBBD) é temporária e reversível 4 e tem uma vantagem sobre química meio de induzir BBBD por ser altamente localizado. FUS induzida BBBD proporciona um meio para investigar os efeitos de uma ampla gama de agentes terapêuticos sobre o cérebro, o que não seria de outro modo ser fornecida ao tecido em concentração suficiente. Enquanto uma vasta gama de parâmetros de ultra-som têm sido bem sucedidos de perturbar o BBB 2,5,7, existem vários passos críticos no procedimento experimental para assegurar o rompimento de sucesso com a segmentação precisas. Este protocolocontornos ol como conseguir guiada por MRI FUS induzidas BBBD em um modelo de rato, com um foco na preparação animal crítica e etapas de manuseio de microbolhas do experimento.

Watch this Scientific Journal Video about MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model at JoVE.com

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

Microbolhas mediada ruptura ultra-som focalizado da barreira sangue-cérebro (BBB) ​​é uma técnica promissora para não-invasivo de entrega de drogas alvo no cérebro<sup&gt; 1-3</sup&gt;. Este protocolo descreve o procedimento experimental para guiada por MRI perturbação BBB transcraniana num modelo de rato.

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 ...

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17.8K visualizações.  Sunnybrook Research Institute. Microbolhas mediada ruptura ultra-som focalizado da barreira sangue-cérebro (BBB) ​​é uma técnica promissora para não-invasivo de entrega de drogas alvo no cérebro 1-3. Este protocolo descreve o procedimento experimental para guiada por MRI perturbação BBB transcraniana num modelo de rato.

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

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multi-modal de imagem de angiogênese em um modelo de rato nu de Câncer de mama utilizando a ressonância magnética, tomografia computadorizada volumétrica e ultra-som

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Medicina

Rat Model of Blood-brain Barrier Disruption to Allow Targeted 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 barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

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 ...

A Model of Chronic Nutrient Infusion in the Rat

Chronic exposure to excessive levels of nutrients is postulated to affect the function of several organs and tissues and to contribute to the development of the many complications associated with obesity and the metabolic syndrome, including type 2 diabetes. To study the mechanisms by which excessive levels of glucose and fatty acids affect the pancreatic beta-cell and the secretion of insulin, we have established a chronic nutrient infusion model in the rat. The procedure consists of catheterizing the right jugular vein and left carotid artery under general anesthesia; allowing a 7-day recuperation period; connecting the catheters to the pumps using a swivel and counterweight system that enables the animal to move freely in the cage; and infusing glucose and/or Intralipid (a soybean oil emulsion which generates a mixture of approximately 80% unsaturated/20% saturated fatty acids when infused with heparin) for 72 hr. This model offers several advantages, including the possibility to finely modulate the target levels of circulating glucose and fatty acids; the option to co-infuse pharmacological compounds; and the relatively short time frame as opposed to dietary models. It can be used to examine the mechanisms of nutrient-induced dysfunction in a variety of organs and to test the effectiveness of drugs in this context.

A protocol for chronic infusions of glucose and Intralipid in rats is described. This model can be used to study the impact of nutrient excess on organ function and physiological parameters.

Um Modelo de Infusão Crónica de nutrientes no Rato

Chronic exposure to excessive levels of nutrients is postulated to  affect the function of several organs and tissues and to contribute to the  ...

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 ...

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol.
This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Measuring Ascending Aortic Stiffness In Vivo in Mice Using Ultrasound

We describe a technique for measuring aortic stiffness from its pressure-diameter relationship in vivo in mice. Aortic diameter is recorded by ultrasound and aortic pressure is measured invasively with a solid-state pressure catheter. Blood pressure is changed incrementally and the resulting diameter is measured.

Medição da aorta ascendente Rigidez<em&gt; In Vivo</em&gt; Em ratos usando ultra-som

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound ...

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 ...

MRI-guided dmPFC-rTMS as a Treatment for Treatment-resistant Major Depressive Disorder

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial prefrontal cortex (dmPFC) in patients with major depressive disorder (MDD). Technicians used a neuronavigation system to process patient MRIs to generate a 3-dimensional head model. The head model was subsequently used to identify patient-specific stimulatory targets. The dmPFC was stimulated daily for 20 sessions. Stimulation intensity was titrated to address scalp pain associated with rTMS. Weekly assessments were conducted on the patients using the Hamilton Rating Scale for Depression (HamD17) and Beck Depression Index II (BDI-II). Treatment-resistant MDD patients achieved significant improvements on both HAMD and BDI-II. Of note, angled, double-cone coil rTMS at 120% resting motor threshold allows for optimal stimulation of deeper midline prefrontal regions, which results in a possible therapeutic application for MDD. One major limitation of the rTMS field is the heterogeneity of treatment parameters across studies, including duty cycle, number of pulses per session and intensity. Further work should be done to clarify the effect of stimulation parameters on outcome. Future dmPFC-rTMS work should include sham-controlled studies to confirm its clinical efficacy in MDD.

Here we outline the procedure for MRI-guided repetitive transcranial magnetic stimulation to the dorsomedial prefrontal cortex as an experimental treatment for major depressive disorder.

Guiada por MRI dmPFC-EMTr como um tratamento para o Transtorno Depressivo Maior resistente ao tratamento

Here we outline the protocol for magnetic resonance imaging (MRI) guided repetitive transcranial magnetic stimulation (rTMS) to the dorsal medial ...

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) ...