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>

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	<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.

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			<td height="21" style="height:21px;width:367px;">Anesthesia instrument</td>
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			<td align="right" style="width:119px;">901806</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Surgical scissors</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">14558-09</td>
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			<td height="21" style="height:21px;width:367px;">Surgical forceps straight tip</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">00108-11</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Surgical forceps angled tip</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">00109-11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Spring scissors</td>
			<td style="width:149px;">Fine Science Tool</td>
			<td style="width:119px;">15000-08</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Nylon suture</td>
			<td style="width:149px;">Braintree Scientific</td>
			<td style="width:119px;">SUT-S 104</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Capillary tubing (Micro-Renathane 0.010&#8221; x 0.005&#8221; per ft.)&#160;</td>
			<td style="width:149px;">Braintree Scientific</td>
			<td style="width:119px;">MRE01050</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Closing suture</td>
			<td style="width:149px;">VWR</td>
			<td style="width:119px;">95057-036</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Isoflurane</td>
			<td style="width:149px;">Piramal</td>
			<td style="width:119px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">2,3,5-Triphenyltetrazolium chloride</td>
			<td style="width:149px;">FisherScientific</td>
			<td style="width:119px;">50-121-8005</td>
		</tr>
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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.remotexs.ntu.edu.sg/files/ftp_upload/54804/54804fig1large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/files/ftp_upload/54804/54804fig2large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

Ultraschall-gesteuerte Mikroinjektion in der Maus Vorderhirnneuronen In Utero In E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

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Neurowissenschaften

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals through the vitreous fluid ensures that they act upon the entire RGC population. Viral vectors, plasmids or short interfering RNAs (siRNAs) can also be delivered to the vitreous chamber in order to infect or transfect retinal cells 5-12. The high tropism of Adeno-Associated Virus (AAV) vectors is beneficial to target RGCs, with an infection rate approaching 90% of cells near the injection site 6, 7, 13-15. Moreover, RGCs can be selectively transfected by applying siRNAs, plasmids, or viral vectors to the cut end of the optic nerve 16-19 or injecting vectors into their target the superior colliculus 10. This allows researchers to study apoptotic mechanisms in the injured neuronal population without confounding effects on other bystander neurons or surrounding glia. RGC apoptosis has a characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a window for experimental manipulations directed against pathways involved in apoptosis. Manipulations that directly target RGCs from the transected optic nerve stump are performed at the time of axotomy, immediately after cutting the nerve. In contrast, when substances are delivered via an intraocular route, they can be injected prior to surgery or within the first 3 days after surgery, preceding the initiation of apoptosis in axotomized RGCs. In the present article, we demonstrate several methods for experimental manipulations after optic nerve transection.

Optic Nerve transection is a widely used model of adult CNS injury. This model is ideal for performing a number of experimental manipulations that target the retina globally or directly target the injured neuronal population of retinal ganglion cells.

Methoden für die experimentelle Manipulationen nach Optic Nerve Durchtrennung im Säuger-ZNS

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. The optic nerve can be ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

Effiziente Gene Lieferung in mehrere ZNS Territories mit In Utero Elektroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Schnelle Micro-Iontophorese von Glutamat und GABA: Ein nützliches Tool, um Synaptic Integration Untersuchen

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

Beurteilung der Gefäß Regeneration im ZNS Verwenden der Maus Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Surgical Method for Virally Mediated Gene Delivery to the Mouse Inner Ear through the Round Window Membrane

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and genetic mechanisms that contribute to hearing loss. Introduction of vectors into the inner ear must be done in a way that widely distributes the agent throughout the cochlea while minimizing injury to the existing structures. This manuscript describes a post-auricular surgical approach that can be used for mouse cochlear therapy using molecular, pharmacologic, and viral delivery to mice postnatal day 10 and older via the round window membrane (RWM). This surgical approach enables rapid and direct delivery into the scala tympani while minimizing blood loss and avoiding animal mortality. This technique involves negligible or no damage to essential structures of the inner and middle ear as well as neck muscles while wholly preserving hearing. To demonstrate the efficacy of this surgical technique, the vesicular glutamate transporter 3 knockout (VGLUT3 KO) mice will be used as an example of a mouse model of congenital deafness that recovers hearing after delivery of VGLUT3 to the inner ear using an adeno-associated virus (AAV-1).

The described post-auricular surgical approach allows rapid and direct delivery into the mouse cochlear scala tympani while minimizing blood loss and animal mortality. This method can be used for cochlear therapy using molecular, pharmacologic and viral delivery to postnatal mice through the round window membrane.

Chirurgische Verfahren zum viral vermittelte Gentransfer in die Maus-Innenohr über die runde Fenstermembran

Gene therapy, used to achieve functional recovery from sensorineural deafness, promises to grant better understanding of the underlying molecular and ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Stereotaktische Infusion von oligomeren Amyloid-beta in die Maus-Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Autonomous and Rechargeable Microneurostimulator Endoscopically Implantable into the Submucosa

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency low-energetic stimulation can help to effectively moderate and alleviate the symptoms of gastric dysmotility. The goal of the research was the development of a miniature, endoscopically implantable device to a submucosal pocket. The implantable device is a fully customized electronic package which was specifically designed for the purpose of experiments in the submucosa. The device is equipped with a lithium-ion battery which can be recharged wirelessly by receiving an incident magnetic field from the charging/transmitting coil. The uplink communication is achieved in a MedRadio band at 432 MHz. The device was endoscopically inserted into the submucosal pocket of a live domestic pig used as an in vivo model, specifically in the stomach antrum. The experiment confirmed that the designed device can be implanted into the submucosa and is capable of bidirectional communication. The device can perform bipolar stimulation of muscle tissue.

The application of high-frequency low-energetic stimulation can alleviate the symptoms of gastric dysmotility. In this research, a miniature, endoscopically implantable and wirelessly rechargeable device which is implanted into a submucosal pocket is presented. Successful both-way communication and stimulation control were achieved during an experiment on live pig.

Autonome und wiederaufladbare Microneurostimulator endoskopisch implantierbare in der Submukosa

Gastric dysmotility can be a sign of common diseases such as longstanding diabetes mellitus. It is known that the application of high-frequency ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Transplantation von chemogenetisch hergestellten kortikalen Interneureuren in frühpostnatale Maushirne

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 &#181;g of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.

Lieferung von Antikörpern in das Murine Gehirn über Konvektionsverstärkte Lieferung

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...