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).

NOTE: Toutes les procédures sont conformes à un protocole animale approuvé par le Comité institutionnel de protection des animaux et l&#39;utilisation (IACUC) à l&#39;Université de Californie du Sud. 1. Préparation pour l&#39;intervention <ol><li> Disposez les outils chirurgicaux et les fournitures nécessaires (stérilisée, articles 1 à 18 dans la liste des matériaux) et d&#39;organiser un microscope à dissection et un fibre optique trans-illuminateur dans une hotte ou sur une table aspirante. </li><li> Placez une souris sexuellement matures (20-30 g, 1 - 3 mois) dans une chambre d&#39;induction avec 4% d&#39;isoflurane pour induire une anesthésie. </li><li> Confirmez l&#39;anesthésie avec une patte et / ou un test queue de pincement. </li><li> Appliquer une pommade pour les yeux de la souris pour éviter la sécheresse des yeux pendant la chirurgie. </li><li> Déplacez la souris pour la platine du microscope. Placer la souris dans le cône de nez du tuyau d&#39;entretien et de positionner latéralement. Fournir une poche chauffante adjacente à l&#39;animal pour aider à maintenir la température du corps. PASE:. Position de la souris dépend de vos préférences personnelles et / ou l&#39;impartialité du chirurgien, côté gauche ou à droite est très bien. </li><li> Réglez l&#39;isoflurane à 2-3% pour l&#39;entretien, et de regarder le taux de respiration. </li><li> Injecter la buprénorphine (0,05 mg / kg) sous-cutanée (de flanc latéral de la jambe) pour la douleur post-opératoire avec une aiguille 25 G. La jambe peut secousse qui est une réaction normale. </li></ol> 2. incision d&#39;ouverture <ol><li> Détecter la nervure souhaitée par palpation, et nettoyer la zone des cheveux avec un rasoir. REMARQUE: Ribs 8-10 sont recommandés depuis nervures 1-7 sont plus proches des poumons et 11-13 sont considérablement plus courte. Compter les côtes devrait commencer par la nervure plus rostrale numéro 1. </li><li> Préparer la zone avec une alternance de povidone-iode et de l&#39;isopropanol (70%) pistonnage (3x est souvent recommandé par les protocoles d&#39;utilisation des animaux). </li><li> Couper une incision de 2 cm à travers la peau, directement au-dessus et parallèlement à la nervure désirée avec des ciseaux de microchirurgie de taille moyenne. INCISe à travers les couches musculaires et adipeuses sous-jacents. </li><li> Placez tous les trois couches (peau, les muscles, la graisse) dans un enrouleur pour exposer la zone chirurgicale, tout en minimisant la taille de l&#39;incision. </li></ol> 3. excision du Rib <ol><li> Couper à travers les muscles intercostaux recouvrant la section désirée de la nervure avec un 5,0 mm scalpel. Position de cette incision autour de 5 mm à proximité de la commune chondrocostale car ce est là la nervure ne est pas aussi forte courbure. Séparez délicatement le muscle de l&#39;os avec pince fine de pointe. </li><li> Pour créer une résection, tout en conservant le périoste chez l&#39;animal, couper à travers le périoste, le long de la longueur de la nervure avec un scalpel 5,0 mm. Soigneusement séparer le périoste de l&#39;os sous-jacent latéralement par pince fine de pointe. Procédez avec prudence, car le périoste est très délicate et a une consistance gélatineuse. </li><li> Suivant faire une coupe transversale à travers l&#39;os à une extrémité avec micro-ciseaux fins. Si nécessaire, mesurer la résectionun réticule dans le microscope ou une jauge de type de règle. Puis soulevez délicatement l&#39;os sur le périoste et couper l&#39;autre extrémité. REMARQUE: Procédez avec prudence. Ce est l&#39;étape la plus délicate, car tirant sans exciser la nervure ci-dessous va déchirer la membrane pleural et provoquer un pneumothorax. Si la membrane est déchirée pleural ce sera facilement évident que l&#39;intérieur de la cavité thoracique devient visible. </li><li> Si le saignement se produit à la suite de la coupe de l&#39;os, appliquer une pression à la fin de coupe avec un tampon de coton-tige pour 4-5 sec pour arrêter le saignement. NOTE: A l&#39;occasion rare qui saignement persiste, il peut être nécessaire de se arrêter et euthanasier l&#39;animal perte de sang importante et compromettront sa reconstitution (sans remplacement des liquides, le volume de sang maximum qui peut être sans risque être perdu est de 10% du volume total de sang ou 7.7 -. 8 ul / g pour une souris de 25 g, ce qui équivaut à environ 180 à 200 pi 9). </li><li> Placer immédiatement la nervure enlevé dans4% PFA pour une analyse ultérieure. REMARQUE: si la résection à la fois de l&#39;os et le périoste doit être effectuée, omettre l&#39;incision du périoste et de la séparation (étape 3.2). Depuis le périoste est très étroitement attaché à la membrane pleurale procéder avec prudence, taquiner la nervure loin de la membrane pleurale avec des pinces pour éviter les déchirures. </li></ol> 4. Fermeture de l&#39;incision <ol><li> Suturer les muscles intercostaux sur le dessus de la douille périoste restant avec sutures 9-0 (2 sutures sont généralement suffisants). Passer les sutures directement au-dessus des extrémités de la nervure de coupe, pour agir comme indicateurs de l&#39;emplacement de chirurgie. </li><li> Retirer l&#39;écarteur. Suturer le muscle sus-jacente et de la graisse avec 9-0 sutures (3-4 sutures sont généralement suffisants). Chez la souris, en particulier avec des couches épaisses de muscle et / ou de la graisse, suturer chaque couche séparément (par exemple, une couche de fils de suture pour la couche musculaire et une couche de fils de suture pour la couche de gras). </li><li> Fermez lepeau avec des sutures 7-0 (4-5 sutures sont généralement suffisants). </li><li> Fixez l&#39;incision avec une suture de la colle, de pincer les bords avec de grandes pinces. </li><li> Sevrer lentement la souris hors isoflurane d&#39;abord se adapter à 1% pendant quelques minutes, puis se éteint. </li><li> Placez la souris sous une lampe de feu et laisser jusqu&#39;à ce que la conscience est retrouvé, 5-10 min. Après reprend connaissance et tout au long de la période de guérison, la souris doit se déplacer et déambuler normalement et ne montrent aucun signe de détresse. </li></ol> 5. Récupération et analyse <ol><li> Assurer la gestion de la douleur post-opératoire, y compris l&#39;administration de la buprénorphine sous une forme orale de gélatine à 0,5 mg / kg toutes les 12 heures pendant 48 heures. L&#39;administration orale ne nécessite pas de maîtriser l&#39;animal qui pourrait causer de la douleur à la région thoracique. </li><li> Maintenir la souris dans une cage avec un accès libre à la nourriture et de l&#39;eau pendant la période de guérison. Après l&#39;incision est sec, les animaux femelles peuvent être co-logés tandis que le mâles doit rester isolé pour éviter les combats. </li><li> Après la période de cicatrisation, suivez la procédure approuvée pour l&#39;euthanasie. Retirer la cage thoracique pour la fixation et l&#39;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

Lors de la première apprendre ce protocole, déterminer où trouver l&#39;incision initiale peut être difficile. Cependant, la pratique sur des souris euthanasiés aide le chirurgien apprendre où placer l&#39;incision initiale et exposer la nervure souhaitée à réséquer. Travailler sur des cadavres améliore également les motricité fine requises pour supprimer la partie thoracique avec ou sans le périoste. En outre, quelqu&#39;un de nouveau à cette procédure pourrait trouver manipuler les outils et fines sutu...

Débilitante blessures squelettiques, l&#39;arthrose chronique et les problèmes graves associés à la chirurgie reconstructive impact sur la productivité économique, bien-être familial, et la qualité de vie. Bien que de petites pauses et des lésions peuvent guérir assez bien, les humains ne sont pas capables de réparer des défauts importants et doivent donc compter sur les procédures de reconstruction pour restaurer la structure et la fonction. Reconstruction greffes allogéniques peut impliquer ou heterogeneic, os morcellized, échafaudages implantés, ou de distraction ostéogenèse. Malheureusement, non seulement il ya des facteurs persistants de morbidité associés à ces traitements, mais la force originale de l&#39;os réparé est rarement atteint. Ainsi, de nouvelles approches cliniques sont nécessaires. Un moyen de développer des méthodes innovantes pour traiter les défauts segmentaires est d&#39;étudier les situations dans lesquelles la réparation à grande échelle se produit naturellement. Amphibiens célèbre peut régénérer éléments squelettiques, tandis que les mammifères sont considérées comme limitées dans eest la capacité. Cependant, depuis le début du 20 e siècle, quelques rapports de la régénération de la nervure humaine ont été publiées suggérant que les humains ne peuvent pas être limitées 1-4. Actuellement ce phénomène est mieux connu par les chirurgiens plasticiens qui utilisent des matériaux de nervure pour la reconstruction mâchoire, le visage et l&#39;oreille, mais il ne est pas plus largement appréciée 5. Afin d&#39;étudier cette réparation plus en détail, nous avons développé un modèle chirurgicale en utilisant la souris. En utilisant ce protocole, les chercheurs peuvent identifier les facteurs innés impliqués et utiliser cette information pour faciliter la cicatrisation osseuse dans d&#39;autres endroits. Il ya plusieurs avantages à utiliser les nervures comme un modèle pour l&#39;étude de la réparation du squelette. Tout d&#39;abord, les nervures entourant fournissent un fixateur naturel (par rapport à la résection du fémur 6,7). Cela diminue le risque de morbidité des fixateurs internes et externes et simplifie la procédure chirurgicale. Deuxièmement, les couches musculaires minces des wa poitrinell prévoir un accès facile et une excellente visibilité qui font le test comparable à la commodité de la voûte du crâne résections 8. Troisièmement, contrairement à la calottes qui forment par ossification dermique, la forme de nervures par ossification endochondrale et croître en longueur via l&#39;extension à plaques de croissance situés à chaque extrémité d&#39;une diaphyse central. Par conséquent, la réparation des nervures peut être plus comparable à la réparation des os longs du squelette appendiculaire. En outre, nous avons trouvé que, par rapport au fémur, le périoste de la nervure est plus épais et peut être plus facilement manipulé. Ainsi, les enquêteurs qui souhaitent doser la réparation osseuse dans le but d&#39;étudier le périoste ou de tester des thérapies cellulaires, des agents pharmacologiques, et / ou échafaudages tissulaires peuvent trouver ce modèle chirurgicale utile. En résumé, ce modèle de résection costale fournit un contexte dans lequel l&#39;étude à grande échelle de réparation naturelle de l&#39;os chez les mammifères comme aucun tel modèle d&#39;usage général qui existe actuellement.

<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

Ce protocole introduit chercheurs à un nouveau modèle de grande échelle réparation osseuse en utilisant la nervure de la souris. La procédure indications suivantes: préparation de l&#39;animal pour la chirurgie, l&#39;ouverture de la paroi thoracique du corps, l&#39;exposition de la nervure souhaitée dans les muscles intercostaux environnantes, excision de la section de nervure souhaitée sans provoquer un pneumothorax, et la fermeture des incisions. Par rapport aux os du squelette appendiculaire, les nervures sont très accessibles. En outre, aucun fixateur interne ou externe ne est nécessaire étant donné que les nervures adjacentes fournissent une fixation naturelle. La chirurgie utilise commercialement fournitures disponibles, est simple à apprendre, et bien toléré par l&#39;animal. La procédure peut être réalisée avec ou sans enlever le périoste environnant et donc la contribution du périoste de réparation peut être évaluée. Les résultats indiquent que si le périoste est retenu, la réparation robuste se produit dans 1-2 mois. Nous nous attendons à ce que l&#39;utilisation de ce protocolestimuler la recherche sur la réparation des côtes et que les conclusions faciliteront le développement de nouveaux moyens de stimuler la réparation osseuse dans d&#39;autres endroits à travers le corps.

Les animaux sont habituellement récupérer rapidement de cette procédure, l&#39;incision guérit bien, et les animaux présentent un comportement normal. La procédure utilise les fournitures disponibles dans le commerce et après la pratique, peut être complété en moins de 1 h. Avant d&#39;utiliser cette procédure pour générer des données expérimentales, il est important d&#39;analyser la résection au point de temps 0 jour pour déterminer si des grands fragments d&#39;os peuvent rester dans la zone de rése...

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.

Une intervention chirurgicale pour résection de la Rib souris: un modèle pour à grande échelle de réparation des os longs

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 vues.  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.

Vidéo: 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.

Une nouvelle approche chirurgicale pour l&#39;administration intratrachéale d&#39;agents bioactifs dans un modèle de souris fœtale

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

Recherche

Médecine

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.

Technique chirurgicale pour la livraison de la moelle épinière des thérapies: Démonstration de procédure à Göttingen miniporcs

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.

Un modèle de la peau de souris fœtale de 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.

L&#39;approche de la souris ronde-fenêtre pour ototoxiques Delivery Agent: Un rapide et fiable Technique d&#39;incitation à Cochlear dégénérescence cellulaire

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 modèle murin de la fistule artério-veineuse échec: la procédure chirurgicale en détail

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.

Angiogenesis chirurgicale en Porcine Allotransplantation Tibial : un nouveau grand Animal OS vascularisé Allotransplantation Composite modèle

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.

Évaluation de la procédure pour effectuer une cystéromie éveillée dans un modèle de souris

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.

Une intervention chirurgicale de l’Administration des médicaments à l’oreille interne dans un Non-Human primates commune Ouistiti (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.

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

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

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.

Évaluation histomorphométrique normalisée de l’arthrose dans un modèle chirurgical de souris

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