Ce travail a été soutenu par la Fondation JB Marshall.

1. Préparer des animaux et de l&#39;équipement pour la procédure <ol><li> Avant de commencer la chirurgie, la préparation du champ opératoire et l&#39;animal. Faire le cathéter carotidien en insérant une aiguille de calibre 23 émoussée dans une extrémité de 12 &quot;de PE50 tube. Couper un biseau d&#39;environ 45 ° à l&#39;extrémité opposée du cathéter. Stériliser le matériel avant l&#39;intervention. Porter un chapeau cheveux, masque chirurgical , et des gants stériles. </li><li> Placez un coussin chauffant sur la surface où l&#39;opération sera effectuée. Activez-le et laissez-le chauffer. Couvrir la zone avec un tampon absorbant banc. </li><li> Vérifiez que vous disposez du mannitol 25% en solution, sans cristaux. Si les cristaux sont présents, les dissoudre en plaçant la fiole dans un bain d&#39;eau chaude à 80 ° C et en agitant périodiquement. </li><li> Noter la masse de l&#39;animal. </li><li> Faire une dilution du propofol pour la livraison à 0,8 mg / kg / min à une vitesse de 0,1 ml / min. </li><li> Placez le rat dans la boîte à induction pendant 3 min. Réglages: 5% d&#39;isoflurane, 2 L / mindébit sur le ventilateur. </li><li> Au sommet d&#39;une serviette jetable, de se raser la nuque et les deux cuisses. </li></ol> 2. Intuber le Rat <ol><li> Placez le dos de l&#39;animal dans la boîte à induction pendant 3 min. </li><li> Préparez votre tube endotrachéal avec un cathéter IV. Pour cette taille et de la souche de rat, nous utilisons un cathéter de calibre 18. Le tube doit être suffisamment grande pour permettre une ventilation adéquate, mais assez petit pour tenir dans la trachée sans causer de dommages. Sortir le pointeau du cathéter en plastique et soigneusement briser le approx aiguille. 1 cm de son extrémité pointue. L&#39;aiguille cariés sera votre stylet. Remettez le aiguille dans le cathéter. </li><li> Accrocher le rat par ses dents sur une surface inclinée. </li><li> Shine a light directement contre le cou de l&#39;animal et la queue entre ses oreilles. </li><li> Insérez une petite spatule env. 3 cm dans l&#39;oropharynx. Soulevez vers le haut tout en tirant doucement la langue hors de la bouche. </li><li> Visualiser l&#39;ouverture de la trachée. Vous aurez soich petit trou s&#39;ouvrent et se ferment à chaque respiration. </li><li> Insérez doucement le cathéter dans le trou. Vous pouvez parfois ressentir le plastique entre le cartilage de la trachée. La trachée est sensible. Ne pas forcer le cathéter. </li><li> Réglez le ventilateur à 1 L / min et 60 bpm et l&#39;isoflurane à 2%. </li><li> Raccorder le cathéter au ventilateur et le tube de bande de la surface de commande. </li><li> Vérifiez que l&#39;animal est anesthésié par l&#39;exécution d&#39;une pincement de l&#39;orteil. </li></ol> 3. Établir propofol IV Anesthésie <ol><li> Fixez le rat en collant les quatre membres et la queue à la surface d&#39;exploitation. </li><li> Frotter le cou rasé et les sites de l&#39;aine avec du savon de chlorhexidine à trois reprises. Puis frotter avec de la chlorhexidine rincer trois fois. </li><li> Fixez une seringue de propofol pour un set de rallonge IV. Monter la seringue sur une pompe à perfusion. </li><li> À l&#39;aide d&#39;un scalpel, couper à travers la peau de la cuisse rasé. Évitez de couper le muscle. </li><li> Ouvrez l&#39;incision avec une forceps pour visualiser la veine fémorale. </li><li> Se détacher du tissu sur l&#39;artère afin d&#39;exposer le navire. </li><li> Insérez un 26 ga Monoject vétérinaire IV cathéter dans la veine. Lorsque l&#39;aiguille perfore la paroi de la veine, vous vous sentirez-il plus facile à pousser. Rétracter l&#39;aiguille et pousser le cathéter dans le vaisseau. </li><li> Commencer la perfusion de propofol à 0,1 ml / min. </li><li> 3-5 min après le début de la perfusion de propofol, passer le gaz d&#39;anesthésie à un mélange de 50% d&#39;O 2 et 50% de N 2 O et couper l&#39;isoflurane. </li></ol> 4. Exposer la bifurcation de l&#39;artère carotide commune <ol><li> Faire une incision médiane au niveau du cou avec une lame de scalpel. </li><li> Couper entre les deux grandes glandes salivaires avec des ciseaux ou d&#39;un scalpel. </li><li> Disséquer loin tissu latéral de la ligne médiane pour exposer l&#39;artère carotide. Utilisez une pince pour ouvrir couches de tissu. Vous pouvez utiliser des ciseaux pour enlever des morceaux qui vous exposent à la pince et de déterminer à Minimally vasculaire. Contrôler le saignement avec de la gaze, cotons-tiges, et la cautérisation si nécessaire. </li><li> Injecter 3 ml solution saline IP s&#39;il ya eu modérée à des saignements abondants. </li><li> Utilisez les rétracteurs de tirer le tissu loin de la carotide. Trouvez la bifurcation. </li><li> Exposer l&#39;artère occipitale juste en aval de la bifurcation. Il cautériser entre la pince et le séparer avec un bistouri électrique. </li><li> Utilisez une pince pour exposer entièrement la bifurcation carotidienne et autant de la CEA que possible. </li><li> Exposer l&#39;artère thyroïdienne supérieure qui bifurque de la CEA distale de l&#39;artère occipitale. Il cautériser entre la pince et le séparer avec un bistouri électrique. </li></ol> 5. Sonder l&#39;artère carotide externe <ol><li> Placez 2 longueurs de 4/0 en soie vertu de l&#39;ECA. </li><li> Prenez 1 longueur de suture avec votre pince hémostatique et rétracter le CEA retour caudale (vers les pieds du rat). </li><li> Prenez la suture autre et attacher autant céphalique (vers la bouche du rat) que possible avecvec un chirurgien noeud. Cela constitue une ligature permanente de sacrifier la CEA. </li><li> Retirez la pince hémostatique premier (celui qui a été d&#39;appliquer une tension caudale) et prendre la suture autre (celui qui est noué autour de la CEA) et appliquer une tension douce crânienne avec la pince hémostatique. </li><li> Attachez un autre noeud du chirurgien lâche (ne garantissent pas - laisser à peine à égalité) en aval de la bifurcation et à proximité de la ligature permanente. </li><li> Rincer avec lidocaïne à 1% pour se détendre l&#39;artère. </li><li> Remplir le cathéter avec du sérum physiologique hépariné. </li><li> Placer un clip récipient temporaire le plus près possible de la bifurcation. Clip pour vaisseau lieu à un angle de maximiser l&#39;espace de cathétérisme. Faites glisser le noeud lâche vers le clip. </li><li> Faire un petit artériotomie (coupe) à la CEA juste en amont de la ligature à l&#39;aide microciseaux. Il devrait y avoir aucun saignement si le clip et la ligature sont sécurisées. </li><li> En utilisant une pince pour contrôler les parois CEA, alimenter l&#39;extrémité biseautée du cathéter dans le vaisseau. </li&gt; <li> Fixer le cathéter avec le noeud de suture lâche. Lier l&#39;artère autour du cathéter. </li><li> Retirez le clip et nettoyer la zone avec un coton-tige. </li><li> Avancer le cathéter à environ 1 mm en aval de la bifurcation. Maintenez l&#39;artère avec une pince proximale du nœud de s&#39;assurer que le navire est immobilisé. </li></ol> 6. Administrer Mannitol <ol><li> Remplissez un 8 &quot;set de rallonge IV et une seringue de 6 ml avec le mannitol sans créer de bulles d&#39;air Conservez ce dans l&#39;incubateur à 37 ° C. Remarque: les travaux rapidement avec le mannitol - il sera facilement précipiter hors de la solution à la température ambiante.. </li><li> Afin de visualiser BBBD, on peut gérer colorant bleu Evans et regardez-le pénétrer dans les zones du cerveau où la barrière a été perturbé. Pour ce faire, donner à l&#39;animal 2 ml / kg de 2% Evans Blue grâce à un nouveau site IV sur la jambe controlatérale à la perfusion de propofol. </li><li> Fixer le prolongateur sur un 5 um filtrer et de le remplir avec du mannitol pour éliminer l&#39;air bubbles. Ensuite, fixez le robinet d&#39;arrêt à ce sans créer de bulles. </li><li> Administrer mannitol à 0,09 ml / sec pendant 25 sec. Surveillez le mannitol pour atteindre l&#39;équilibre avec le flux dans la carotide commune à un point juste en amont de l&#39;ICA. Il devrait apparaître à impulsions à la fréquence cardiaque du rat. Si le mannitol coule simplement vers le bas vers le cœur que vous avez vaincu la pression artérielle du rat. Cela peut causer des complications potentiellement mortelles et BBBD pauvres. </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=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>

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Il existe quelques moyens de maximiser l&#39;efficacité de BBBD. Il est important de minimiser le saignement pendant la phase de coupe-bas. La pression artérielle et la fréquence cardiaque peut être affectée par des saignements importants et ces facteurs sont connus pour affecter le degré de BBBD 13. Le saignement peut être réduite en utilisant des sutures pour ligaturer les gros vaisseaux, tels que la thyroïdienne supérieure et les artères occipitales, qui doivent être divisées. En outre, l&#39;...

<table border="1"><tbody><tr><td> Nom du produit </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commenter </td></tr><tr><td> Longue rat Evans </td><td> Harlan Laboratories </td><td></td><td> 210-250 g, mâle </td></tr><tr><td> PE 50 Tubes </td><td> Beckton-Dickinson </td><td></td><td></td></tr><tr><td> Calibre 18 x 2.5 &quot;cathéter IV </td><td> Terumo </td><td></td><td> Pour tube HE </td></tr><tr><td> 30 &quot;prolongation IV énonce </td><td> Abbott </td><td></td><td></td></tr><tr><td> Cathéter de calibre 26 vétérinaire IV </td><td> Monoject </td><td></td><td></td></tr><tr><td> Colorant bleu Evans </td><td> Sigma </td><td> E2129 </td><td></td></tr><tr><td> Bipolaire </td><td> Codman </td><td></td><td></td></tr><tr><td> Filtrer, 5 um </td><td> Braun </td><td></td><td></td></tr></tbody></table>

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

Les cellules endothéliales de jonctions serrées le long de la membrane basale et pieds astrocytaires entourent extrémité vaisseaux sanguins cérébraux pour former la barrière hémato-encéphalique 1. La barrière exclut sélectivement les molécules du croisement entre le sang et le cerveau en fonction de leur taille et de charge. Cette fonction peut entraver la fourniture de traitements pour les troubles neurologiques. Un certain nombre de médicaments chimiothérapeutiques, par exemple, ne sera pas efficace traverser la barrière hémato-encéphalique pour atteindre les cellules tumorales 2. Ainsi, l&#39;amélioration de l&#39;administration de médicaments à travers la barrière hémato-encéphalique est un domaine d&#39;intérêt. Les méthodes les plus répandues pour améliorer la délivrance de médicaments au cerveau sont directement perfusion cérébrale et barrière hémato-encéphalique rupture 3. Des garanties directes liées à la perfusion intra-cérébrales que les thérapies atteignent le cerveau, mais cette méthode a une capacité limitée pour disperser le médicament 4. Rupture de la barrière hémato-encéphalique (BBBD) almédicaments à bas découler directement de l&#39;appareil circulatoire dans le cerveau et donc d&#39;atteindre plus efficacement les cellules tumorales dispersées. Trois méthodes de rupture de la barrière comprennent rupture de la barrière osmotique, rupture de la barrière pharmacologique et ultrasons focalisés avec des microbulles. Perturbation osmotique, mis au point par Neuwelt, utilise une solution hypertonique de mannitol 25% qui déshydrate les cellules de la barrière hémato-encéphalique les obligeant à se rétrécir et perturber leurs jonctions serrées. Rupture de la barrière peut également être réalisé avec des composés pharmacologiquement vasoactifs tels que l&#39;histamine 5 et 6 bradykinine. Cette méthode, cependant, est sélective principalement pour la barrière hémato-tumorale 7. En outre, la RMP-7, un analogue du peptide bradykinine, a été jugée inférieure par rapport tête-à-tête avec BBBD osmotique avec le mannitol 25% 8. Une autre méthode, ultrasons focalisés (FUS) en conjonction avec microbulles agents de contraste ultrasonores, a également été montréde manière réversible ouvrir la barrière hémato-encéphalique 9. En comparaison avec le FUS, cependant, le mannitol 25% a une longue histoire de tolérance chez des patients humains qui en fait un outil éprouvé pour la recherche translationnelle 10-12. Afin d&#39;accomplir BBBD, le mannitol doit être livré à un taux élevé directement dans la circulation artérielle du cerveau. Chez l&#39;homme, un cathéter endovasculaire est guidé vers le cerveau où la rapidité, le débit direct ne peut être accompli. Ce protocole modèles humaine BBBD aussi étroitement que possible. Suite à la réduction jusqu&#39;à la bifurcation de l&#39;artère carotide commune, un cathéter est inséré rétrograde dans la CEA et utilisés pour apporter le mannitol directement dans l&#39;artère carotide interne (ACI) la circulation. Propofol et de N 2 O anesthésie sont utilisés pour leur capacité à maximiser l&#39;efficacité de 13 rupture de la barrière. Si exécuté correctement, cette procédure a la capacité de manière sûre, efficace et réversible ouvrir la barrière hémato-encéphalique et improvisatione l&#39;administration de médicaments qui ne sont pas habituellement atteignent le cerveau 8,13,14.

<img alt="Figure 1" src="/files/ftp_upload/50019/50019fig1.jpg" /> Figure 1. Visualisation rupture de la barrière hémato-encéphalique par l&#39;intermédiaire de colorant bleu Evans extravasation. Colorant bleu Evans est un pigment qui se lie à l&#39;albumine et n&#39;est pas extravasation dans le cerveau, dans des conditions physiologiques. Rupture de la barrière hémato-encéphalique d&#39;un côté du cerveau permet de bleu d&#39;Evans d&#39;entrer et d...

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

Rupture de la barrière hémato-encéphalique facilite la prestation de certains médicaments vers le cerveau. Mannitol livré intra-artérielle se rétrécit cellules entourant les vaisseaux sanguins afin de perturber physiquement la barrière.

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 ...

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19.3K vues.  Vanderbilt University School of Medicine. Rupture de la barrière hémato-encéphalique facilite la prestation de certains médicaments vers le cerveau. Mannitol livré intra-artérielle se rétrécit cellules entourant les vaisseaux sanguins afin de perturber physiquement la barrière.

Vidéo: 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.

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 ...

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Médecine

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 ...

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 ...

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 carotidienne Balloon Modèle de lésion pour tester Therapeutics Rénovation Anti-vasculaires

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 Submandibulaire Gland instillation et Localized Fractionnée Irradiation dans un modèle de rat de salivaire Hyposécrétion

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.

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) ...

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.

Mesure de la contractilité isométrique de l’artère mésentérique de souris à l’aide de fil myographie

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

Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model

Myocardial infarction is one of the leading causes of death and disability worldwide, and there is an urgent need for novel cardioprotective or regenerative strategies. An essential component of drug development is determining how a novel therapeutic is to be administered. Physiologically relevant large animal models are of critical importance in assessing the feasibility and efficacy of various therapeutic delivery strategies. Due to their similarities to humans in cardiovascular physiology, coronary vascular anatomy, and heart weight to body weight ratio, swine is one of the preferred species in the preclinical evaluation of new therapies for myocardial infarction. The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. After percutaneously induced myocardial infarction, female landrace swine received treatment with novel agents through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump. The procedures employed for each technique are reproducible, resulting in reliable cardioactive drug delivery. These models can be easily adapted to suit individual study designs, and each of these delivery techniques can be used to investigate a variety of possible interventions. Therefore, these methods are a useful tool for translational scientists pursuing novel biological approaches in cardiac repair following myocardial infarction.

The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. Female landrace swine received treatment through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump.