Name: mouse microsurgery infusion technique for targeted substance delivery into the cns via the internal carotid artery
Uploaded: 2017-01-31T14:00:00.000+00:00
Duration: 6 min 41 s
Description: Watch this Scientific Journal Video about Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery at JoVE.com

We would like to thank Dr. Lei Chen (Icahn School of Medicine at Mount Sinai, NY) who first established the use of this model in our laboratory, and to Dr. Gretchen Wolff (German Cancer Research Center, Heidelberg, Germany) for disseminating the technique in our laboratory. Supported in part by HL126559, DA039576, MH098891, MH63022, MH072567, DA027569, and NSC 2015/17/B/NZ7/02985.

Die Verfahren in dem folgenden Protokoll beteiligt wurden von der University of Miami Institutional Animal Care und Use Committee (IACUC) zugelassen. Darüber hinaus werden alle Verfahren in Einrichtungen werden von der Gesellschaft für Evaluierung und Akkreditierung von Labor Animal Care International (AAALAC) genehmigt durchgeführt. 1. Herstellung von Mäusen für Chirurgie <ol><li> Anesthetize Maus mit Isofluran mit Sauerstoff gemischt, ein Labor, Anästhesie-System. Verwenden Sie Isofluran bei zwischen 4-5% und Sauerstoffstrom bei 2 l / min auf die kommerzielle Maschine einstellen (siehe Werkstoff-Tabelle). Bringen Sie das Tier zu der Operation Oberfläche, unter einem Stereomikroskop und Sustain Anästhesie einen Nasenkegel mit (verwenden Isofluran Einstellung 1,5-2,5 und Sauerstoffstrom bei 2 l / min). <ol><li> Stellen Sie sicher, dass die Maus Atemfrequenz liegt bei etwa 1 bis 2 Atmung / sec ohne Keuchen. Darüber hinaus gewährleisten, dass das Tier nicht Whiskers Stimulation nicht aufweist Reaktion und Pedal reflex (toe Prise). Überwachen Sie die Atemfrequenz und Aufwand während der Operation, mindestens alle 5 Minuten. Folgen Sie spezifische Institutional Animal Care und Use Committee und Veterinär Richtlinien zur Bekämpfung von Nagetieren Anästhesie Überwachung. </li></ol></li><li> Einen Tropfen ophthalmische Schmiermittel auf jedes Auge einen sterilen Tupfer, um mit Trockenheit zu verhindern. Verabreichung von entzündungshemmenden und schmerzlindernden Beschwerden zu lindern empfohlen. </li><li> Mit dem Tier auf dem Rücken liegend, halten Anästhesie einen Nasenkegel verwenden. </li><li> Reinigen Sie den Nackenbereich des Tieres durch Abwischen der Fläche, die dreimal mit Ethanol 70% und Chlorhexidin. Rasur der OP-Bereich des Tieres unter Verwendung einer Rasierklinge (siehe unten). </li></ol> 2. Dissektion der Arteria carotis communis (CCA) <ol><li> Führen Sie die gesamte mikrochirurgisches Verfahren unter einem Stereomikroskop. Mit chirurgische Scheren und Pinzetten führen eine flache Mittelschnitt im Nacken, von oberhalb des Brustbeins auf unter ter Backe (ca. 3 bis 4 cm). </li><li> Mit einer Pinzette vorsichtig getrennten Fett- und Bindegewebe, um die Luftröhre aus. </li><li> Legen Sie ein Kissen (rundes Objekt, etwa 0,5 cm im Durchmesser) auf der Rückseite des Halses der Maus, um den Hals zu verlängern, das weiterhin den Bereich auszusetzen. </li><li> Trennen Sie das Gewebe entweder eine Geweberetraktor oder Haken. </li><li> Auf der linken Seite der Trachea des Tieres, tweeze sorgfältig das Bindegewebe neben den linken CCA zu belichten. </li><li> Mit einer Pinzette vorsichtig entfernen alle Bindegewebe der CCA Gabelung, und den Beginn der externen und internen Arteria carotis zu belichten. </li></ol> 3. Vorbereitung des CCA für Substanz Infusion <ol><li> Legen Sie zwei Segmente von Nylonfaden (ca. 1 cm jeweils) unter A. carotis externa (ECA), mit einer Pinzette. </li><li> Legen Sie ein dauerhafter Knoten am höchsten Punkt möglich von der ECA. </li><li> Am tiefsten Punkt möglich des ECA, unmittelbar über dem CCA Bifurkation Orteinen abnehmbaren Knoten. Dieser Knoten sollte locker sein im Vergleich zu dem oberen Knoten. </li><li> Schließen Sie die CCA einen Behälter Clip, am tiefsten Punkt. </li><li> Schließen Sie die A. carotis interna (ICA) mit einem Schiff Clip. </li><li> Mit microdissection Feder Schere führen Sie einen kleinen Schnitt in der ECA (ca. 2 mm), zwischen den beiden Knoten. </li></ol> 4. Substanz Infusion über ICA <ol><li> Zubauen ein Infusionssystem. Bringen Sie 6 Zoll Kapillarschlauch (ideal Abmessungen: 2,5 mm x 1,2 mm) auf eine Tuberkulin-Spritze 250 &amp; mgr; l der Substanz vorsichtig und legen Sie die Kapillarspitze in den Einschnitt infundiert (unter anderem Medikamente, Krankheitserreger, extrazellulären Vesikeln) ausgeführt werden in Schritt 3.6. </li><li> Weiter die Kapillare nach unten rutschen, bis sie einen Mittelpunkt zwischen der Gabelung erreicht und den Clip des CCA zu schließen. </li><li> Binden Sie den unteren ECA Knoten. Stellen Sie sicher, dass der Knoten locker genug ist Kapillare Fluidität und dicht genug, um zu verhindern, dass leakinG. </li><li> Entfernen Clip aus dem ICA. </li><li> Durch leichten Druck auf den Spritzenkolben zu ermöglichen Substanz Infusion etwa 10 &amp; mgr; l pro Sekunde. </li></ol> 5. Nach Infusion Verfahren <ol><li> Platz Clip auf der ICA zurück. </li><li> Entfernen Sie vorsichtig die Kapillarschlauchs. </li><li> Binden Sie den unteren ECA Knoten vollständig. </li><li> Entfernen Sie Clips aus dem ICA und der CCA. </li></ol> 6. Incision Closing und postoperative Pflege <ol><li> Entfernen Aufroller oder Gewebehaken und Kissen. </li><li> Saubere Nahtbereich mit einer sterilen Kochsalzlösung. </li><li> Schließen Sie den Schnitt mit Nylonfaden / Nadel und Pinzette. </li><li> Verabreichen entzündungshemmende und analgetische postoperative Beschwerden zu lindern. </li><li> Tier in den Käfig auf Heizkissen gelegt für mindestens 1 Stunde und Monitor Erholung. </li></ol>

<ol>
	<li>Chen, L., Swartz, K. R., Toborek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+microport+technique+for+applications+in+cerebrovascular+research.">Vessel microport technique for applications in cerebrovascular research.</a> J Neurosci Res. 87 (7), 1718-1727 (2009).</li><li>Frisella, W. 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=Intracranial+injection+of+recombinant+adeno-associated+virus+improves+cognitive+function+in+a+murine+model+of+mucopolysaccharidosis+type+VII.">Intracranial injection of recombinant adeno-associated virus improves cognitive function in a murine model of mucopolysaccharidosis type VII.</a> Mol Ther. 3 (3), 351-358 (2001).</li><li>Wei, L., Erinjeri, J. P., Rovainen, C. M., Woolsey, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collateral+growth+and+angiogenesis+around+cortical+stroke.">Collateral growth and angiogenesis around cortical stroke.</a> Stroke. 32 (9), 2179-2184 (2001).</li><li>Wu, 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=Targeted+delivery+of+methotrexate+to+epidermal+growth+factor+receptor-positive+brain+tumors+by+means+of+cetuximab+(IMC-C225)+dendrimer+bioconjugates.">Targeted delivery of methotrexate to epidermal growth factor receptor-positive brain tumors by means of cetuximab (IMC-C225) dendrimer bioconjugates.</a> Mol Cancer Ther. 5 (1), 52-59 (2006).</li><li>Pignataro, G., Studer, F. E., Wilz, A., Simon, R. P., Boison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotection+in+ischemic+mouse+brain+induced+by+stem+cell-derived+brain+implants.">Neuroprotection in ischemic mouse brain induced by stem cell-derived brain implants.</a> J Cereb Blood Flow Metab. 27 (5), 919-927 (2007).</li><li>Sugiyama, Y., Kusuhara, H., Suzuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+and+biochemical+analysis+of+carrier-mediated+efflux+of+drugs+through+the+blood-brain+and+blood-cerebrospinal+fluid+barriers:+importance+in+the+drug+delivery+to+the+brain.">Kinetic and biochemical analysis of carrier-mediated efflux of drugs through the blood-brain and blood-cerebrospinal fluid barriers: importance in the drug delivery to the brain.</a> J Control Release. 62 (1-2), 179-186 (1999).</li><li>Pardridge, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+and+gene+delivery+to+the+brain:+the+vascular+route.">Drug and gene delivery to the brain: the vascular route.</a> Neuron. 36 (4), 555-558 (2002).</li><li>Vantyghem, S. A., Postenka, C. O., Chambers, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrous+cycle+influences+organ-specific+metastasis+of+B16F10+melanoma+cells.">Estrous cycle influences organ-specific metastasis of B16F10 melanoma cells.</a> Cancer Res. 63 (16), 4763-4765 (2003).</li><li>Huang, R. Q., 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=Efficient+gene+delivery+targeted+to+the+brain+using+a+transferrin-conjugated+polyethyleneglycol-modified+polyamidoamine+dendrimer.">Efficient gene delivery targeted to the brain using a transferrin-conjugated polyethyleneglycol-modified polyamidoamine dendrimer.</a> FASEB J. 21 (4), 1117-1125 (2007).</li><li>Liu, R., Martuza, R. L., Rabkin, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracarotid+delivery+of+oncolytic+HSV+vector+G47Delta+to+metastatic+breast+cancer+in+the+brain.">Intracarotid delivery of oncolytic HSV vector G47Delta to metastatic breast cancer in the brain.</a> Gene Ther. 12 (8), 647-654 (2005).</li><li>Kumar, P., 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=Transvascular+delivery+of+small+interfering+RNA+to+the+central+nervous+system.">Transvascular delivery of small interfering RNA to the central nervous system.</a> Nature. 448 (7149), 39-43 (2007).</li><li>Wrobel, J. K., Wolff, G., Xiao, R., Power, R. F., Toborek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+Selenium+Supplementation+Modulates+Growth+of+Brain+Metastatic+Tumors+and+Changes+the+Expression+of+Adhesion+Molecules+in+Brain+Microvessels.">Dietary Selenium Supplementation Modulates Growth of Brain Metastatic Tumors and Changes the Expression of Adhesion Molecules in Brain Microvessels.</a> Biol Trace Elem Res. , (2015).</li></ol>

The authors have nothing to disclose.

Die Infusions Mikrochirurgie hier beschrieben wurde , erwies sich bei der Bereitstellung von exogenen Substanzen verschiedener biologischer Funktionen in das ZNS sehr erfolgreich zu sein, verhindert unerwünschte Verbreitung im ganzen Körper 1, 12. Störung der Blut-Hirn-Schranke ist ein pathologisches Merkmal mehrerer ZNS zusammenhängenden Krankheiten; daher die Beurteilung der Beziehung von exogenen Substanzen mit dem Blut-Hirn-Schranke ist von großer Bedeutung und Interesse. Diese Operation vorgestellte Modell verursacht begrenzte Trauma für die Tiere und ist mit sehr niedrigen Sterblichkeit 1, 12 verbunden. Darüber hinaus ist das Verfahren nicht mit der ZNS - Funktion oder zerebralen Blutfluß 1, 12 stören. Der wichtigste Aspekt dieses Verfahrens ist es, die richtigen Schnitt in der ECA auszuführen, die nur das ermöglichen sollteEinsetzen des Kapillarrohres. Ein breiterer Schnitt verursacht undichte des Stoffes infundiert werden, eine andere ECA Operation auf der gegenüberliegenden Seite zu erfordern. Um diese Situation zu vermeiden, sollte die Kapillarschlauchs einen größeren Durchmesser als der Schnitt haben, und auch eine scharfe Kante haben sollten, um seinen Eingang durch die Arterie zu erleichtern. In Bezug auf den technischen Aspekt ist, ein voll ausgebildetes Personal in der Lage diese Operation innerhalb von 20 Minuten durchzuführen. Die wesentliche Einschränkung dieser Technik ist die Möglichkeit der Infusion nur einmal durch die gleiche ICA. Für wiederholte Substanzzuführsegment in das ZNS, ein Gefäß Mikroport verwendet werden muss , und wurde vorher 1 .Das Technik hier hat das Potential , beschrieben in den beschriebenen Untersuchungen über neue positive und negative Wechselwirkungen innerhalb der BBB, sowie ihrer Unterstützung zu liefern Pathogenen , Drogen und physiologische Mittel in das Gehirn.

In - vivo - Modellen des zentralen Nervensystems (ZNS) Erkrankungen erfordern eine effektive Bereitstellung von exogenen Substanzen, wie Medikamente, Krankheitserreger oder Exosomen, in das Gehirn. Daher sollte eine ideale Übermittlungsmethode minimalem Trauma für das Tier veranlassen, um die Integrität des neuronalen Netzes zu erhalten, und eine hohe Stoffkonzentrationen im Gehirn 1 erzielen. Mehrere chirurgische Methoden der lokalen Substanzabgabe beschrieben wurden, einschließlich intra-Mantel, intrazerebrale und intraventrikuläre Injektionen oder Implantate 2, 3, 4, 5. Diese Ansätze sind jedoch als traumatisches zum ZNS und ermöglichen die Verabreichung von nur geringen Mengen der Substanz von Interesse. Außerdem wurde vorgeschlagen , dass exogene Substanzen können schnell durch die Zerebrospinalflüssigkeit 6 entfernt werden , </sup&gt; und eine geringe Penetrationsbereich zum Hirnparenchym wurde 7 beobachtet, wenn die oben genannten Techniken eingesetzt werden. Systemische Liefermethoden, wie orale, pulmonale, subkutane und intravenöse Wege, sind häufiger in Tiermodellen verwendet, obwohl sie bei der Bereitstellung der Substanzen in das ZNS geringe Wirksamkeit aufweisen, aufgrund von anderen Organen Aufnahme 8, 9. Daher erfordern diese Verabreichungswege erhöhten Dosen der Substanzen verabreicht, 10 das Risiko von Nebenwirkungen und Toxizität zu, 11. Hier beschreiben wir eine Maus Infusionsmikrotechnik, die effektiv Substanzen direkt in das Gehirn über die Arteria carotis interna liefert. Zusätzlich zu der Abgabe an das ZNS Targeting, ist diese Technik nicht die normale physiologische Barrieren umgehen und ist daher von großer Bedeutung für biological Prozesse in den Passagen von Therapeutika oder Krankheitserreger in das Gehirn beteiligt.

<table><tbody><tr><td>Anesthesia instrument</td><td>Vetequip</td><td>901806</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tool</td><td>14558-09</td><td></td></tr><tr><td>Surgical forceps straight tip</td><td>Fine Science Tool</td><td>00108-11</td><td></td></tr><tr><td>Surgical forceps angled tip</td><td>Fine Science Tool</td><td>00109-11</td><td></td></tr><tr><td>Spring scissors</td><td>Fine Science Tool</td><td>15000-08</td><td></td></tr><tr><td>Nylon suture</td><td>Braintree Scientific</td><td>SUT-S 104</td><td></td></tr><tr><td>Capillary tubing (Micro-Renathane 0.010&amp;rdquo; x 0.005&amp;rdquo; per ft.)&amp;nbsp;</td><td>Braintree Scientific</td><td>MRE01050</td><td></td></tr><tr><td>Closing suture</td><td>VWR</td><td>95057-036</td><td></td></tr><tr><td>Isoflurane</td><td>Piramal</td><td></td><td></td></tr><tr><td>2,3,5-Triphenyltetrazolium chloride</td><td>FisherScientific</td><td>50-121-8005</td><td></td></tr></tbody></table>

mouse microsurgery infusion technique for targeted substance delivery into the cns via the internal carotid artery

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous substances into the brain. These exogenous substances may induce injurious impact or constitute therapeutic strategy. The most common delivery methods of exogenous substances into the brain are based on systemic deliveries, such as subcutaneous or intravenous routes. Although commonly used, these approaches have several limitations, including low delivery efficacy into the brain. In contrast, surgical methods that locally deliver substances into the CNS are more specific and prevent the uptake of the exogenous substances by other organs. Several surgical methods for CNS delivery are available; however, they tend to be very traumatic. Here, we describe a mouse infusion microsurgery technique, which effectively delivers substances into the brain via the internal carotid artery, with minimal trauma and no interference with normal CNS functionality.

Die Maus Infusions Mikrochirurgie hier beschriebene Technik ist sehr vielseitig und verwendet wurde , verschiedene Stoffe zu liefern , direkt in das Gehirn, einschließlich der Lieferung von Tumorzellen in einem repräsentativen Modells der Hirnmetastasenbildung 1, 12. Diese Technik ist auch geeignet, um die pathologischen Aspekte verschiedener Krankheitserreger im ZNS zu beurteilen. In einem Mausmodell der HIV-Infektion wurde die Infusions Chirurgie verwendet viralen Partikel direkt in den CCA zu injizieren. Wir fanden, dass 7 Tage nach der Operation, die für ZNS-HIV durch Echtzeit-PCR-positiv war. Weiterhin war die ipsilaterale Hemisphäre Infektion 6-10 Falten höher als die kontralateralen Hemisphäre (Abbildung 2). Ein anderer geeigneter Ansatz der Infusions Chirurgie hier beschrieben wird, ist zu liefern<html> extrazellulären Vesikeln (ECV), vor allem Exosomen, in das ZNS. Abbildung 3 zeigt die Gegenwart von CD63, einem Marker für Exosomen, markiert mit grün fluoreszierendem Protein (GFP) in der ipsilateralen Gehirn einer Maus 24 Stunden nach der Infusion. <img alt="Abbildung 1" src="/files/ftp_upload/54804/54804fig1.jpg" /> Abbildung 1: Schematische Darstellung der Infusion über die Arterie carotis interna. Im Einsatz (a) und (b) zeigen die Lokalisierung der oberen und unteren Knoten verbunden sind; (c) stellt den kleinen Schnitt in der ECA, durch welche das Kapillarrohr eingeführt wird (graue Linie). <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54804/54804fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54804/54804fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <html> 4804fig2.jpg &quot;/&gt; Abbildung 2: HIV - Nachweis durch Real-time PCR nach Viral Infusion über die Arteria carotis interna. Die Balken zeigen Mittelwerte HIV - DNA geerntet von Mäusen , 7 Tage die Infusion über die linke Arteria carotis interna posten. Die Fehlerbalken zeigen die Standardabweichung. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54804/54804fig2large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54804/54804fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 3" src="/files/ftp_upload/54804/54804fig3.jpg" /> Abbildung 3: Immunfluoreszenz von Maus Brain Section Depicting GFP-markierten CD63, ein exosome Marker, nach ECV Infusion über die Arteria carotis interna. Das repräsentative Bild zeigt eine Maus Gehirn microvessel mit CD63 exprimieren ECV (grün) verbunden. Maßstabsbalken: 50 &amp; mgr; m.erhalten = &quot;_ blank&quot;&gt; Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.

Watch this Scientific Journal Video about Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery at JoVE.com

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Maus Mikroinfusionstechnik für Zielsubstanz Lieferung in das ZNS<i&gt; über</i&gt; Die Arteria carotis interna

Animal models of central nervous system (CNS) diseases and, consequently, blood-brain barrier disruption diseases, require the delivery of exogenous ...

mouse-microsurgery-infusion-technique-for-targeted-substance-delivery

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

10.8K Aufrufe.  University of Miami. The present protocol describes a mouse microsurgery infusion technique, which effectively delivers substances directly into the brain via the internal carotid artery.

Serie: Mouse Microsurgery Infusion Technique for Targeted Substance Delivery into the CNS via the Internal Carotid Artery

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Intracarotid Cancer Cell Injection zu produzieren Mausmodelle von Hirnmetastase

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

Forschung

Krebsforschung

Targeting of Deep Brain Structures with Microinjections for Delivery of Drugs, Viral Vectors, or Cell Transplants

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an injecting needle produces during its trajectory is a major concern especially in the mouse brain for not only the brain is small but also sometimes multiple injections are needed.  We show here a method to produce glass capillary needles with a 50-&mu;m lumen which significantly reduces the brain damage and allows a precise targeting into the rodent brain. This method allows a delivery of small volumes (from 20 to 100 nl), reduces bleeding risks, and minimizes passive diffusion of drugs into the brain parenchyma. By using different size of capillary glass tubes, or changing the needle lumen, several types of substances and cells can be injected. Microinjections with a glass capillary tube represent a significant improvement in injection techniques and deep brain targeting with minimal collateral damage in small rodents.

In this article, we show a method to make glass capillary needles with a 50-&mu;m lumen. This technique significantly reduces the brain damage, minimizes passive diffusion of drugs and allows a precise targeting into the rodent brain.

Targeting von Deep Brain Strukturen mit Mikroinjektionen für die Lieferung von Drogen, virale Vektoren oder Zelltransplantaten

Microinjections into the brain parenchyma are important procedures to deliver drugs, viral vectors or cell transplants.  The brain lesion that an ...

Neurowissenschaften

Construction and Implantation of a Microinfusion System for Sustained Delivery of Neuroactive Agents.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with high precision while minimizing localized trauma and inflammation. Also, the ability to customize the system to accommodate animals of different species and sizes is desirable. This video presentation demonstrates the construction of an infusion system that can be fitted to any particular research animal. The delivery microcannula diameter is approximately 10-fold smaller than most infusion cannulas presently used. This translates into enhanced accuracy and reduced trauma to the brain region under study. The delivery cannula can also be sculpted to fit the contour of the surface of the animal's skull, thereby allowing closure of the scalp incision neatly over the infusion system, precluding the need for a skull-mounted pedestal, reducing risk of infection, and ensuring a greater level of comfort to the animal. The system is assembled in an air-free environment and requires the researcher to fashion glass micropipettes with a heat source. These construction methods require special skills that are best acquired, if not in person, using video instruction.  (This article is based on work first reported in J Neurosci Methods. 2008 Jan 30;167(2):213-20. Epub 2007 Aug 28.).

As neuroscience inquiry becomes more sophisticated, investigation of brain structures and circuitry requires improved levels of accuracy and higher resolution. We have developed a method for the preparation and implantation of a chronic infusion system within the brain utilizing a borosilicate microcannula with a tip diameter of 50 microns.

Bau-und Implantation eines Mikroinfusion System für Nachhaltige Lieferung von Neuroactive Agents.

Sustained delivery of neuroactive agents is widely used in neuroscience, but poses many technical challenges. It is necessary to deliver the agent with ...

Biologie

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

Maus-Modell der Intraluminal MCAO: Hirninfarkt Evaluation durch Cresylviolett Staining

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

Medizin

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.

Rat Modell der Blut-Hirn-Schranke Störung zu Neurovascular Therapeutics Gezielte zulassen

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

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural microenvironment and stromal cell interaction, cannot be entirely replicated with in vitro assays; thus, animal models are critical for investigating and understanding the effects of therapeutic intervention. However, most brain tumor xenografting methods do not produce brain metastases consistently in terms of the time frame and tumor burden. Brain metastasis models generated by intracardiac injection of cancer cells can result in unintended extracranial tumor burden and lead to non-brain metastatic morbidity and mortality. Although intracranial injection of cancer cells can limit extracranial tumor formation, it has several caveats, such as the injected cells frequently form a singular tumor mass at the injection site, high leptomeningeal involvement, and damage to brain vasculature during needle penetration. This protocol describes a mouse model of brain metastasis generated by internal carotid artery injection. This method produces intracranial tumors consistently without the involvement of other organs, enabling the evaluation of therapeutic agents for brain metastasis.

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Most brain metastasis mouse models are complicated by systemic metastases confounding analysis of mortality and therapeutic intervention outcomes. Presented here is a protocol for internal carotid injection of cancer cells that produces consistent intracranial tumors with minimal systemic tumors.

Modellierung der Hirnmetastasierung durch Injektion von Krebszellen in die innere Halsschlagader

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

Heterotope Mucosal-Transplantationsverfahren für Direkt Drug Delivery zum Gehirn bei Mäusen

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Halsschlagader Infusionen für pharmakokinetischen und pharmakodynamischen Analyse von Taxanen in Mäusen

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

<meta charset="utf8"></head><body>Reparatur der Arteria carotis communis nach Injektion in einem Mausmodell

Facilitating Repeat Intracarotid Injections in Mouse Models by a Novel Injection Site Repair Technique

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there is an urgent need to develop methods for intracarotid injections in mouse models, including methods to repair the carotid artery in mice after injection to allow for subsequent injections. We developed a method of intracarotid injection in a mouse model to deliver therapeutics into the internal carotid artery (ICA) with two alternative procedures. 
During injection, the needle is inserted into the common carotid artery (CCA) after tying a suture around the external carotid artery (ECA) and injected therapeutics are delivered into the ICA. Following injection, the common carotid artery (CCA) can be ligated, which limits the number of intracarotid injections to one. The alternative procedure described in this article includes a modification where intracarotid artery injection is followed by injection site repair of the CCA, which restores blood flow within the CCA and avoids the complication of cerebral ischemia seen in some mouse models. 
We also compared the delivery of bone marrow-derived human mesenchymal stem cells (BM-hMSCs) to intracranial tumors when delivered through intracarotid injection with and without injection site repair following the injection. Delivery of BM-hMSCs does not differ significantly between the methods. Our results demonstrate that injection site repair of the CCA allows for repeat injections through the same artery and does not impair the delivery and distribution of injected material, thus providing a model with greater flexibility that more closely emulates intracarotid injection in humans.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Weiterentwicklung der Glioblastombehandlung durch intraarterielle Verabreichungsstrategien für onkolytische Viren und microRNAs

Repair of the intracarotid artery in a mouse model after injection returns blood flow to the artery without negatively impacting the distribution of the injected material. Injection site repair facilitates subsequent injections through the same artery and prevents cerebral ischemia in mouse strains that lack a complete Circle of Willis.

Erleichterung wiederholter Intrakarotis-Injektionen in Mausmodellen durch eine neuartige Reparaturtechnik an der Injektionsstelle

Given recent advances in the delivery of novel antitumor therapeutics using endovascular selective intraarterial delivery methods in neuro-oncology, there ...

Maus Mikroinfusionstechnik für Zielsubstanz Lieferung in das ZNS

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Intracarotid Cancer Cell Injection zu produzieren Mausmodelle von Hirnmetastase

Targeting von Deep Brain Strukturen mit Mikroinjektionen für die Lieferung von Drogen, virale Vektoren oder Zelltransplantaten

Bau-und Implantation eines Mikroinfusion System für Nachhaltige Lieferung von Neuroactive Agents.

Maus-Modell der Intraluminal MCAO: Hirninfarkt Evaluation durch Cresylviolett Staining

Rat Modell der Blut-Hirn-Schranke Störung zu Neurovascular Therapeutics Gezielte zulassen

Modellierung der Hirnmetastasierung durch Injektion von Krebszellen in die innere Halsschlagader

Heterotope Mucosal-Transplantationsverfahren für Direkt Drug Delivery zum Gehirn bei Mäusen

Halsschlagader Infusionen für pharmakokinetischen und pharmakodynamischen Analyse von Taxanen in Mäusen

Erleichterung wiederholter Intrakarotis-Injektionen in Mausmodellen durch eine neuartige Reparaturtechnik an der Injektionsstelle

Erleichterung wiederholter Intrakarotis-Injektionen in Mausmodellen durch eine neuartige Reparaturtechnik an der Injektionsstelle