Les auteurs tiennent à remercier Shawna Rideout-Gros et Garces Alexandra pour leur aide avec le soin des animaux, et Ping Wu, pour son aide technique. Prise en charge de ce travail a été fourni par le National Institutes of Health sous la concession no. EB003268, et le Canada des chaires de recherche du programme.

1. L&#39;échographie et l&#39;IRM de configuration Une IRM-compatible à trois axes du système d&#39;ultrasons focalisés a été utilisée dans cette étude (FUS Instruments, Inc, Toronto, Ontario, Canada). Deux transducteurs à ultrasons différents ont été utilisés: un dans la maison construite 551,5 kHz sphérique centrée transducteur (rayon de courbure = 60 mm, diamètre extérieur = 75 mm, diamètre intérieur = 20 mm), et une MHz 1,503, 8-secteur de tableau avec intégré PZT hydrophone (Imasonic Inc, Voray-sur-L&#39;Orgnon, France), entraîné comme un élément sphérique seul transducteur focalisé (FN = 0,8, ouverture = 10 cm). Une IRM-compatible PVDF récepteur 8 a été utilisé pour enregistrer les émissions acoustiques lorsque le transducteur 551,5 kHz a été utilisé. Si l&#39;équipement est utilisé, il est suggéré de: <ol><li> Sélectionnez une manière appropriée, calibrée transducteur à ultrasons avec une fréquence centrale de la fourchette de 0,25 MHz -1,5 et un nombre f (rayon de courbure / ouverture) de 1 ou moins. </li><li> Placez un bain d&#39;eau filled tiède, dégazé, de l&#39;eau déminéralisée sur le lit d&#39;un T 1,5 ou 3 T IRM. Le bain d&#39;eau doit avoir une plaque supérieure, qui peut contenir les animaux. Monter la sonde d&#39;échographie dans le réservoir sur une scène IRM positionnement compatible à trois axes ou d&#39;un système 9, face à la surface de l&#39;eau. </li><li> Exécutez le câble du transducteur à l&#39;équipement de conduite ultrasons à travers un panneau de pénétration. </li><li> Générer le signal de commande du transducteur en utilisant un générateur de fonctions et amplificateur de puissance. Utilisation d&#39;un circuit externe appariement à minimiser la puissance électrique réfléchi. </li><li> Connaissant la longueur focale du transducteur, placer l&#39;accent transducteur à la surface de l&#39;eau et sonifier la surface de l&#39;eau pour générer une fontaine d&#39;eau visible. Étapes 1.6 et 1.7 décrivent comment enregistrer le focus à l&#39;aide de la fontaine d&#39;eau généré. Une méthode plus précise pour localiser le foyer, ce qui implique une sonication absorbant la chaleur de gel et de l&#39;imagerie de l&#39;élévation de température résultant à la mise au point avec l&#39;IRM, peut être trouvé dans 9 &lt;/ Sup&gt;. </li><li> Marquez l&#39;emplacement du foyer à l&#39;aide d&#39;un marqueur qui sera visible sur les images IRM. Cela peut être fait en utilisant une plaque avec un trou au centre qui permettra de combler avec de l&#39;eau lorsqu&#39;il est placé sur la mise au point. Le trou rempli d&#39;eau sera visible sur l&#39;IRM et fournir des coordonnées dans deux plans focaux, tandis que le coordonnée dans la direction axiale du transducteur peut être déterminée à partir de la surface de l&#39;eau. </li><li> Grâce à une séquence alignement 3-plan, l&#39;image du marqueur mise au point et enregistrer l&#39;emplacement de l&#39;accent mis dans le système de coordonnées IRM. </li><li> Pour surveiller les émissions acoustiques, monter un 8 hydrophone compatible IRM qui est utilisé comme un détecteur de cavitation passive (PCD) dans le bain d&#39;eau dirigé au foyer du transducteur, ou utiliser un transducteur avec hydrophone intégré. </li><li> Faites passer les câbles de la CPD pour une carte de la portée qui est suffisamment rapide pour capturer les éclats de jusqu&#39;à 10 ms à PRF jusqu&#39;à 2 Hz (par exemple ATS460, AlazarTech, Pointe-Claire, Québec, Canada). Les câbles doivent être mis à la pénétrationtration panneau ou blindage RF pour minimiser le bruit. </li></ol> 2. Préparation des animaux <ol><li> Anesthésier les animaux au moyen de gaz isofluorane. Depuis isofluorane a un effet sur ​​rupture de la BHE 10, les animaux devraient être retirés des gaz 10 minutes avant le début de l&#39;expérience. Pommade oculaire lubrifiant Lieu de chaque oeil au début de l&#39;anesthésie pour protéger la cornée de la dessiccation ou de blessure. </li><li> Utiliser un rasoir électrique raser la fourrure du haut de la tête de l&#39;animal et le cou, puis retirez la fourrure restant en utilisant une crème dépilatoire (par exemple Nair, Church &amp; Dwight Co., Princeton, NJ, Etats-Unis) et rincer le cuir chevelu avec un savon doux et de l&#39;eau. </li><li> Préparer la queue avec un gommage bétadine suivi par un lavage Bridine. Effectuer une wipedown final à l&#39;alcool afin de visualiser la veine de la queue. </li><li> Insérer un cathéter de calibre 22 avec robinet 3 voies dans la veine de la queue et rincer avec un mélange de saline / héparine (33 U / ml) pour éviter la formation de caillotsdans le cathéter. </li><li> Livrer anesthésiques injectables (40-50 mg de kétamine / kg, 10 mg / kg de xylazine) par injection intramusculaire, et enlever l&#39;animal de l&#39;isofluorane. </li><li> Placez le dos animal anesthésié sur le système de positionnement par ultrasons avec le haut de la tête de contact du bain-marie à travers un trou dans la plaque supérieure (Fig.1). Une petite quantité de gel à ultrasons peut être appliquée à la partie supérieure de la tête de l&#39;animal afin de minimiser le risque de bulles d&#39;air piégées. </li><li> Collez les jambes pour le système de positionnement. La tête peut être maintenue en place en utilisant soit un bar morsure, si disponible, ou un ruban placé fermement sur le menton. </li><li> Couvrir l&#39;animal avec une serviette et faire circuler l&#39;eau dans une couverture afin de le garder au chaud. </li></ol> 3. Target Selection <ol><li> Acquérir de référence axiale T 2-pondérées et T 1-pondérées des images IRM du cerveau. Si vous utilisez un 1,5 T dédié d&#39;IRM et de taille d&#39;une tête bobine de surface RF-recevoir, adaptée scun des paramètres pourrait être: Pondérée T 2: FSE, TE = 60 ms, TR = 2000 ms Pondérée T 1: FSE, TE = 10 ms, TR = 500 ms </li><li> Sélectionnez la cible de la T 2-pondérées des analyses, en évitant les ventricules et la ligne médiane du cerveau, et en sélectionnant une profondeur mi-cerveau. </li><li> Déplacer le focus transducteur à l&#39;emplacement cible. </li></ol> 4. Préparation des microbulles Microbulles Definity (Lantheus Medical Imaging, MA, Etats-Unis) sont utilisés par plusieurs groupes pour le FUS médiation microbulles induites BBBD 2,5,7. Les doses appropriées pour d&#39;autres types de microbulles peuvent être trouvés dans la littérature 11,12. <ol><li> Activer les microbulles Definity et lentement élaborer un petit volume dans une seringue de 1 ml à l&#39;aide d&#39;une aiguille de calibre 18. </li><li> Retirer l&#39;air emprisonné dans la seringue en faisant bouger le piston d&#39;avant en arrière. Ne touchez pas la seringue comme cela peut casser les bulles. </li><li> Diluer le Definity dans des conditions normales<html> une solution saline dans un rapport de 10:1 à une solution saline Definty par injection lentement le volume requis de Definity dans une seringue d&#39;une solution saline. Si une prestation de perfusion est utilisé, l&#39;Definity peut être diluée, à 50:1 ou 100:1. </li><li> Inverser doucement la seringue pour bien mélanger le Definity et une solution saline jusqu&#39;à un aspect encore est atteint. Inversion douce de la seringue immédiatement avant l&#39;injection peut également être nécessaire si les bulles ont commencé à flotter hors de la suspension. </li><li> Calculer le volume de la dose requise en fonction de 0,02 mL / kg de Definity, soit 0,2 mL / kg de la solution (à 10:1 de dilution). </li></ol> 5. Livraison ultrasons <ol><li> Définissez les paramètres de sonication, en utilisant de faibles éclats de cycles d&#39;usage, et non sonications ondes continues. Paramètres de sonication appropriées à 0,5 MHz sont MPa 0,23 en pression in situ, 10 coups ms à 1 Hz PRF, 2 min. temps de sonication totale. A 1,5 MHz appropriée des pressions in situ tomber autour de 0,45 à 0,5 MPa. </li></ol>_content &quot;&gt; pressions appropriées à différentes fréquences peut être estimée à l&#39;aide d&#39;un index mécanique de 0,46 13. <ol start="2"><li> Vérifiez que la tête de l&#39;animal est toujours couplé à l&#39;eau. </li><li> Injecter des microbulles lentement dans le cathéter dans la veine caudale et rincer avec 0,5 ml solution saline normale. Commencer la sonication simultanément avec le début de l&#39;injection. </li></ol> 6. Évaluation IRM des résultats du traitement <ol><li> Après la sonication, injecter un agent de contraste IRM (par exemple 0,2 ml / kg Omniscan, GE Healthcare) par l&#39;intermédiaire du cathéter dans la veine caudale suivie d&#39;un rinçage de 0,5 ml solution saline. </li><li> Effectuer pondération T 1 jusqu&#39;à ce que la crête d&#39;imagerie de contraste s&#39;est écoulé. Les sites sonication qui ont été avec succès perturbé montrera une meilleure valorisation que le tissu environnant. </li><li> Effectuer T 2-imagerie pondérée pour vérifier l&#39;œdème. Signal de haute sur les sites de sonication est indicative de l&#39;œdème. </li></ol> 7. ReprésentantRésultats Agents de contraste IRM peut être livré avec succès à travers la BHE en utilisant des ultrasons focalisés et la diffusion des microbulles. La figure 2 montre pré et post-classique FUS T 1-w images. Figure 2B montre un contraste amélioré (CE) T 1-w image avec distinctes ouvertures focaux dans quatre endroits soniqués. Endroits sonication 1 et 2 montrent un rehaussement particulièrement lumineux. En Fig.3 emplacements 1 et 2 peuvent également être vu de correspondre avec T 2-w signal élevé, indiquant un œdème. La mesure de T 2-w oedème peut parfois être plus facilement visualisés sur des tranches sagittales. La figure 4 montre CE-T 1-T 2 w et-w coupes sagittales par endroits sonication 1 et 3. L&#39;œdème est visible à l&#39;emplacement 1, mais pas l&#39;emplacement 3. L&#39;analyse spectrale des données capturées émissions acoustiques (Fig.5) peut montrer des émissions harmoniques et / ou sous / ultra émissions d&#39;harmoniques lorsque cavitation stable se produit. HarmonICS peuvent aussi survenir à partir de tissus non-linéarités, tandis que les émissions sous et ultraharmonic ne peut se produire comme un résultat de 14 l&#39;activité bulle. A des pressions plus élevées des émissions à large bande indiquant la cavitation inertielle peuvent également être détectées. Cependant ceux-ci ont été associés à de plus grandes quantités de rouge extravasations de globules et microlésions que sonications sans cavitation inertielle 11. L&#39;utilisation de fréquences sonication des résultats plus élevés dans les ouvertures plus localisées dues à la taille plus petite tache focale. La figure 6 montre que les fréquences plus élevées peuvent être utilisées pour créer de plus petites régions de l&#39;ouverture. Cela permet étude des effets à mi-cerveau avec moins de quasi-crâne effets. <img alt="Figure 1." src="/files/ftp_upload/3555/3555fig1.jpg" /> Figure 1. Configuration expérimentale. <spune classe = &quot;pdflinebreak&quot;&gt; <img alt="Figure 2." src="/files/ftp_upload/3555/3555fig2.jpg" /> Figure 2. Pré (à gauche) et après traitement (droite) T 1-w images d&#39;un cerveau de rat montrant l&#39;amélioration à quatre endroits sonication. <img alt="Figure 3." src="/files/ftp_upload/3555/3555fig3.jpg" /> Figure 3. Pré (à gauche) et après traitement (droite) T 2-w images d&#39;un cerveau de rat (même animal que Fig.2) montrant T 2-w oedème à des endroits sonication 1 et 2. <img alt="Figure 4." src="/files/ftp_upload/3555/3555fig4.jpg" /> Figure 4. Post-traitement sagittale T 1-w (à gauche) et T 2-w (à droite) des images du cerveau de rat même que les figures. 2 et 3. L&#39;ouverture à l&#39;emplacement 1 (à gauche) est en corrélation avec T 2-w oedème (à droite). Localisation La figure 3 montre un œdème ouverture (à gauche), mais pas T 2-w. </p&gt; <img alt="Figure 5." src="/files/ftp_upload/3555/3555fig5.jpg" /> Figure 5. Du spectre des fréquences à partir des données saisies au cours d&#39;une salve unique de 10 ms à 551,5 kHz. La fréquence fondamentale (f 0) ainsi que les harmoniques (2 f 0) et sous ou ultraharmonics (0,5 f 0, f 0 1,5) sont visibles. <img alt="Figure 6." src="/files/ftp_upload/3555/3555fig6.jpg" /> Figure 6. Post-traitement CE-T 1-w axiale (à gauche) et sagittale (à droite) des images à partir d&#39;un cerveau de rat soniquée dans quatre endroits au 1,503 MHz. Ouvertures BBB à cette fréquence sont considérés comme plus localisée.

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K. et R. Hynynen Chopra sont co-fondateurs de FUS Instruments, Inc R. Chopra, A. et K. Waspe Hynynen sont actionnaires dans des instruments du FUS, Inc K. Hynynen reçoit le soutien de la recherche à partir FUS instruments, Inc

Préparation des animaux et de microbulles sont les aspects les plus critiques de cette procédure. Les cheveux sur la tête de l&#39;animal doivent être complètement enlevées pour éviter d&#39;atténuer le faisceau d&#39;ultrasons. Le BBB peut être perturbé sous anesthésie isofluorane, cependant, il devient plus difficile de réaliser une ouverture constante. Les microbulles doit toujours être manipulé avec soin et de petit calibre, les aiguilles de grand diamètre doivent être ut...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Small Animal Système à ultrasons focalisés </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

Ultrasons focalisés (FUS) la perturbation de la barrière hémato-encéphalique (BHE) est une technique de plus en plus d&#39;une enquête pour contourner le 1-5 BBB. La BHE est un obstacle important à des traitements pharmaceutiques de troubles du cerveau car elle limite le passage des molécules de la vascularisation dans le tissu cérébral de molécules inférieur à environ 500 Da en taille 6. FUS induit des perturbations BBB (BBBD) est temporaire et réversible 4 et a un avantage sur des moyens chimiques d&#39;induire BBBD en étant très localisée. FUS induit BBBD fournit un moyen pour étudier les effets d&#39;une large gamme d&#39;agents thérapeutiques sur le cerveau, ce qui ne serait autrement pas livrable pour le tissu à une concentration suffisante. Bien qu&#39;un large éventail de paramètres échographiques ont fait leurs preuves à perturber le BBB 2,5,7, il ya plusieurs étapes critiques dans la procédure expérimentale afin de perturber le succès avec un ciblage précis. Ce protocolecontours ol comment parvenir à l&#39;IRM-guidées FUS induites BBBD dans un modèle de rat, avec un accent sur la préparation des animaux critique et les étapes de manipulation de microbulles de l&#39;expérience.

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

Microbulles médiée par la perturbation des ultrasons focalisés de la barrière hémato-encéphalique (BHE) est une technique prometteuse pour les non-invasive administration ciblée de médicaments dans le cerveau<sup&gt; 1-3</sup&gt;. Ce protocole décrit la procédure expérimentale pour guidée par IRM BBB perturbations transcrânienne dans un modèle de rat.

Guidée par IRM rupture de la barrière hémato-encéphalique en utilisant transcrânienne d&#39;ultrasons focalisés dans un modèle de rat

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

mri-guided-disruption-blood-brain-barrier-using-transcranial-focused

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17.8K vues.  Sunnybrook Research Institute. Microbulles médiée par la perturbation des ultrasons focalisés de la barrière hémato-encéphalique (BHE) est une technique prometteuse pour les non-invasive administration ciblée de médicaments dans le cerveau 1-3. Ce protocole décrit la procédure expérimentale pour guidée par IRM BBB perturbations transcrânienne dans un modèle de rat.

Vidéo: 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-modale d&#39;imagerie de l&#39;angiogenèse dans un modèle de rat nu des métastases osseuses du cancer du sein par imagerie par résonance magnétique, la tomodensitométrie et volumétrique ultrasons

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

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Médecine

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.

Modèle Rat de rupture de la barrière hémato-encéphalique pour permettre ciblée Therapeutics neurovasculaire

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.

Un modèle d&#39;infusion de nutriments chronique chez le rat

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.

Étirer dans le cerveau cellules endothéliales microvasculaires (CEND) comme<em&gt; In Vitro</em&gt; Traumatisme modèle du cerveau de la barrière hémato-encéphalique

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.

Mise en place d&#39;un<em&gt; In Vitro</em&gt; Modèle de Rat barrière hémato-encéphalique (BHE): un regard sur le Bureau imperméabilité et de transport médié par le récepteur

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.

Mesure aortique ascendant Rigidité<em&gt; In Vivo</em&gt; Chez la souris en utilisant des ultrasons

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.

Oxygène-glucose privation et réoxygénation comme un<em&gt; In Vitro</em&gt; Ischémie-reperfusion modèle pour étudier la dysfonction barrière hémato-encéphalique

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.

Guidée par IRM dmPFC-SMTr comme un traitement pour trouble dépressif majeur résistant au traitement

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.

Essais in vitro pour évaluer la formation et la perturbation du vaisseau en forme de barrière de sang-cerveau

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.

Procédé amélioré pour la création d’un In Vitro barrière hémato - encéphalique modèle basé sur les cellules endothéliales du cerveau porcin

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) ...