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

Dit werk werd gedeeltelijk ondersteund door de Amerikaanse Vereniging voor Heelkunde van het Hand Pilot Award en NIH / NIAMS 1K01AR068386-01 (naar AEL) en NIAMS / NIH P30AR061307.

De Universitaire Commissie Animal Research aan de Universiteit van Rochester keurden alle dierproeven. Ten-12 weken oude vrouwelijke C57BL / 6J muizen werden gebruikt. 1. Voorbereiding van de dieren voor flexorpees Surgery (~ 15 min) <ol><li> Autoclaaf chirurgische instrumenten te steriliseren, draag steriele handschoenen in, en handhaven van een steriele operatiekamer veld. </li><li> Verdoven de muis via intraperitoneale injectie (ip) met een volume van ketamine (80 mg / kg) en xylazine (...

<ol>
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W. <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+flexor+tendon+surgery:+twenty-five+years+of+progress.">Development of flexor tendon surgery: twenty-five years of progress.</a> J Hand Surg [Am]. 25, 214-235 (2000).</li><li>Bunnell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+tendons+in+the+fingers+and+description+of+two+new+instruments.">Repair of tendons in the fingers and description of two new instruments.</a> Surg Gynecol Obstet. 26, 103-110 (1918).</li><li>Aydin, 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=Single-stage+flexor+tendoplasty+in+the+treatment+of+flexor+tendon+injuries.">Single-stage flexor tendoplasty in the treatment of flexor tendon injuries.</a> Acta Orthop Traumatol Turc. 38, 54-59 (2004).</li><li>Gelberman, R. H., Steinberg, D., Amiel, D., Akeson, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+chemotaxis+after+tendon+repair.">Fibroblast chemotaxis after tendon repair.</a> J Hand Surg Am. 16, 686-693 (1991).</li><li>Lundborg, G., Rank, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+intrinsic+healing+of+flexor+tendons+based+upon+synovial+fluid+nutrition.">Experimental intrinsic healing of flexor tendons based upon synovial fluid nutrition.</a> J Hand Surg Am. 3, 21-31 (1978).</li><li>Aoki, M., Kubota, H., Pruitt, D. L., Manske, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+and+histologic+characteristics+of+canine+flexor+tendon+repair+using+early+postoperative+mobilization.">Biomechanical and histologic characteristics of canine flexor tendon repair using early postoperative mobilization.</a> J Hand Surg Am. 22, 107-114 (1997).</li><li>Kim, H. 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=Technical+and+biological+modifications+for+enhanced+flexor+tendon+repair.">Technical and biological modifications for enhanced flexor tendon repair.</a> J Hand Surg Am. 35, 1031-1037 (2010).</li><li>Aoki, M., Manske, P. R., Pruitt, D. L., Kubota, H., Larson, B. 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. 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=The+cellular+biology+of+flexor+tendon+adhesion+formation:+an+old+problem+in+a+new+paradigm.">The cellular biology of flexor tendon adhesion formation: an old problem in a new paradigm.</a> Am J Pathol. 175, 1938-1951 (2009).</li></ol>

De chirurgische procedure voor een muizenmodel van volledige doorsnijding en reparatie van de flexor digitorum longus pees wordt in deze studie. Daarnaast een nieuwe toepassing van concentratie kleine celpopulaties met cytologie centrifuge blijkt, waardoor kwantitatieve immunocytochemische analyse van de cellulaire omgeving tijdens flexorpees genezing. Dit model van flexorpeesherstel toont een reproduceerbare genezende reactie, die kan worden gebruikt om wijzigingen in het genezingsproces behulp knockout modellen of far...

Flexor pezen in de hand werken samen met de flexor spieren van de onderarm en digitale omhulsels flexie van de cijfers en grijpfunctie van de hand mogelijk. Flexorpezen lopen langs de palmaire aspect van de hand; deze relatief oppervlakkige locatie resulteert vaak in verwondingen aan de flexorpezen tijdens trauma aan de kant. Pezen genezen door middel van een littekenweefsel respons in plaats van regeneratie van normaal peesweefsel 1. Hoewel dit littekenweefsel biedt continuïteit van de pees, wordt de functie drastisch afgenomen ten opzichte van een gezonde pees. Pees-littekenweefsel composieten worden gekenmerkt door een verminderde mechanische eigenschappen 1, waardoor het gerepareerde pezen meer kans op scheuren. Bovendien littekenweefsel mist de organisatie van het natieve collageen pees vezelstructuur, resulterend in een toename van de pees grootte en massa. Gezien de anatomische beperkingen van de pees-hulseenheid zelfs bescheiden toename pees grootte kunnen drastisch rodeUCE de glijdende functie van de pees, en dus digit bereik van de beweging en handfunctie. Voorafgaand aan de verwondingen 1960 om de pezen, met name in zone II van de hand, niet routinematig gerepareerd vanwege de ernstige complicaties bij de genezing die zich tijdens deze reparatie 2. Dit gebied van de hand werd aangeduid als &#39;niemandsland&#39; 3. Echter, zijn verbeteringen in chirurgische technieken, hechtdraad patronen en fysiotherapie revalidatie protocollen sterk verbeterde resultaten van flexorpees reparaties 2. Ondanks deze vooruitgang, tot 40% van reparaties leiden tot voldoende adhesie formatie handfunctie 4 belemmeren. Daarom is een biologische benadering bij genezing verbeteren. Helaas, zeer weinig bekend over de pees genezingsproces op cellulair en moleculair niveau. Derhalve was het doel een murine model dat kan worden gebruikt om de fundamentele understandin ontwikkelen ter verbeteringg de cellulaire en moleculaire componenten van buigpees genezing en littekenvorming respons, als middel om nieuwe therapeutische targets te identificeren om genezing te verbeteren. Grotere diermodellen zijn instrumenteel in het bevorderen van begrip van de flexorpees genezingsproces geweest. Honden en konijnen studies hebben aangetoond dat zowel de intrinsieke als extrinsieke herstellend vermogen van pezen 5,6, het belang van vroegtijdige gecontroleerde passieve beweging minimaliseren adhesies opzichte van immobilisatie 7, evenals de effecten van verschillende patronen op de hechtdraad genezingsproces 8 , 9. Bovendien heeft het hondenmodel bruikbaar bij testen translationele weefselmanipulatie benaderingen genezing 10 verbeteren. Er zijn echter een aantal belangrijke voordelen in het gebruik van een muizenmodel opzichte van een groot diermodel, zoals de relatieve kosten, beschikbaarheid van muis specifieke reagentia en het gemak van generating globaal Knock-outs of weefsel-specifieke schrapping / overexpressie constructen. Bovendien is de functionele overeenkomst tussen menselijk en muizen ten opzichte flexorpezen 11 geven de potentiële bruikbaarheid bij de ontwikkeling van een muismodel. Ontwikkeling van een muizenmodel van flexorpees doorsnijding en reparatie bootst vele aspecten van klinische genezing, met inbegrip van de vorming van overvloedige littekenweefsel en verminderde mechanische eigenschappen. De hier beschreven model is een echte herhaling van klinische praktijk door doorsnijden van de FDL de myotendinous knooppunt om de reparatieplaats beschermen. Bovendien heeft dit model geen rekening met de bijdrage van synoviale cellen huls wordt genezing, aangezien er geen synoviale schede die het middengedeelte van de pees waar de reparatie plaatsvindt. Ondanks deze beperkingen heeft dit model het voordeel van het genereren bereik van beweging te beperken adhesies, die nog moet worden aangetoond bij muizenmodellen die meer closely benaderen de klinische scenario. Dit model is gebruikt om knock-out muismodellen 12,13 beoordelen en om verschillende farmacologische benaderingen om de genezing 14-17 te verbeteren testen. Histologische analyses van dit model, middels immunohistochemie en in situ hybridisatie, kunnen belangrijke inzichten in de lokalisatie van belangrijke genen en eiwitten bieden tijdens de genezing. Echter, histologie geeft slechts een dwarsdoorsnede ruimtelijke analyse en maakt kwantificering niet mogelijk het gehele weefsel. Flowcytometrie is een meer kwantitatieve benadering, maar slechts een zeer beperkt aantal cellen kunnen worden geïsoleerd uit de genezing peesweefsel in het muismodel, en dit aantal verder af tijdens fixatie, permeabilisatie en wasstappen. Neemt dit in rekening flowcytometrie wordt een benadering onhaalbaar als gevolg van het aantal dieren dat nodig zou zijn. Een alternatieve methode moet de meerderheid van dit kleine celpopulatie behouden omverder karakteriseren genezing milieu. De methode die wordt gebruikt om dit te bereiken, hier afgebeeld, houdt de concentratie van de geïsoleerde cellen via cytologie centrifugeren op een glasplaatje, gevolgd door immunocytochemie. In de onderhavige studie EDU (5-ethynyl-2&#39;deoxyuridine een thymidine analoog) incorporatie en naderhand coderen werd gebruikt om de relatieve proliferatieve toestand van cellen te bepalen op de genezing plaats. Deze benadering kan worden toegepast om de effectiviteit van farmacologische behandelingen testen celproliferatie, gen knock-out of overexpressie, of verschillende celpopulaties identificeren en te kwantificeren.

<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>
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			<td height="21" style="height:21px;">Clear nail polish</td>
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murine flexor tendon injury and repair surgery

Pees verbindt het skelet spieren en botten, het vergemakkelijken van de beweging van bijna het hele lichaam. In de hand, flexorpezen (FTS) maken buiging van de vingers en de algemene handfunctie. Verwondingen aan de FTS komen vaak voor, en bevredigende genezing wordt vaak aangetast als gevolg van teveel littekenweefsel en verklevingen tussen de pees en het omringende weefsel. Er is echter weinig bekend over de moleculaire en cellulaire componenten van FT reparatie. Daartoe een muizenmodel van FT reparatie die vele aspecten van genezing bij de mens, waaronder verminderde beweeglijkheid recapituleert en verminderde mechanische eigenschappen, ontwikkeld en eerder beschreven. Hier een diepgaande demonstratie van deze chirurgische procedure verschaft, waarbij doorsnijding en daaropvolgende reparatie van de flexor digitorum longus (FDL) pees in de achterpoot van muizen. Deze techniek kan worden gebruikt om lineage analyse van verschillende celtypen te voeren, de effecten van gen winst of verlies-van-functie getest, en de effi testenciëntie van farmacologische interventies in het genezingsproces. Er zijn echter twee belangrijke beperkingen aan dit model: i) het FDL pees in het middengedeelte van het muizen achterpoot, waarbij de doorsnijding en reparatie optreden, wordt niet omgeven door een synoviale schede. Derhalve Dit model houdt geen rekening met de mogelijke bijdrage van de mantel om de littekenvorming proces. ii) Om de integriteit van de reparatieplaats beschermen, is de FT vrijgegeven op het myotendinous knooppunt, het verminderen van de mechanische krachten van de pees, waarschijnlijk bijdragen aan de grotere littekenvorming. Isolatie van voldoende cellen van het granulatieweefsel van de FT tijdens het genezingsproces voor flowcytometrische analyse moeilijk gebleken; cytologie centrifugatie aan deze cellen concentraat is een alternatieve methode, en zorgt voor het genereren van celpreparaten, waarop immunofluorescentie labeling kan worden uitgevoerd. Met deze methode kwantificering van cellen of eiwitten van belang tijdens FT genezing mogelijk.

Flexor digitorum longus (FDL) spieren in de kuit, werkt om de cijfers van de muis achterpoot buigen via de flexorpees (blauwe omtrek in figuur 1A en histologisch figuur 2A), die proximaal loopt van de myotendinous knooppunt en eindigt in de distale vingerkootjes. In dit model van flexorpees genezing, is de FDL pees doorgesneden en gerepareerd in het midden van de voet, proximaal van de vertakking in de cijfers van de achterpoot (rode pijlen, Figu...

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

Murine Flexor Tendon Injury and Repair Surgery

Flexorpezen in de hand worden vaak gewond, wat leidt tot een verminderde handfunctie. Echter, het littekenweefsel genezende reactie niet goed gekarakteriseerd. Een muizenmodel van flexorpees genezing is hier gedemonstreerd. Dit model kan algemeen begrip van het genezingsproces te verbeteren en therapeutische benaderingen om de genezing te verbeteren beoordelen.

Murine flexorpeesletsel en Repair Surgery

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.

Acute en chronische Tactile Sensory Testing na ruggenmergletsel bij ratten

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

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.

Een Contusive model van eenzijdige cervicale ruggenmerg letsel met behulp van de Oneindige 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.

Een nieuw muizenmodel van endovasculaire aortaaneurismareparatie

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.

Gecontroleerde Corticale Impact Model voor traumatisch hersenletsel

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.

Een muis Foetale Skin Model van 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.

Een muizenmodel van slagaderrestenose: technische aspecten van Femorale Wire 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.

Muizenmodel van Intestinale ischemie-reperfusie schade

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.

Reproduceerbare Arteriële ontbossing en erosie Letsel door infrarenale abdominale aorta vastklemmen in een Murine Model

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.

Evaluatie van stamcellen therapieën in een bilaterale Patellaire pees letsel Model bij 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.

Het genereren van een lymfkliertest Orthotopic uitgezaaide borst kanker Model en het uitvoeren van lymfkliertest radicale mastectomie

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