We thank members of the Mariani lab for critical reading of the manuscript. Our funding sources were: the Baxter Medical Scholar Research Fellowship (to M.K.S.), USC undergraduate fellowships and the Provost, Dean Joan M. Schaeffer, and Rose Hills Fellowships, (to M.K.S.). We also acknowledge a CIRM BRIDGES fellowship through Pasadena City College (to T.T.T). and the James H. Zumberge Research and Innovation Fund, the USC Regenerative Medicine Initiative, and the NIAMS NIH under Award Number R21AR064462 (to F.V.M).

HINWEIS: Alle Verfahren sind in Übereinstimmung mit einem von der Institutional Animal Care und Verwenden Committee (IACUC) an der University of Southern California genehmigten Tier Protokoll. 1. Vorbereitung für die Chirurgie <ol><li> Legen Sie die notwendigen chirurgischen Werkzeuge und Materialien (sterilisiert, Einträge 1 bis 18 in der Materialliste) und vereinbaren Sie einen Binokular und einen Lichtwellenleiter transLicht in einer Haube oder einem Wartungstisch. </li><li> Legen Sie eine geschlechtsreif Maus (20 - 30 g, 1-3 Monate alt) in einer Induktionskammer mit 4% Isofluran Anästhesie zu induzieren. </li><li> Bestätigen Anästhesie mit einer Pfote und / oder Schwanz Prise Test. </li><li> Bewerben Salbe die Augen der Maus die zu Augentrockenheit während der Operation zu vermeiden. </li><li> Bewegen Sie die Maus auf den Mikroskoptisch. Platzieren Sie die Maus in die Bugnase des Wartungsschlauch und seitlich zu positionieren. Geben Sie eine Erwärmung Beutel neben dem Tier zur Aufrechterhaltung der Körpertemperatur. NICHTE:. Position der Maus hängt von persönlichen Vorlieben und / oder Händigkeit des Chirurgen, der rechten oder linken Seite ist in Ordnung. </li><li> Passen Isofluran zu 2-3% für die Wartung, und beobachten Sie die Atemfrequenz. </li><li> Injizieren Buprenorphin (0,05 mg / kg) subkutan (Seitenflanke des Beins) zum postoperativen Schmerz mit einer 25 G-Nadel. Das Bein kann zucken, welche eine normale Reaktion ist. </li></ol> 2. Öffnungs Incision <ol><li> Ermitteln Sie die gewünschte Rippe durch Palpation, und löschen Sie den Bereich der Haare mit einem Rasierer. HINWEIS: Rippen 8-10 werden empfohlen, da Rippen 1-7 sind näher an der Lunge und 11-13 sind deutlich kürzer. Zählen der Rippen sollte mit den meisten rostral Rippe als Nummer 1 beginnen. </li><li> Bereiten Sie die Gegend mit wechsel PVP-Jod und Isopropanol (70%) Abtupfen (3x wird oft von Tiernutzung Protokolle empfohlen). </li><li> Schneiden Sie ein 2 cm Schnitt durch die Haut, direkt über und parallel mit mittelgroßen Mikroscheren gewünschten Rippe. IncisE durch die darunter liegende Muskulatur und Fettschichten. </li><li> Legen Sie alle drei Schichten (Haut, Muskeln, Fett) in eine Rückziehvorrichtung, um das Operationsfeld freizulegen, bei gleichzeitiger Minimierung der Größe des Einschnitts. </li></ol> 3. Ausschneiden des Rib <ol><li> Durch die Zwischenrippenmuskeln, die über dem gewünschten Abschnitt der Rippe mit einer 5,0 mm Skalpell. Position diesen Schnitt um 5 mm proximal der chondrocostal gemeinsame da hier die Rippe ist nicht so stark gekrümmten. Trennen Sie vorsichtig die Muskeln von den Knochen mit feiner Spitze Pinzette. </li><li> Um eine Resektion zu schaffen unter Beibehaltung der Knochenhaut in der Tier, schneiden durch die Knochenhaut, über die Länge der Rippe mit einer 5,0 mm Skalpell. Trennen Sie vorsichtig das Periost von der darunter liegenden Knochen seitlich feine Spitze Pinzette. Gehen Sie mit Vorsicht, da die Knochenhaut ist sehr empfindlich und hat eine gelartige Konsistenz. </li><li> Weiter stellen Sie einen Querschnitt durch den Knochen an einem Ende mit feinen Mikro Schere. Bei Bedarf messen die Resektion mitein Fadenkreuz im Mikroskop oder einem Lineal Typ Wertangabe. Dann heben Sie die Knochen von der Knochenhaut und schneiden Sie das andere Ende. HINWEIS: Gehen Sie mit Vorsicht. Dies ist der heikelste Schritt, wie oben ziehen, ohne Auslösen der Rippe unter die Pleura Membran reißen und zu einem Pneumothorax. Wenn der pleuralen Membran zerrissen wird dies ohne weiteres ersichtlich sein, wie das Innere der Brusthöhle sichtbar werden. </li><li> Wenn die Blutung als Folge des Schneidens des Knochens auftritt, wenden Sie Druck an der Schnittende mit einem Wattestäbchen für 4-5 Sekunden, um die Blutung zu stoppen. HINWEIS: In den seltenen Fällen, dass Blutung anhält, kann es notwendig sein, zu stoppen und das Tier einschläfern als signifikante Blutverlust wird die Erholung gefährden (ohne Flüssigkeitsersatz ist die maximale Blutvolumen, die sicher verloren gehen können 10% des gesamten Blutvolumens oder 7.7 -. 8 ul / g für eine 25 g Maus entspricht dies rund 180 bis 200 &amp; mgr; 9). </li><li> Sofort legen Sie die entfernt Rippe in4% PFA für eine spätere Analyse. HINWEIS: Wenn die Resektion von sowohl dem Knochen und der Knochenhaut ist durchgeführt werden soll, lassen Sie den Knochenhaut Schnitt und Trennung (Schritt 3.2). Da die Knochenhaut ist sehr eng an die Pleura Membran angebracht gehen vorsichtig, Hänseleien der Rippe von der Pleura-Membran mit einer Pinzette, um Risse zu vermeiden. </li></ol> 4. Schließen der Inzision <ol><li> Nähen Sie die Zwischenrippenmuskeln über die Spitze des verbliebenen periostalen Hülle mit 9-0 Nahtmaterial (2 Nähte sind in der Regel ausreichend). Zeigen die Fäden direkt über die Schnittenden der Rippe, die als Indikatoren für die Chirurgie Ort handeln. </li><li> Entfernen Sie die Rückziehvorrichtung. Naht der darüberliegenden Muskeln und Fett mit 9-0 Nahtmaterial (3-4 Nähte sind in der Regel ausreichend). In Mäusen, die mit besonders dicken Schichten von Muskulatur und / oder Fett, Naht jede Ebene (beispielsweise 1-Schicht von Nähten für die Muskelschicht und 1 Schicht von Nähten für die Fettschicht). </li><li> Schließen Sie dieHaut mit 7-0 Nahtmaterial (4-5 Nähte sind in der Regel ausreichend). </li><li> Sichern Sie den Schnitt mit Nahtkleber, Kneifen die Ränder zusammen mit großen Zangen. </li><li> Langsam zu entwöhnen Sie die Maus aus Isofluran durch zunächst für einige Minuten Einstellung auf 1% und schalten Sie. </li><li> Platzieren Sie die Maus unter einer Wärmelampe und verlassen, bis das Bewusstsein wiedererlangt, 5 - 10 min. Nach Bewusstsein wiedererlangt und während der Heilungsphase sollte die Maus bewegen und normal ambulate und zeigen keine Anzeichen von Leiden. </li></ol> 5. Wiederaufnahme und Analyse <ol><li> Geben postoperativen Schmerztherapie einschließlich der Anwendung von Buprenorphin in einer mündlichen Gelatineform bei 0,5 mg / kg alle 12 Stunden für 48 Stunden. Die orale Verabreichung erfordert keine Zurückhalten der Tiere, die Schmerzen für den Brustbereich verursachen könnten. </li><li> Pflegen Sie die Maus in einen Käfig mit freiem Zugang zu Futter und Wasser während der Heilungsphase. Nachdem der Schnitt trocken ist, können weibliche Tiere zusammengebracht werden, während Männers müssen getrennt bleiben, um Kämpfe zu vermeiden. </li><li> Nach der Einheilzeit, folgen Sie dem zugelassenen Verfahren für die Euthanasie. Entfernen Sie den Brustkorb zur Fixierung und Analyse. </li></ol>

<ol>
	<li>Philip, S. J., Kumar, R. J., Menon, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+study+of+rib+regeneration+following+costectomy+in+adolescent+idiopathic+scoliosis.">Morphological study of rib regeneration following costectomy in adolescent idiopathic scoliosis.</a> Eur Spine J. 14 (8), 772-776 (2005).</li><li>Munro, I. R., Guyuron, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-rib+cranioplasty.">Split-rib cranioplasty.</a> Ann Plast Surg. 7 (5), 341-346 (1981).</li><li>Taggard, D. A., Menezes, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Successful+use+of+rib+grafts+for+cranioplasty+in+children.+Pediatric+neurosurgery.">Successful use of rib grafts for cranioplasty in children. Pediatric neurosurgery.</a> 34 (3), 149-155 (2001).</li><li>Head, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+Regeneration+fo+the+Ribs:+A+problem+in+thoracic+surgery.">Prevention of Regeneration fo the Ribs: A problem in thoracic surgery.</a> Archives of Surgery. 14 (6), 1215-1221 (1927).</li><li>Kawanabe, Y., Nagata, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+of+costal+cartilage+harvest+for+total+auricular+reconstruction:+part+I.+Avoidance+and+prevention+of+intraoperative+and+postoperative+complications+and+problems.">A new method of costal cartilage harvest for total auricular reconstruction: part I. Avoidance and prevention of intraoperative and postoperative complications and problems.</a> Plastic and reconstructive surgery. 117 (6), 2011-2018 (2006).</li><li>Cheung, K. M., 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=An+externally+fixed+femoral+fracture+model+for+mice.">An externally fixed femoral fracture model for mice.</a> Journal of orthopaedic research : official publication of the Orthopaedic Research Society. 21 (4), 685-690 (2003).</li><li>Matthys, R., Perren, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+fixator+for+use+in+the+mouse.">Internal fixator for use in the mouse.</a> Injury. 40, S103-S109 (2009).</li><li>Cooper, G. M., 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=Testing+the+critical+size+in+calvarial+bone+defects:+revisiting+the+concept+of+a+critical-size+defect.">Testing the critical size in calvarial bone defects: revisiting the concept of a critical-size defect.</a> Plastic and reconstructive surgery. 125 (6), 1685-1692 (2010).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ask+the+Vet.">Ask the Vet.</a> JAX NOTES. 499, (2005).</li><li>Flecknell, P. A., Roughan, J. V., Stewart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+oral+buprenorphine+('buprenorphine+jello')+for+postoperative+analgesia+in+rats--a+clinical+trial.">Use of oral buprenorphine ('buprenorphine jello') for postoperative analgesia in rats--a clinical trial.</a> Laboratory animals. 33 (2), 169-174 (1999).</li><li>Rigueur, D., Lyons, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+skeletal+staining.">Whole-mount skeletal staining.</a> Methods in molecular biology. 1130, 113-121 (2014).</li><li>Evans, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+somitic+cells+to+the+avian+ribs.">Contribution of somitic cells to the avian ribs.</a> Developmental biology. 256 (1), 114-126 (2003).</li><li>Colnot, C., Thompson, Z., Miclau, T., Werb, Z., Helms, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+fracture+repair+in+the+absence+of+MMP9.">Altered fracture repair in the absence of MMP9.</a> Development. 130 (17), 4123-4133 (2003).</li><li>Lu, C., 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=Cellular+basis+for+age-related+changes+in+fracture+repair.">Cellular basis for age-related changes in fracture repair.</a> Journal of orthopaedic research : official publication of the Orthopaedic Research Society. 23 (6), 1300-1307 (2005).</li><li>Zilberman, Y., Gafni, Y., Pelled, G., Gazit, Z., Gazit, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+imaging+in+bone.">Bioluminescent imaging in bone.</a> Methods in molecular biology. 455, 261-272 (2008).</li><li>Pelled, G., Gazit, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+using+osteocalcin-luciferase.">Imaging using osteocalcin-luciferase.</a> Journal of musculoskeletal. 4 (4), 362-363 (2004).</li><li>Elefteriou, F., Yang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+mouse+models+for+bone+studies--strengths+and+limitations.">Genetic mouse models for bone studies--strengths and limitations.</a> Bone. 49 (6), 1242-1254 (2011).</li><li>Srour, M. 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=Natural+large-scale+regeneration+of+rib+cartilage+in+a+mouse.">Natural large-scale regeneration of rib cartilage in a mouse.</a> J. Bone Miner. , (2014).</li></ol>

The authors have nothing to disclose

Wenn erst zu lernen, dieses Protokoll, zu bestimmen, wo die anfänglichen Schnitt zu finden kann eine Herausforderung sein. , Praxis auf eingeschläfert Mäusen hilft jedoch dem Chirurgen erfahren, wo die anfänglichen Schnitt legen und setzen die gewünschte Rippe reseziert werden. Die Arbeit an Kadavern verbessert auch die feinmotorischen Fähigkeiten erforderlich, um den Rippenabschnitt mit oder ohne Knochenhaut zu entfernen. Zudem könnte jemand neu in diesem Verfahren finden die Manipulation der feinen Werkzeuge un...

Schwächenden Skelettverletzungen, chronische Arthrose, und die schwere Probleme mit der Wiederherstellungschirurgie verbunden Auswirkungen der wirtschaftlichen Produktivität, Familie Wohlbefinden und Lebensqualität. Während kleine Pausen und Läsionen können ziemlich gut heilen, sind die Menschen nicht in der Lage zu reparieren große Defekte und muss daher auf rekonstruktive Eingriffe angewiesen, um Struktur und Funktion wiederherzustellen. Die Rekonstruktion kann es sich um allogene Transplantate oder heterogeneic, morcellized Knochen implantiert Gerüste oder Distraktionsosteogenese. Leider gibt es nicht nur hartnäckig Morbidität Faktoren mit diesen Behandlungen aber die ursprüngliche Festigkeit des reparierten Knochens selten erreicht verbunden. So werden neue klinische Ansätze erforderlich. One-way, um innovative Methoden, um Segmentdefekte zu behandeln entwickeln zu Situationen, in denen groß angelegte Reparatur kommt in der Natur zu studieren. Amphibien können bekanntlich Skelettelemente zu regenerieren, während Säugetiere sind als begrenzt in thist die Fähigkeit. Da jedoch der Beginn des 20. Jahrhunderts, einige wenige Berichte über die Regeneration der menschlichen Rippe wurden veröffentlicht hindeutet, dass Menschen nicht so beschränkt 1-4 werden. Derzeit wird dieses Phänomen am besten plastischen Chirurgen, die Rippenmaterial für Kiefer-, Gesichts- und Gehör Rekonstruktion verwenden bekannt, aber es ist nicht mehr weit geschätzt 5. Um dieses Reparatur genauer zu untersuchen, haben wir ein chirurgisches Modell unter Verwendung der Maus entwickelt. Mit diesem Protokoll können die Forscher die angeborenen Faktoren zu identifizieren und diese Informationen benutzen, um Skelettheilung an anderen Standorten zu erleichtern. Es gibt viele Vorteile, um mit den Rippen als Modell für die Untersuchung der Skelettreparatur. Zunächst werden die umgebenden Rippen eine natürliche Fixateur (verglichen mit der Resektion des Femurs 6,7). Dies verringert das Morbiditätsrisiko der internen und externen Fixatoren und vereinfacht die chirurgische Prozedur. Zweitens sind die dünnen Muskelschichten der Brust wawerde sorgen für einfachen Zugang und gute Sicht, die der Test vergleichbar mit dem Komfort der Schädel Resektionen 8 zu machen. Drittens, im Gegensatz zu der, die von Calvarien intramembranous Ossifikation Rippen Form durch endochondrale Ossifikation bilden und über Verlängerung an Wachstumszonen an jedem Ende eines zentralen Diaphyse angeordnet wachsen in der Länge. Daher kann die Reparatur der Rippen mehr vergleichbar Reparatur der langen Röhrenknochen der Gliedmaßenskelett sein. Darüber hinaus haben wir festgestellt, dass im Vergleich zu dem Femur, die Knochenhaut der Rippe dicker und kann leichter gehandhabt werden. So können Forscher, die zur Knochenreparatur zum Zweck der Untersuchung der Knochenhaut oder Testzelltherapien, pharmakologische Wirkstoffe und / oder Gewebegerüste testen möchten finden diese Operationsmodell sinnvoll. Zusammenfassend stellt diese Rippenresektion Modell ein Rahmen ist, um natürliche großen Knochenreparatur bei Säugetieren zu studieren, da kein solches Modell im allgemeinen Gebrauch derzeit existiert.

<table border="0" cellpadding="0" cellspacing="0" width="896">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Name of Reagent/ Equipment</td>
			<td style="width:160px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:304px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">Medium sized micro-dissection scissors (Vannas-T&#252;bingen Spring Scissors 5 mm)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Fine micro-dissection scissors (Vannas Spring Scissors - 2mm Cutting Edge)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">15000-04</td>
			<td>curved tip is beneficial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Micro-scalpel 5.0 mm</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>10315-12</td>
			<td>other fine scalpels can be substituted</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Dumont 55 forceps</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">11295-51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Retractor&#160;</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>17004-05</td>
			<td>adjustability is convenient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Micro-needle holders</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">12060-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">9.0 nylon sutures (Ethilon), taper point best</td>
			<td style="width:160px;">Ethicon</td>
			<td>2819G or similar</td>
			<td>taper point best but reverse cutting is also good</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">7.0 prolene sutures (Prolene)</td>
			<td style="width:160px;">Ethicon</td>
			<td>8700H or similar</td>
			<td>6-0 can be used too, needle point can vary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Large forceps (Adson Forceps)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">11006-12</td>
			<td>other brands are fine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Lubricant Eye Ointment (Akwa Tears)</td>
			<td style="width:160px;">Akorn</td>
			<td style="width:157px;">17478-062-35</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Suture glue (GLUture Topical Tissue Adhesive)</td>
			<td style="width:160px;">Abbot</td>
			<td style="width:157px;">32046-01</td>
			<td>has excellent working time</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Shaver</td>
			<td style="width:160px;">Wahl</td>
			<td>9918-6171 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Clamp lamp</td>
			<td>Zoo Med</td>
			<td style="width:157px;">LF-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Infrared Bulb, 75W</td>
			<td style="width:160px;">Zoo Med</td>
			<td>RS-75</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:275px;">RC2 Rodent Anesthesia System</td>
			<td style="width:160px;">VetEquip&#160;</td>
			<td style="width:157px;">922100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">IsoFlo (Isoflurane)</td>
			<td style="width:160px;">Abbot</td>
			<td style="width:157px;">05260-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Buprenorphine (Buprenex)</td>
			<td style="width:160px;">Reckitt Benckiser</td>
			<td style="width:157px;">12496-0757-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Betadine</td>
			<td style="width:160px;">Purdue Frederick</td>
			<td>67618015017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Flavored Gelatin, raspberry</td>
			<td style="width:160px;">Jell-O</td>
			<td>B000E1FYL0</td>
			<td>made up firm, to the consistency of &#39;jigglers&#39;</td>
		</tr>
	</tbody>
</table>

a surgical procedure for resecting the mouse rib: a model for large-scale long bone repair

Dieses Protokoll stellt die Forscher ein neues Modell für große Knochenreparatur unter Verwendung der Maus Rippe. Das Verfahren beschreibt die folgenden: Vorbereitung des Tieres für die Chirurgie, Öffnen des Brustkörperwand, indem die gewünschte Rippe aus den umliegenden Zwischenrippenmuskeln, Ausschneiden des gewünschten Abschnitt der Rippe, ohne einen Pneumothorax, und Schließen der Schnitte. Im Vergleich zu den Knochen des Gliedmaßenskelett, die Rippen sind sehr zugänglich. Darüber hinaus ist kein interner oder externer Fixateur notwendig, da die benachbarten Rippen eine natürliche Fixierung. Die Operation nutzt kommerziell verfügbare Angebot, ist einfach zu lernen, und durch das Tier gut vertragen. Das Verfahren kann mit oder ohne Entfernung des umgebenden Knochenhaut erfolgen, und somit ist der Beitrag der Knochenhaut zu reparieren beurteilt werden kann. Die Ergebnisse zeigen, dass, wenn die Knochenhaut gehalten wird, in 1 tritt robust Reparatur - 2 Monate. Wir gehen davon aus, dass die Verwendung dieses Protokolls wirdFörderung von Forschung in die Rippe Reparatur und dass die Ergebnisse wird die Entwicklung von neuen Wegen, um die Knochenreparatur in anderen Orten rund um den Körper zu stimulieren, zu erleichtern.

Tiere in der Regel schnell wieder von diesem Verfahren, der Schnitt heilt gut, und die Tiere zeigen normales Verhalten. Die Prozedur verwendet handelsübliche Versorgung und nach dem Training, kann in weniger als 1 Stunde durchgeführt werden. Bevor Sie dieses Verfahren, um experimentelle Daten zu generieren, ist es wichtig, die Resektion am Tag 0 Zeitpunkt zu analysieren, um festzustellen, ob große Knochenfragmente könnte innerhalb der Resektion Zone bleiben. Ein Weg, um die richtige Vorgehensweise zu beurteilen ist,...

Watch this Scientific Journal Video about A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair at JoVE.com

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

Ein chirurgischer Eingriff zur Resektion der Maus Rib: Ein Modell für Großlangknochenreparatur

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

a-surgical-procedure-for-resecting-mouse-rib-model-for-large-scale

Research

JoVE Journal

Medicine

13.0K Aufrufe.  Keck School of Medicine, University of Southern California. The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

Serie: A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

A Novel Surgical Ansatz intratracheale Verabreichung von Wirkstoffen in einem Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Forschung

Medizin

Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs

This is a compact visual description of a combination of surgical technique and device for the delivery of (gene and cell) therapies into the spinal cord. While the technique is demonstrated in the animal, the procedure is FDA-approved and currently being used for stem cell transplantation into the spinal cords of patients with ALS. While the FDA has recognized proof-of-principle data on therapeutic efficacy in highly characterized rodent models, the use of large animals is considered critical for validating the combination of a surgical procedure, a device, and the safety of a final therapy for human use. The size, anatomy, and general vulnerability of the spine and spinal cord of the swine are recognized to better model the human. Moreover, the surgical process of exposing and manipulating the spinal cord as well as closing the wound in the pig is virtually indistinguishable from the human. We believe that the healthy pig model represents a critical first step in the study of procedural safety.

Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application.

Operationstechnik für Spinal Cord Lieferung von Therapien: Demonstration der Vorgehensweise in Göttingen Minischweine

This is a  compact visual description of a combination of surgical technique and device  for the  delivery of (gene and cell) therapies into the spinal ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Eine Maus fetalen Hautmodell von Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

Die Maus Round-Fenster-Ansatz für Ototoxische Agent-Lieferung: eine schnelle und zuverlässige Technik zur Induktion Cochlear Zelldegeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

A Novel Mausmodell der arteriovenöse Fistel Ausfall: Der chirurgische Eingriff im Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Chirurgische Angiogenese in porcinen tibiale Allotransplantation: eine neue große Tierknochen durchblutet zusammengesetzte Allotransplantation Modell

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

Auswertung der Prozedur für die Durchführung einer wachen Zystometrie in einem Mausmodell

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among ...

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus

We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane.

Ein chirurgischer Eingriff für die Verwaltung der Medikamente an das Innenohr in eine nicht-menschliche Primaten Weißbüschelaffe (Callithrix Jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

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.

Messung der isometrischen Kontraktilität der Maus mesenterialen Arterie mit Draht Myographie

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

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

Standardisierte histomorphometrische Bewertung von Osteoarthritis in einem chirurgischen Mausmodell

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...