Questo lavoro è stato sostenuto dalla Fondazione Marshall JB.

1. Preparare animale e l&#39;attrezzatura per la procedura <ol><li> Prima di iniziare l&#39;intervento chirurgico, preparare l&#39;area chirurgica e l&#39;animale. Rendere il catetere carotide inserendo un ago di calibro 23 smussata in un&#39;estremità 12 &quot;di PE50 tubi. Tagliare uno smusso di circa 45 ° alla estremità opposta del catetere. Sterilizzare le attrezzature prima della procedura. Indossare un cofano capelli, maschera chirurgica e guanti sterili. </li><li> Posizionare una piastra elettrica sulla superficie dove l&#39;intervento verrà eseguito. Attivare e lasciarlo riscaldare. Coprire l&#39;area con un tampone assorbente panca. </li><li> Verificare di avere il 25% di mannitolo in soluzione senza cristalli. Se sono presenti cristalli, scioglierle ponendo la provetta in un bagno di acqua calda a 80 ° C e agitando periodicamente. </li><li> Registrare la massa dell&#39;animale. </li><li> Effettuare una diluizione propofol per la consegna a 0,8 mg / kg / min ad una velocità di 0,1 ml / min. </li><li> Posizionare il topo nella casella induzione per 3 min. Regolazioni: 5% isoflurano, 2 L / minportata sul ventilatore. </li><li> Sulla cima di un asciugamano usa e getta, la barba al collo e due cosce. </li></ol> 2. Intubare il Ratto <ol><li> Posizionare il retro animale nella scatola induzione per 3 min. </li><li> Prepara il tuo tubo endotracheale con un catetere IV. Per queste dimensioni e ceppo di ratti, si usa un 18 ga catetere. Il tubo deve essere sufficientemente grande da consentire un flusso d&#39;aria, ma abbastanza piccolo da entrare nella trachea senza causare danni. Estrarre il ago del catetere di plastica e con attenzione rompere il ca ago. 1 cm dalla punta affilata. L&#39;ago bunted sarà lo stilo. Rimettere il ago nel catetere. </li><li> Appendere il topo dai denti su una superficie angolata. </li><li> Shine a luce direttamente sul collo dell&#39;animale e allineati tra le sue orecchie. </li><li> Inserire un ca piccola spatola. 3 centimetri nel dell&#39;orofaringe. Sollevare verso l&#39;alto, mentre tirando delicatamente la lingua fuori dalla bocca. </li><li> Visualizzare l&#39;apertura della trachea. Potrai seforo ea piccolo aprire e chiudere con ogni respiro. </li><li> Inserire delicatamente catetere nel foro. A volte si può sentire la plastica entra la cartilagine della trachea. La trachea è sensibile. Non forzare il catetere. </li><li> Impostare il ventilatore a 1 L / min e 60 bpm e l&#39;isoflurano al 2%. </li><li> Collegare il catetere al ventilatore e il tubo nastro alla superficie operativa. </li><li> Verificare che l&#39;animale è anestetizzato eseguendo un pizzico punta. </li></ol> 3. Stabilire Propofol Anestesia IV <ol><li> Fissare il topo con nastro adesivo tutti e quattro gli arti e la coda alla superficie operativa. </li><li> Scrub al collo rasato e siti inguine con clorexidina sapone tre volte. Poi strofinare con clorexidina lavare tre volte. </li><li> Collegare una siringa di propofol a un set di prolunga IV. Montare la siringa su una pompa per infusione. </li><li> Utilizzando un bisturi, tagliare la pelle della coscia rasata. Evitare di tagliare muscolare. </li><li> Aprire l&#39;incisione con la forzaps per visualizzare la vena femorale. </li><li> Tirare via il tessuto sopra l&#39;arteria per esporre il vaso. </li><li> Inserire un ga 26 Monoject veterinario IV catetere nella vena. Quando l&#39;ago perfora la parete della vena vi sentirete diventa più facile da spingere. Ritrarre l&#39;ago e spingere il catetere nel vaso. </li><li> Avviare l&#39;infusione di propofol a 0.1 ml / min. </li><li> 3-5 minuti dopo l&#39;inizio dell&#39;infusione propofol, passare il gas anestesia ad una miscela di 50% O 2 e 50% N 2 O e spegnere il isoflurano. </li></ol> 4. Esporre la biforcazione della carotide comune <ol><li> Effettuare una incisione mediana al collo con lama di bisturi. </li><li> Tagliare tra le due grandi ghiandole salivari con le forbici o bisturi. </li><li> Dissect lontano tessuto lateralmente alla linea mediana per esporre l&#39;arteria carotide. Usare pinze per aprire strati di tessuto. È possibile usare le forbici per rimuovere pezzi che si espongono con la pinza e determinare di essere minimally vascolare. Controllare l&#39;emorragia con una garza, tamponi di cotone, e cauterizzazione se necessario. </li><li> Iniettare 3 IP ml di soluzione salina, se non vi è stata da moderata a gravi emorragie. </li><li> Utilizzare divaricatori per estrarre il tessuto dalla carotide. Trova la biforcazione. </li><li> Esporre l&#39;arteria occipitale appena distalmente alla biforcazione. Cauterize esso tra pinza e separarlo con un elettrocauterizzazione. </li><li> Utilizzare pinze per esporre completamente la biforcazione carotidea e quanto più possibile l&#39;ECA. </li><li> Esporre l&#39;arteria tiroidea superiore si dirama la Corte dei conti distale all&#39;arteria occipitale. Cauterize esso tra pinza e separarlo con un elettrocauterizzazione. </li></ol> 5. Cateterismo dell&#39;arteria carotide esterna <ol><li> Mettere 2 lunghezze di 4/0 di seta sotto la Corte dei conti. </li><li> Prendete 1 lunghezza di sutura con la pinza emostatica e ritrarre la Corte dei conti torna caudalmente (verso i piedi del ratto). </li><li> Prendete l&#39;altra sutura e cravatta fino cranialmente (verso la bocca del ratto) possibile wesima di un chirurgo nodo. Questo forma una legatura permanente a sacrificare la Corte dei conti. </li><li> Rimuovere la pinza emostatica primo (quello che è stato l&#39;applicazione di tensione caudale) e afferrare la sutura altro (quello che si annoda intorno alla Corte dei conti) e applicare leggera tensione cranialmente con la pinza emostatica. </li><li> Tie un altro nodo del chirurgo vagamente (non garantiscono - lasciano poco legato) distale alla biforcazione e prossimale alla legatura permanente. </li><li> Irrigare con 1% di lidocaina per rilassarsi l&#39;arteria. </li><li> Riempi il tuo catetere con soluzione fisiologica eparinizzata. </li><li> Posizionare una clip temporanea vaso più vicino possibile alla biforcazione. Posto fermaglio nave ad un angolo di massimizzare lo spazio per la cateterizzazione. Far scorrere il nodo sciolto verso la clip. </li><li> Fai un piccolo arteriotomia (taglio) in ECA appena prossimale alla legatura con microforbici. Non ci dovrebbero essere sanguinamento se la clip e la legatura sono al sicuro. </li><li> Utilizzando pinze per controllare le pareti ECA, alimentare la estremità smussata del catetere nel vaso. </li&gt; <li> Fissare il catetere con il nodo allentato sutura. Legare l&#39;arteria intorno al catetere. </li><li> Rimuovere la clip e pulire la zona con un batuffolo di cotone. </li><li> Avanzare il catetere di circa 1 mm distalmente alla biforcazione. Tenere l&#39;arteria con pinze prossimali al nodo di garantire la nave è immobilizzato. </li></ol> 6. Somministrare mannitolo <ol><li> Riempire un 8 &quot;set di estensione IV e una siringa da 6 ml con il mannitolo senza creare bolle d&#39;aria Conservare questo in incubatore a 37 ° C. Nota: il lavoro rapidamente con mannitolo - sarà facilmente precipitare dalla soluzione a temperatura ambiente.. </li><li> Al fine di visualizzare BBBD, si può somministrare Evans colorante blu e vederlo entrare nelle zone del cervello dove la barriera è stata interrotta. Per fare questo, dare all&#39;animale 2 ml / kg del 2% Blu di Evans attraverso un nuovo sito IV sulla gamba controlaterale alla infusione di propofol. </li><li> Collegare il set di prolunga a un micron 5 Eliminare filtro e riempirlo con il mannitolo per rimuovere le bolle d&#39;ariaBles. Quindi, fissare questo al rubinetto senza creare bolle. </li><li> Somministrare mannitolo a 0,09 ml / sec per 25 sec. Guardare per il mannitolo per raggiungere l&#39;equilibrio con il flusso nella carotide comune in un punto appena prossimale al ICA. Dovrebbe apparire a impulsi con la frequenza cardiaca del ratto. Se il mannitolo scorre semplicemente verso il cuore aver superato la pressione del ratto. Ciò può causare complicanze potenzialmente letali e BBBD poveri. </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outwitting+the+blood-brain+barrier+for+therapeutic+purposes:+osmotic+opening+and+other+means.">Outwitting the blood-brain barrier for therapeutic purposes: osmotic opening and other means.</a> Neurosurgery. 42, 1083 (1998).</li><li>Kroll, R. A., Pagel, M. A., Muldoon, L. L., Roman-Goldstein, S., 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=Increasing+volume+of+distribution+to+the+brain+with+interstitial+infusion:+dose,+rather+than+convection,+might+be+the+most+important+factor.">Increasing volume of distribution to the brain with interstitial infusion: dose, rather than convection, might be the most important factor.</a> Neurosurgery. 38, 746-754 (1996).</li><li>Butt, A. M., Jones, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+histamine+and+antagonists+on+electrical+resistance+across+the+blood-brain+barrier+in+rat+brain-surface+microvessels.">Effect of histamine and antagonists on electrical resistance across the blood-brain barrier in rat brain-surface microvessels.</a> Brain research. 569, 100-105 (1992).</li><li>Inamura, T., Black, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bradykinin+selectively+opens+blood-tumor+barrier+in+experimental+brain+tumors.">Bradykinin selectively opens blood-tumor barrier in experimental brain tumors.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 14, 862-870 (1994).</li><li>Matsukado, K., 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=Enhanced+tumor+uptake+of+carboplatin+and+survival+in+glioma-bearing+rats+by+intracarotid+infusion+of+bradykinin+analog,+RMP-7.">Enhanced tumor uptake of carboplatin and survival in glioma-bearing rats by intracarotid infusion of bradykinin analog, RMP-7.</a> Neurosurgery. 39, 125 (1996).</li><li>Kroll, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+drug+delivery+to+intracerebral+tumor+and+surrounding+brain+in+a+rodent+model:+a+comparison+of+osmotic+versus+bradykinin+modification+of+the+blood-brain+and/or+blood-tumor+barriers.">Improving drug delivery to intracerebral tumor and surrounding brain in a rodent model: a comparison of osmotic versus bradykinin modification of the blood-brain and/or blood-tumor barriers.</a> Neurosurgery. 43, 879 (1998).</li><li>Kinoshita, M., McDannold, N., Jolesz, F. A., Hynynen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+localized+delivery+of+Herceptin+to+the+mouse+brain+by+MRI-guided+focused+ultrasound-induced+blood-brain+barrier+disruption.">Noninvasive localized delivery of Herceptin to the mouse brain by MRI-guided focused ultrasound-induced blood-brain barrier disruption.</a> Proceedings of the National Academy of Sciences. 103, 11719-11723 (2006).</li><li>Neuwelt, E. A., Dahlborg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+administered+in+conjunction+with+osmotic+blood-brain+barrier+modification+in+patients+with+brain+metastases.">Chemotherapy administered in conjunction with osmotic blood-brain barrier modification in patients with brain metastases.</a> Journal of neuro-oncology. 4, 195-207 (1987).</li><li>Doolittle, N. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficacy+of+a+multicenter+study+using+intraarterial+chemotherapy+in+conjunction+with+osmotic+opening+of+the+blood-brain+barrier+for+the+treatment+of+patients+with+malignant+brain+tumors.">Safety and efficacy of a multicenter study using intraarterial chemotherapy in conjunction with osmotic opening of the blood-brain barrier for the treatment of patients with malignant brain tumors.</a> Cancer. 88, 637-647 (2000).</li><li>Guillaume, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-arterial+chemotherapy+with+osmotic+blood-brain+barrier+disruption+for+aggressive+oligodendroglial+tumors:+results+of+a+phase+I+study.">Intra-arterial chemotherapy with osmotic blood-brain barrier disruption for aggressive oligodendroglial tumors: results of a phase I study.</a> Neurosurgery. 66, 48 (2010).</li><li>Remsen, L. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+anesthetic+choice,+PaCO2,+and+other+factors+on+osmotic+blood-brain+barrier+disruption+in+rats+with+brain+tumor+xenografts.">The influence of anesthetic choice, PaCO2, and other factors on osmotic blood-brain barrier disruption in rats with brain tumor xenografts.</a> Anesthesia &amp; Analgesia. 88, 559-559 (1999).</li><li>Nilaver, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+herpesvirus+and+adenovirus+to+nude+rat+intracerebral+tumors+after+osmotic+blood-brain+barrier+disruption.">Delivery of herpesvirus and adenovirus to nude rat intracerebral tumors after osmotic blood-brain barrier disruption.</a> Proceedings of the National Academy of Sciences. 92, 9829 (1995).</li><li>Fortin, D., McCormick, C. I., Remsen, L. G., Nixon, R., Neuwelt, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unexpected+neurotoxicity+of+etoposide+phosphate+administered+in+combination+with+other+chemotherapeutic+agents+after+blood-brain+barrier+modification+to+enhance+delivery,+using+propofol+for+general+anesthesia,+in+a+rat+model.">Unexpected neurotoxicity of etoposide phosphate administered in combination with other chemotherapeutic agents after blood-brain barrier modification to enhance delivery, using propofol for general anesthesia, in a rat model.</a> Neurosurgery. 47, 199 (2000).</li><li>Neuwelt, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+CNS+lymphoma+treated+with+osmotic+blood-brain+barrier+disruption:+prolonged+survival+and+preservation+of+cognitive+function.">Primary CNS lymphoma treated with osmotic blood-brain barrier disruption: prolonged survival and preservation of cognitive function.</a> Journal of clinical oncology. 9, 1580 (1991).</li><li>Remsen, L. G., Trail, P. A., Hellstr&#246;m, I., Hellstr&#246;m, K. E., Neuwelt, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+delivery+improves+the+efficacy+of+a+tumor-specific+doxorubicin+immunoconjugate+in+a+human+brain+tumor+xenograft+model.">Enhanced delivery improves the efficacy of a tumor-specific doxorubicin immunoconjugate in a human brain tumor xenograft model.</a> Neurosurgery. 46, 704 (2000).</li><li>Neuwelt, E. A., Pagel, M. A., Kraemer, D. F., Peterson, D. R., Muldoon, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+chemoprotection+without+compromise+of+chemotherapy+efficacy+in+a+rat+brain+tumor+model.">Bone marrow chemoprotection without compromise of chemotherapy efficacy in a rat brain tumor model.</a> Journal of Pharmacology and Experimental Therapeutics. 309, 594-599 (2004).</li></ol>

Nessun conflitto di interessi dichiarati.

Ci sono pochi mezzi di massimizzare l&#39;efficacia di BBBD. È importante ridurre al minimo il sanguinamento durante la fase di cut-down. La pressione sanguigna e la frequenza cardiaca può essere influenzata da sanguinamento sostanziale e questi fattori sono noti per influenzare il grado di BBBD 13. Sanguinamento può essere ridotto utilizzando suture per legare vasi grandi, come la tiroide superiore e arterie occipitali, che devono essere divisi. Inoltre, elettrocauterizzazione può essere utilizzato per d...

<table border="1"><tbody><tr><td> Nome del materiale </td><td> Azienda </td><td> Numero di catalogo </td><td> Commento </td></tr><tr><td> Lungo Evans ratto </td><td> Harlan Laboratories </td><td></td><td> 210-250 g, di sesso maschile </td></tr><tr><td> PE 50 Tubi </td><td> Beckton-Dickinson </td><td></td><td></td></tr><tr><td> Calibro 18 x 2.5 &quot;catetere IV </td><td> Terumo </td><td></td><td> Per tubo endotracheale </td></tr><tr><td> 30 &quot;estensione IV imposta </td><td> Abbott </td><td></td><td></td></tr><tr><td> 26 calibro catetere IV veterinario </td><td> Monoject </td><td></td><td></td></tr><tr><td> Evans colorante blu </td><td> Sigma </td><td> E2129 </td><td></td></tr><tr><td> Bipolare </td><td> Codman </td><td></td><td></td></tr><tr><td> Filtro, 5 micron </td><td> Braun </td><td></td><td></td></tr></tbody></table>

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

Cellule endoteliali con giunzioni strette insieme con la membrana basale e piedi finali astrociti circondano vasi sanguigni cerebrali per formare la barriera emato-encefalica 1. La barriera esclude selettivamente molecole di attraversare tra il sangue e il cervello in base alla loro dimensione e carica. Questa funzione può impedire l&#39;erogazione di terapie per disturbi neurologici. Un certo numero di farmaci chemioterapici, per esempio, non viene effettivamente attraversare la barriera emato-encefalica per raggiungere le cellule tumorali 2. Quindi, migliorare l&#39;erogazione di farmaci attraverso la barriera sangue-cervello è una zona di interesse. I metodi più diffusi per migliorare la consegna di farmaci al cervello sono diretti infusione cerebrale e barriera emato-encefalica interruzione 3. Diretti garanzie infusione intracerebrale che le terapie arrivano al cervello, tuttavia, questo metodo ha una limitata capacità di disperdere il farmaco 4. Emato-encefalica perturbazione barriera (BBBD) alfarmaci bassi di fluire direttamente dal sistema circolatorio nel cervello e quindi più efficace raggiungere dispersi cellule tumorali. Tre metodi di interruzione barriera includono interruzioni osmotica barriera, barriera interruzione farmacologica, e ultrasuoni focalizzati con microbolle. Interruzione osmotica, introdotta da Neuwelt, utilizza una soluzione ipertonica di 25% mannitolo che disidrata le cellule della barriera sangue-cervello facendoli restringersi e distruggere le loro giunzioni strette. Interruzione barriera può anche essere realizzato con composti farmacologicamente vasoattive quali l&#39;istamina e bradichinina 5 6. Questo metodo, tuttavia, è selettivo principalmente per il cervello-tumorale barriera 7. Inoltre, RMP-7, un analogo del peptide bradichinina, è risultata essere inferiore rispetto testa a testa con BBBD osmotico con 25% di mannitolo 8. Un altro metodo, ultrasuoni focalizzati (FUS) in combinazione con agenti di microbolle di contrasto ad ultrasuoni, è stato anche dimostratoper aprire reversibilmente la barriera emato-encefalica 9. Rispetto al FUS, però, il 25% mannitolo ha una storia più lunga di sicurezza in pazienti umani che lo rende uno strumento collaudato per la ricerca traslazionale 10-12. Al fine di realizzare BBBD, mannitolo devono essere consegnati a un tasso elevato direttamente nella circolazione arteriosa del cervello. Negli esseri umani, un catetere endovascolare è guidato al cervello dove rapida, flusso diretto può essere realizzato. Questo protocollo modelli umana BBBD più possibile. A seguito di un cut-down alla biforcazione della carotide comune, un catetere viene inserito retrogrado nella CCE e usati per fornire mannitolo direttamente nell&#39;arteria carotide interna (ICA) circolazione. Propofol e N 2 O anestesia vengono utilizzati per la loro capacità di massimizzare l&#39;efficacia di interruzione barriera 13. Se eseguito correttamente, questa procedura ha la capacità di in modo sicuro, efficace e reversibile aprire la barriera emato-encefalica e improvvisazionee l&#39;erogazione di farmaci che ordinariamente non raggiungono il cervello 8,13,14.

<img alt="Figura 1" src="/files/ftp_upload/50019/50019fig1.jpg" /> Figura 1. Visualizzazione ematoencefalica interruzione barriera via stravaso Evans colorante blu. Evans blue colorante è un pigmento che si lega all&#39;albumina e non stravaso nel cervello in condizioni fisiologiche. Perturbazione della barriera ematoencefalica su un lato del cervello Evans blue permette di entrare e macchiare l&#39;azzurro emisfero interrotto mentre l&#39;emisfero non perturb...

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

Emato-encefalica perturbazione barriera favorisce l&#39;erogazione di alcuni farmaci al cervello. Mannitolo consegnato intra-arteriosa cellule restringe i vasi sanguigni circostanti, al fine di distruggere fisicamente la barriera.

Modello Rat di emato-encefalica Turbativa Barriera per consentire mirato Therapeutics neurovascolare

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 Views.  Vanderbilt University School of Medicine. Emato-encefalica perturbazione barriera favorisce l'erogazione di alcuni farmaci al cervello. Mannitolo consegnato intra-arteriosa cellule restringe i vasi sanguigni circostanti, al fine di distruggere fisicamente la barriera.

Video: 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.

MRI-guidato Interruzione della barriera emato-encefalica transcranica con ultrasuoni focalizzati in un modello murino

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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Medicina

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.

Stretch in microvascolari cerebrali cellule endoteliali (CEND) come<em&gt; In Vitro</em&gt; Trauma cranico modello della barriera ematoencefalica

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.

Istituzione di un<em&gt; In Vitro</em&gt; Modello di Rat barriera emato-encefalica (BBB): A Focus on BBB impermeabilità e Trasporti recettore-mediata

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.

L&#39;ossigeno-glucosio Deprivazione e riossigenazione come<em&gt; In Vitro</em&gt; Ischemia-riperfusione Injury modello per lo studio barriera sanguigna del cervello Erettile

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.

Un ratto carotidea Pallone Lesioni modello per verificare Therapeutics ritocca Anti-vascolari

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 ghiandola sottomandibolare instillazione e localizzati frazionato irradiazione in un modello murino di salivare Ipofunzione

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.

In vitro dosaggi per valutare la formazione e la rottura del vaso come la barriera emato-cerebrale

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.

Migliorato il metodo per la creazione di un In Vitro barriera emato - encefalica modello basato sulle cellule endoteliali Porcine cervello

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.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

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.