Name: murine flexor tendon injury and repair surgery
Uploaded: 2016-09-19T14:00:00
Description: Watch this Scientific Journal Video about Murine Flexor Tendon Injury and Repair Surgery at JoVE.com

Diese Arbeit wurde teilweise von der amerikanischen Gesellschaft für Handchirurgie Pilot-Award und NIH / NIAMS 1K01AR068386-01 (zu AEL) und NIAMS / NIH P30AR061307 unterstützt.

Die Universität Ausschuss für Tierforschung an der Universität von Rochester genehmigt alle Tierversuche. Ten-12 Wochen alte weibliche C57BL / 6J-Mäuse wurden verwendet. 1. Vorbereitung der Tiere für Beugersehne Chirurgie (~ 15 min) <ol><li> Autoklav chirurgische Instrumente zu sterilisieren, tragen sterile Handschuhe im gesamten Gebäude, und ein steriles Operationsfeld halten. </li><li> Anästhesieren die Maus über intraperitoneale Injektion (ip) mit einem Volumen von Ketamin (80 m...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Work+of+flexion+after+flexor+tendon+repair+according+to+the+placement+of+sutures.">Work of flexion after flexor tendon repair according to the placement of sutures.</a> Clin Orthop Relat Res. , 205-210 (1995).</li><li>Zhao, 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=Award+for+Outstanding+Orthopaedic+Research:+Engineering+flexor+tendon+repair+with+lubricant,+cells,+and+cytokines+in+a+canine+model.">Award for Outstanding Orthopaedic Research: Engineering flexor tendon repair with lubricant, cells, and cytokines in a canine model.</a> Clin Orthop Relat Res. 472, 2569-2578 (2014).</li><li>Wong, J., Bennett, W., Ferguson, M. W., McGrouther, D. 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E., 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=Bone+marrow-derived+matrix+metalloproteinase-9+is+associated+with+fibrous+adhesion+formation+after+murine+flexor+tendon+injury.">Bone marrow-derived matrix metalloproteinase-9 is associated with fibrous adhesion formation after murine flexor tendon injury.</a> PloS one. 7, e40602 (2012).</li><li>Lee, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parathyroid+hormone+1-34+enhances+extracellular+matrix+deposition+and+organization+during+flexor+tendon+repair.">Parathyroid hormone 1-34 enhances extracellular matrix deposition and organization during flexor tendon repair.</a> J Orthop Res. 33, 17-24 (2015).</li><li>Geary, M. B., 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=Systemic+EP4+Inhibition+Increases+Adhesion+Formation+in+a+Murine+Model+of+Flexor+Tendon+Repair.">Systemic EP4 Inhibition Increases Adhesion Formation in a Murine Model of Flexor Tendon Repair.</a> PloS one. 10, e0136351 (2015).</li><li>Loiselle, A. E., 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=Development+of+antisense+oligonucleotide+(ASO)+technology+against+Tgf-beta+signaling+to+prevent+scarring+during+flexor+tendon+repair.">Development of antisense oligonucleotide (ASO) technology against Tgf-beta signaling to prevent scarring during flexor tendon repair.</a> J Orthop Res. 33, 859-866 (2015).</li><li>Orner, C. A., Geary, M. B., Hammert, W. C., O'Keefe, R. J., Loiselle, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+and+short-duration+Matrix+Metalloproteinase+9+Inhibition+does+not+affect+adhesion+formation+during+murine+flexor+tendon+healing.">Low-dose and short-duration Matrix Metalloproteinase 9 Inhibition does not affect adhesion formation during murine flexor tendon healing.</a> Plast Reconstr Surg. , (2016).</li><li>Loiselle, A. E., 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=Remodeling+of+murine+intrasynovial+tendon+adhesions+following+injury:+MMP+and+neotendon+gene+expression.">Remodeling of murine intrasynovial tendon adhesions following injury: MMP and neotendon gene expression.</a> J Orthop Res. 27, 833-840 (2009).</li><li>Tsubone, T., 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=Effect+of+TGF-beta+inducible+early+gene+deficiency+on+flexor+tendon+healing.">Effect of TGF-beta inducible early gene deficiency on flexor tendon healing.</a> J Orthop Res. 24, 569-575 (2006).</li><li>Beason, D. P., Kuntz, A. F., Hsu, J. E., Miller, K. S., Soslowsky, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+multiple+tendon+injury+models+in+the+mouse.">Development and evaluation of multiple tendon injury models in the mouse.</a> J Biomech. 45, 1550-1553 (2012).</li><li>David, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tendon+repair+is+compromised+in+a+high+fat+diet-induced+mouse+model+of+obesity+and+type+2+diabetes.">Tendon repair is compromised in a high fat diet-induced mouse model of obesity and type 2 diabetes.</a> PloS one. 9, e91234 (2014).</li><li>Wong, J. 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Das chirurgische Verfahren für ein Mausmodell der vollständigen Durchtrennung und die Reparatur der digitorum longus Flexor wird in dieser Studie vorgestellt. Zusätzlich wird eine neuartige Anwendung der Konzentration kleiner Zellpopulationen mit Zytologie Zentrifuge gezeigt, was eine quantitative immunocytochemische Analyse der zellulären Umgebung während der Beugesehnenheilung. Dieses Modell der Beugesehnenreparatur zeigt eine reproduzierbare Heilungsreaktion, die verwendet werden können Veränderungen in den He...

Flexor Sehnen in der Hand arbeiten gemeinsam mit den Beugemuskeln des Unterarms und digitale Scheiden Beugung der Ziffern und Greiffunktion der Hand zu ermöglichen. Beugesehnen entlang der Palmarseite der Hand laufen; Diese relativ oberflächliche Lage führt oft zu Verletzungen an den Beugesehnen während eines Traumas auf der Hand. Sehnen heilen durch eine Reaktion von Narbengewebe statt Regeneration von normalen Sehnengewebe 1. Während diese Narbengewebe Kontinuität der Sehne bereitstellt, wird Funktion verringert sich dramatisch auf gesunde Sehne relativ. Tendon-Narbengewebe Komposite werden durch beeinträchtigte mechanische Eigenschaften 1, dadurch die reparierten Sehnen eher zu Bruch zu machen. Darüber hinaus mangelt es Narbengewebe, die Organisation der nativen Sehnenkollagen Faserstruktur, was zu einer Erhöhung der Sehne Größe und Masse. die anatomischen Einschränkungen der Sehnenscheide Einheit, selbst eine bescheidene Zunahme der Sehne Größe drastisch rot GegebenUCE die Gleitfunktion der Sehne und damit der Bewegung und Handfunktion einstelligen Bereich. Vor der Verletzungen von 1960 an die Beugesehnen, insbesondere solche , in Zone II der Hand wurden nicht routinemäßig wegen der schweren Komplikationen bei der Heilung repariert , die 2 mit diesen Reparaturen entstanden sind . Dieser Bereich der Hand wurde als &quot;Niemandsland&quot; 3 bezeichnet. Allerdings Verbesserungen in der chirurgischen Techniken, Nahtmuster und physikalische Therapie Rehabilitation Protokolle haben sich dramatisch Ergebnisse der Beugesehne Reparaturen verbessert 2. Trotz dieser Fortschritte bis zu 40% der Reparaturen führen in ausreichender Adhäsionsbildung Handfunktion 4 zu behindern. Daher wird eine biologische Ansatz erforderlich Heilung zu verbessern. Leider ist nur sehr wenig auf zellulärer und molekularer Ebene über die Sehne Heilungsprozess bekannt. Somit war das Ziel, ein Mausmodell zu entwickeln, die verwendet werden könnten, die grundlegende understandin zu verbesserng der zellulären und molekularen Komponenten der flexor tendon Heilung und Narbenbildung Antwort als Mittel neue therapeutische Targets zu identifizieren, die Heilung zu verbessern. Größere Tiermodelle haben dazu beigetragen, bei der Förderung Verständnis des Prozesses der Heilung Beugesehne. Hunde- und Kaninchen Studien haben sowohl die intrinsische als auch extrinsische Heilungsfähigkeit der Beugesehnen 5,6 gezeigt, die Bedeutung einer frühzeitigen passiven Bewegung gesteuert bei der Minimierung von Adhäsionen in Bezug auf die Immobilisierung 7, sowie die Auswirkungen unterschiedlicher Nahtmuster auf den Heilungsprozess 8 9. Darüber hinaus hat das canine Modellansätze in Prüfung translatorische gewebe Engineering nützlich gewesen 10 die Heilung zu verbessern. Allerdings gibt es mehrere wichtige Vorteile in einem murinen Modell relativ zu einem Großtiermodell verwenden, einschließlich der relativen Kosten, Verfügbarkeit von murinen spezifischen Reagenzien und die Leichtigkeit des Erzeugens globalen Knock-outs oder gewebespezifische Löschen / Überexpression Konstrukte. Darüber hinaus zeigen die funktionellen Ähnlichkeiten zwischen Mensch und Mäusen in Bezug auf Beugesehnen 11 die potentiellen Nutzen eines Mausmodells zu entwickeln. Entwicklung eines Mausmodell der Beugesehne durchtrennt und die Reparatur imitiert viele Aspekte der klinischen Heilung, einschließlich der Bildung von Narbengewebe reichlich vorhanden und die Beeinträchtigung der mechanischen Eigenschaften. Das hier beschriebene Modell ist keine echte Reprise der klinischen Praxis aufgrund Trennung der FDL am Muskel-Sehnen-Übergang, um die Reparaturstelle zu schützen. Darüber hinaus ist Konto dieses Modell nicht für den Beitrag der Synovialscheide Zellen zur Heilung Antwort, da es keine Synovialscheide ist der Mittelteil der Sehne bedeckt, wo die Reparatur erfolgt. Trotz dieser Einschränkungen hat dieses Modell den Vorteil der Erzeugung Bewegungsbereich beschränkende Verwachsungen, die noch in Mausmodellen, die mehr clos nachgewiesen werden muss,ely das klinische Szenario nähern. Dieses Modell wurde verwendet, Knock-out - Modelle Maus 12,13, zu bewerten und verschiedene pharmakologische Ansätze zu testen Heilung 14-17 zu verbessern. Die histologische Analysen dieses Modells, Immunhistochemie und in-situ-Hybridisierung können wichtige Einblicke in die Lokalisierung von Schlüsselgenen und Proteinen während der Heilung bieten. Jedoch stellt die Histologie nur eine Querschnitts räumliche Analyse und Quantifizierung nicht während des gesamten Gewebes ermöglichen. Flow ist Zytometrie eine quantitative Ansatz, aber nur eine sehr begrenzte Anzahl von Zellen aus der Heilungssehnengewebe im Mausmodell, isoliert werden, und diese Zahl wird weiter verringert während der Fixierung, Permeabilisierung und Waschschritte. Wenn man dies in Rechnung, um, Durchflusszytometrie eine undurchführbar Ansatz wird aufgrund der Anzahl der Tiere, die erforderlich wäre. Ein alternatives Verfahren ist notwendig, um die Mehrheit der kleinen Zellpopulation zu erhalten, umweiter zu den Heilungs milieu charakterisieren. Das verwendete Verfahren, dies zu erreichen, ist hier dargestellt, beinhaltet Konzentration der isolierten Zellen über Zentrifugation Zytologie auf einen Glasträger, gefolgt von Immunzytochemie. In der vorliegenden Studie EdU (5-Ethinyl-2&#39;deoxyuridine, ein Thymidin-Analogon) Einbau und nachfolgende Markierung wurde verwendet, um die relativen proliferative Zustand der Zellen bei der Heilungsstelle zu bestimmen. Dieser Ansatz kann die Wirksamkeit von pharmakologischen Behandlungen auf die Zellproliferation, Gen-Knock-out oder Überexpression oder zur Identifizierung und Quantifizierung verschiedenen Zellpopulationen zu testen, angewendet werden.

<table border="0" cellpadding="0" cellspacing="0" width="1047">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6J mice&#160;</td>
			<td>Jackson Laboratories</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine</td>
			<td>Hospira</td>
			<td>NDC# 0409-2051-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine</td>
			<td>Lloyd Inc.</td>
			<td>NDC# 61311-482-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenorphine</td>
			<td>Par Pharmaceutical Inc.</td>
			<td>NDC# 42023-179-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.9% sodium chloride irrigation</td>
			<td>Hospira</td>
			<td>NDC# 0409-6138-03</td>
			<td>For preparation of ketamine/xylazine and buprenorphine solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1ml syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30G needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone-Iodine solution</td>
			<td>Aplicare</td>
			<td>82-226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puralube vet opthalmic ointment</td>
			<td>Dechra Veterinary Products</td>
			<td>NDC# 17033-211-38</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Portable balance 200g</td>
			<td>Ohaus</td>
			<td>SP202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15124-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle holders</td>
			<td>Fine Science Tools</td>
			<td>91201-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro needle holders</td>
			<td>Fine Science Tools</td>
			<td>12061-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-0 nylon sutures</td>
			<td>Ethicon</td>
			<td>668G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8-0 microsurgery nylon sutures</td>
			<td>Ethicon</td>
			<td>2808G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab-Line histology slide warmer</td>
			<td>Barnstead International</td>
			<td>26025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytospin method</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase Type I, lyophilized</td>
			<td>Life Technologies&#160;</td>
			<td>1700-017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>Cell Signaling Technologies</td>
			<td>9998S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X PBS</td>
			<td>Thermo Fisher</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytology funnels</td>
			<td>Fisher HealthCare</td>
			<td>10-354</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HistoBond+ microscope slides</td>
			<td>VWR</td>
			<td>16005-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytospin 2 centrifuge</td>
			<td>Shandon</td>
			<td>SH-CYTO2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunocytochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide staining tray with black lid</td>
			<td>IHC World</td>
			<td>M920-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Click-iT Plus EdU Imaging Kit</td>
			<td>Life Technologies&#160;</td>
			<td>C10639</td>
			<td>Includes EdU and&#160; Hoeschst 33342</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immedge hydrophobic barrier pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Diamond mounting medium</td>
			<td>Thermo Fisher</td>
			<td>P36970</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass coverslips 24x50mm #1.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clear nail polish</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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murine flexor tendon injury and repair surgery

Tendon verbindet der Skelettmuskulatur und Knochen, Bewegung fast den gesamten Körper zu erleichtern. In der Hand ermöglichen Beugesehnen (FTS) Beugung der Finger und die allgemeine Funktion der Hand. Verletzungen FTS sind häufig, und eine zufriedenstellende Heilung wird oft durch übermäßige Narbengewebe und Verwachsungen zwischen der Sehne und des umgebenden Gewebes beeinträchtigt. Es ist jedoch wenig über die molekularen und zellulären Bestandteile des FT Reparatur bekannt. Zu diesem Zweck ist ein murines Modell FT Reparatur, die viele Aspekte der Heilung beim Menschen, einschließlich gestörter Bewegungsbereich und verringerte mechanische Eigenschaften rekapituliert, entwickelt und oben beschrieben wurde. Hier eine eingehende Demonstration dieses chirurgischen Verfahrens ist vorgesehen, an denen transection und anschließende Reparatur des Flexor digitorum longus (FDL) Sehne im Maus-Hinterpfote. Diese Technik kann verwendet werden, lineage Analyse verschiedener Zelltypen zu führen, um die Auswirkungen von Gen-Gewinn oder Verlust-of-Funktion zu bewerten und die Effi zu testensamkeit von pharmakologischen Interventionen in den Heilungsprozess. Allerdings gibt es zwei primäre Einschränkungen zu diesem Modell: i) die FDL-Sehne im mittleren Teil des murinen Hinterpfote, wo der Durchtrennung und Reparatur auftreten, wird nicht von einer Synovialscheide umgeben. Daher ist dieses Modell nicht berücksichtigt den möglichen Beitrag der Hülle zur Narbenbildung Prozess. ii) Um die Integrität der Reparaturstelle zu schützen, wird die FT am Muskel-Sehnen-Übergang freigegeben, um die mechanischen Kräfte der Sehne abnimmt, wahrscheinlich zu einer erhöhten Narbenbildung beitragen. Isolation von ausreichender Zellen aus dem Granulationsgewebe des FT während des Heilungsprozesses zur Analyse durchflusszytometrischen hat sich als schwierig; Zytologie Zentrifugation diese Zellen zu konzentrieren, ist eine alternative Methode verwendet wird, und ermöglicht es zur Erzeugung von Zellpräparationen, auf dem Immunofluoreszenz Kennzeichnung durchgeführt werden kann. Bei diesem Verfahren wird die Quantifizierung von Zellen oder Proteine ​​von Interesse während FT Heilung möglich.

Der Flexor digitorum longus (FDL) Muskel, in der Wade befindet, wirkt die Ziffern der Maus Hinterpfote über die Beugesehne (umrissen in blau in 1A und histologisch in 2A gezeigt) zu biegen, die sich von der Muskel - Sehnen läuft proximal Kreuzung und endet in den Endphalangen. In diesem Modell der Beugesehnenheilung wird der FDL - Sehne durchtrennt und in der Mitte Fuß, proximal der Bifurkation in den Ziffern der Hinterpfote (rote Pfeile, Abbi...

Watch this Scientific Journal Video about Murine Flexor Tendon Injury and Repair Surgery at JoVE.com

Murine Flexor Tendon Injury and Repair Surgery

Flexor Sehnen in der Hand sind häufig verletzt, zu einer Beeinträchtigung der Funktion der Hand führt. Allerdings ist die Narbengewebeheilungsreaktion nicht gut charakterisiert. Ein Mausmodell der Beugesehnenheilung wird hier demonstriert. Dieses Modell kann allgemeine Verständnis des Heilungsprozesses zu verbessern und therapeutische Ansätze zu bewerten, um die Heilung zu verbessern.

Murine Beugersehne Verletzung und Reparatur Chirurgie

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers ...

The over all goal of this surgical procedure is to enhance the understanding of the cellular and molecular processes of scar and adhesion formation during flexor tendon healing. This method can help answer key questions about the flexor tendon healing process, such as which cell types contribute to scar formation and repair. The main advantage of this technique is that transgenic and knock out mice can be used to better understand the healing process.This technique may identify potential biological targets for improving the outcomes of flexor tendon healing as it facilitates the investigation of these poorly designed cell populations that contribute to scarring. After confirming a lack of response to toe pinch, apply a ophthalmic ointment to the animals eyes, and clip the fur from the entire hind limb. Sterilize the surgical site with sequential povedone iodine and 70%ethanol scrubs, and locate the flexor digitorum longus, or FDL tendon within the medial aspect of the calf.Next, make a 0.5 to once centimeter incision in the skin to expose the tendon. Use the forceps to separate the FDL tendon from the surrounding tissue and trace the tendon up to the miotendinous junction. Using spring scissors, release the tendon at the junction, taking care to avoid the posterior tibial artery.Then close the skin with 5-0 nylon sutures. Now transfer the mouse under a stereo microscope and use spring micro scissors to make a three millimeter incision over the posterolateral aspect of the hind paw. Use the forceps to gently retract the surrounding soft tissue and muscle, taking care to minimize the tissue damage, and identify the FDL tendon.Then gently raise the tendon and use micro scissors to completely transect the tissue. Using 8.0 sutures in a modified Kessler pattern, ligate the ends of the FDL tendon. Periodically hydrating the tendon with saline throughout the procedure if necessary.When the ends have been sutures, return the soft tissue and muscle over the tendon and use 5.0 nylon sutures to close the incision. Then place the mouse on a 37 degrees Celsius slide warmer with monitoring until fully recovered. 24 hours before the experimental end point, inject the mice with 100 micro liters of EDU.The next day, locate the repaired tendon as just demonstrated, and harvest the transected tissue. Isolating the granulated tissue surrounding the healing tendon is a critical step for allowing the eventual aminocytochemical analysis. And care must be taken to minimize the inclusion of peripheral tissues.Place the tendon in a petri dish containing one milliliter of collagenase. Then, mince the tissues with the scalpel followed by trituration of the fragments with several passes through an 18 gauge needle. Transfer the slurry into 1.5 milliliter centrifuge tubes and digest the tendon pieces for one hour and 37 degrees Celsius with shaking.At the end of the digestion, centrifuge the tissue suspensions and resuspend the pellets in 500 micro liters of three percent BSA in PBS. Next, filter the cells through a 70 micron strainer to remove the debris and large pieces of undigested tendon. And place the cells in a 37 degrees Celsius incubator.Transfer the undigested tendon pieces to new, 1.5 milliliter micro centrifuge tubes containing one milliliter of fresh collagenase, and mix the pieces by pipe heading. After another hour of digestion with shaking, spin down the tissue pieces again, and resuspend the pellets in 500 microliters of BSA supplemented PBS. Then filter the suspension through a new 70 micron cell strainer into the tube of cells collected after the first digestion.Count the cells and spin them down by centrifugation. Then resuspend the pellet at a three to six times 10 to the fourth cells per 100 microliters of PBS supplemented with BSA concentration. To detect EDU incorporation by immunocytochemistry, first use a hydrophobic barrier pen to circle the cells on the slides and immediately fix the samples with 150 microliters of three percent paraformaldehyde and PBS at room temperature.After 15 minutes, rinse the cells with two five minute washes in 150 microliters of BSA supplemented PBS. Then permeableize the samples with 0.5%triton X 100 in PBS for 20m minutes. After washing as just demonstrated, add 150 microliters of freshly prepared EDU reaction cocktail to the cells for a 30 minute incubation at room temperature.Then wash the cells again and counter stain the samples with 150 microliters of Hoechst. After 30 minutes at room temperature, add one to two drops of antifade mounting medium to the cells, and carefully cover each sample with the cover slip, taking care to avoid bubbles. Cure the mounting medium over night in the dark at room temperature.Then seal the cover slips with clear nail polish. Finally, image the slides on a fluorescent microscope using 40X magnification. In a successful repair, the granulation tissue displays a greatly increased cellularity compared to the uninjured tendon.In this image, a histological example of a ruptured flexor tendon repair is shown. EDU labeling highlights only those cells that actively proliferate during the 24 hours following the EDU pulse in life mice. Facilitating a quantitative assessment of the proliferation within the granulation tissue of the healing tendon.After watching this video you should have a good understanding of how to perform the flexor tendon injury and repair surgery in mice, to better understand the cell populations that contribute to scar formation in the flexor tendon during healing.

murine-flexor-tendon-injury-and-repair-surgery

Research

JoVE Journal

Medicine

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Akute und chronische taktile Sensorische Testung nach Verletzungen des Rückenmarks bei der Ratte

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

Forschung

Medizin

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

Ein Modell von gequetscht Einseitige Cervical Spinal Cord Injury Mit dem Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Ein neues Mausmodell der endovaskuläre Aortenaneurysma Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Controlled Cortical Impact-Modell für traumatische Hirnverletzung

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

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 ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

Ein Mausmodell von arterieller Restenose: Technische Aspekte der Femoral Draht Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Murine Modell der intestinalen Ischämie-Reperfusionsschaden

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

Reproduzierbare Arterielle Denudation Verletzung von Infra Abdominal Aortic Spannen in einem Mausmodell

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

Bewertung von Stammzelltherapien in einem bilateralen Patellasehne Verletzungen Modell bei Ratten

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

Generieren eines murinen orthotopen metastasierendem Brustkrebs Krebs Modells und Durchführung von murinen radikale Mastektomie

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...