Die Autoren bedanken sich finanzielle Unterstützung aus dem National Health and Medical Research Council (NHMRC) von Australien (Projektstipendium ID: 632512) bestätigt. Louise Dunn wurde durch eine NHMRC Early Career Fellowship und Christina Bursill von einem National Heart Foundation Career Development Fellowship unterstützt.

1. Herstellung von Schienen und Okklusivverbänden <ol><li> Gliederung 10 mm Kreise auf 0,5 mm dicken Silikon-Folien und Verwendung einer Schere oder einem Biopsiestanze zu Silikon Festplatten erstellen. </li><li> Center ein 5 mm Biopsiestanze in der Mitte des 10 mm Kreis und drücken Sie sie fest, um ein Loch, um einen &quot;Donut&quot;-ähnliche Scheibe, die wie eine Schiene verwendet werden Formular zu erstellen. </li><li> Gliederung 10 mm Kreise auf einem transparenten Okklusivverband wie Opsite und mit einer Schere kreisförmigen Dressings erstellen. </li></ol> 2. Versuchstiere <ol><li> Erhalten Tier Ethikkommission Zulassung für alle Experimente, die ausgeführt werden. </li><li> Mit 8 Wochen alt (22-26 Gramm) männlich C57BL/6J-Mäusen von einem kommerziellen Züchter (zB The Jackson Laboratories). </li><li> Halten Mäuse in Standard-Bedingungen von 21 ° C und eine 12 Stunden Licht-Dunkel-Zyklus mit freiem Zugang zu Futter und Wasser. </li><li> (Optional): Diabetes kann in 6 bis 7 Wochen alten Mäusen durch einen Bolus intraperitoneale inje induziert werdenktion von 165 mg / kg Streptozotocin (in Citratpuffer, pH 4,5), die mit Hyperglykämie durch regelmäßige Blutzuckermessung (AccuChek glucometer) bestätigt. Diabetische Mäuse haben Polyurie und so ihr Bettzeug müssen öfter gewechselt werden, um Feuchtigkeit zu beseitigen und ihre Gewichte sollten engmaschig überwacht werden. </li></ol> 3. Anästhesie und Operative Vorbereitung <ol><li> Induce Vollnarkose mit Isofluran 5% in 100% Sauerstoff (Flussrate 1 L / min) und Aufrechterhaltung einer Narkose mit 1-3% Isofluran. </li><li> Sicherstellen, dass die tiefe Pedal Reflexe der Maus werden unterdrückt und platzieren Sie den Mauszeiger in der Bauchlage. </li><li> Bereiten Sie den operativen Bereich durch Entfernen Fell mit Schermaschinen von der Basis des Halses zu 3 cm weiter nach unten den Rücken und zwischen den beiden Schulterblättern. </li><li> Eine leichte Anwendung der Enthaarungscreme kann nicht länger als 2 min angewendet werden. Wet Mullkompressen kann verwendet werden, um alle zu gewährleisten und Sahne restlichen Fell entfernt wird. </li><li> Wischen Sie die Haut mit einemAlkoholtupfer und zwei Anwendungen von 10% PVP-Jod (Betadine) und drapieren die Maus. </li></ol> 4. Exzision und Schienung Wound <ol><li> Schematische Darstellung des murinen Wundheilung Modell ist in Abbildung 1 dargestellt. </li><li> Verwenden Sie eine sterile 4 mm Biopsiestanze zwei kreisförmige Muster für die Wunde auf beiden Seiten der Maus die Mittellinie auf der Höhe der Schultern skizzieren (siehe Abbildungen 2a und 2b). </li><li> Verwenden gezackten Zange, um die Haut in der Mitte der Umriss und Iris Schere, um ein Full-Dicke Wunde, die durch das subkutane Gewebe (Abb. 2c bis 2d), einschließlich der Panniculus carnosus (Abbildung 2e) erstreckt erstellen heben, und Verbrauchsteuern die kreisförmiges Stück Gewebe. </li><li> Wiederholen des Prozesses für die Wunde auf der anderen Seite der Mittellinie (Fig. 2f). </li><li> Entfernen Kunststoff-Schutzschicht überzogen von jeder Seite des Silikons Schiene. </li><li> Bewerben cyanoacrylate Klebstoff (zB Sekundenkleber oder Krazy Glue) auf der einen Seite aus einem Silikon-Schiene. </li><li> Center die Schiene über der Wunde (Abbildung 2g) und Anker die Schiene mit unterbrochenen 6-0 Nylonnaht die Positionierung (Abbildung 2h) zu gewährleisten. </li><li> Wiederholen Sie den Vorgang auf der Schienung andere Wunde. </li><li> Wenn zu dieser Zeit erforderlich sein sollte, gelten die therapeutische Verbindung, die getestet werden (bis zu 100 ul) einer Wunde (2i) und dem Fahrzeug an die andere. </li><li> Decken Sie die Wunde mit einer transparenten Okklusivverband (z. B. OpSite) (2j-2k). </li><li> Ein Lineal ist unter den Schienen gelegt und eine mikroskopische Aufnahme gemacht (Abb. 2l). </li></ol> 6. Postoperative Behandlung <ol><li> Carprofen (5 mg / kg) einmal täglich über subkutane Injektion für post-operative Schmerzlinderung verabreicht. </li><li> Nach der Operation Tiere werden einzeln in Käfigen gehalten und auf Wärme mats bis vollständig erholt. </li><li> Überwachen Tiere zweimal täglich für Manifestationen von Schmerzen und Gewichtsverlust. Wir beobachteten keine groben Verhaltens Displays von Schmerzen oder Gewichtsverlust. </li></ol> 7. Wound Messung und Behandlung <ol><li> Die Wunde kann täglich gemessen werden. </li><li> Induce Vollnarkose mit 5% Isofluran-Gas (Flussrate 1 L / min), und dann sicherstellen, dass die tiefen sensorischen Reflexe der Maus unterdrückt werden mit 1-3% Isofluran. </li><li> Vorsichtig schälen die Okklusivverband mit einer Pinzette. </li><li> Verwenden chirurgischen Bremssättel, um die Wunde Durchmesser messen. Wir nehmen den Mittelwert aus drei Messungen entlang der X-, Y-und Z-Achse (Abb. 2m-2o) sowie eine Mikrofotografie für zukünftige Referenz (Abbildung 2p). </li><li> Optional: Zu diesem Zeitpunkt kann das Tier für Durchblutung mittels Laser Doppler Imager (Abbildung 2q) beurteilt werden. </li><li> Re-Anwendung der therapeutischen Verbindung und der Fahrzeugsteuerung kann Leistung seinmed. an dieser Stelle. </li><li> Eine saubere transparent Okklusivverband wird dann neu aufgetragen und die Tiere warm gehalten, bis sie vollständig erholt. </li><li> Hinweis: Wenn Schienen nicht ordnungsgemäß gesichert, die Wunde wird leicht schrumpfen (Abbildung 2r). </li></ol> 8. Die histologische Analyse <ol><li> Euthanize die Mäuse nach 10 Tagen mit einer Überdosis Narkose. </li><li> Mit einer Pinzette und einem Skalpell entfernen Sie die Nähte und vorsichtig abziehen der Schiene. </li><li> Verwenden Irisschere eine breite durchgehende Exzision um und unter der Wunde zu schaffen und Inkubieren des Gewebes in 4% Paraformaldehyd in Phosphat gepufferter Salzlösung (PBS) bei 4 ° C über Nacht. </li><li> Übertragen Gewebe bis 17% Saccharose in PBS-Lösung für weitere 24 Stunden bei 4 ° C und entfernen Sie überschüssige Lösung durch Abtupfen sorgfältig auf das Gewebe und in OCT-Verbindung und frieren bei -80 ° C. </li><li> Hämatoxylin und Eosin-Färbung kann die gewickelte Struktur und Epithelzellen Spalt zu visualisieren. Neovascularisation durch immunhistochemische Analysen überprüft werden, um die Anzahl der Kapillaren (mit von Willebrand-Faktor) zu bestimmen. </li></ol>

<ol>
	<li>Sen, C. 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=Human+skin+wounds:+a+major+and+snowballing+threat+to+public+health+and+the+economy.">Human skin wounds: a major and snowballing threat to public health and the economy.</a> Wound Repair. 17, 763-771 (2009).</li><li>Sen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+essentials:+let+there+be+oxygen.">Wound healing essentials: let there be oxygen.</a> Wound Repair Regen. 17, 1-18 (2009).</li><li>Gurtner, G. C., Werner, S., Barrandon, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> Nature. 453, 314-321 (2008).</li><li>Lindblad, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Considerations+for+selecting+the+correct+animal+model+for+dermal+wound-healing+studies.">Considerations for selecting the correct animal model for dermal wound-healing studies.</a> J. Biomater. Sci. Polym. Ed. 19, 1087-1096 (2008).</li><li>Grose, R., Werner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound-healing+studies+in+transgenic+and+knockout+mice.">Wound-healing studies in transgenic and knockout mice.</a> Mol. Biotechnol. 28, 147-166 (2004).</li><li>Reid, R. R., Said, H. K., Mogford, J. E., Mustoe, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+wound+healing:+pursuing+surgical+models+in+transgenic+and+knockout+mice.">The future of wound healing: pursuing surgical models in transgenic and knockout mice.</a> J. Am. Coll. Surg. 199, 578-585 (2004).</li><li>Fang, R. C., Mustoe, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+wound+healing:+utility+in+transgenic+mice.">Animal models of wound healing: utility in transgenic mice.</a> J. Biomater. Sci. Polym. Ed. 19, 989-1005 (2008).</li><li>Wong, V. W., Sorkin, M., Glotzbach, J. P., Longaker, M. T., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+approaches+to+create+murine+models+of+human+wound+healing.">Surgical approaches to create murine models of human wound healing.</a> J. Biomed. Biotechnol. 2011, 969618 (2011).</li><li>Galiano, R. D., Michaels, J. t., Dobryansky, M., Levine, J. P., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+reproducible+murine+model+of+excisional+wound+healing.">Quantitative and reproducible murine model of excisional wound healing.</a> Wound Repair. 12, 485-492 (2004).</li><li>Galiano, R. D., 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=Topical+vascular+endothelial+growth+factor+accelerates+diabetic+wound+healing+through+increased+angiogenesis+and+by+mobilizing+and+recruiting+bone+marrow-derived+cells.">Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells.</a> The American Journal of Pathology. 164, 1935-1947 (2004).</li><li>Thangarajah, H., 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+molecular+basis+for+impaired+hypoxia-induced+VEGF+expression+in+diabetic+tissues.">The molecular basis for impaired hypoxia-induced VEGF expression in diabetic tissues.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 13505-13510 (2009).</li><li>Raza, A., Bayles, C., Biebel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+Bacterial+Growth+and+Moisture+Handling+Properties+of+Transparent+Dressings:+3M+Tegaderm+Transparent+Dressing,+3M+Tegaderm+HP+Transparent+Dressing,+and+Opsite+IV3000+Transparent+Dressing.">Investigation of Bacterial Growth and Moisture Handling Properties of Transparent Dressings: 3M Tegaderm Transparent Dressing, 3M Tegaderm HP Transparent Dressing, and Opsite IV3000 Transparent Dressing.</a> Smith and Nephew Report. , (1998).</li><li>Chung, T. Y., Peplow, P. V., Baxter, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Testing+photobiomodulatory+effects+of+laser+irradiation+on+wound+healing:+development+of+an+improved+model+for+dressing+wounds+in+mice.">Testing photobiomodulatory effects of laser irradiation on wound healing: development of an improved model for dressing wounds in mice.</a> Photomed. Laser Surg. 28, 589-596 (2010).</li></ol>

Die Autoren haben nichts zu offenbaren.

Dies ist eine experimentelle Mausmodell der kutanen Wundheilung. Ein wesentliches Merkmal dieses Modells ist die Verwendung von Silikon-Schienen gewickelt Kontraktion zu verhindern, so dass Reepithelisierung und neuem Gewebe auftreten, so dass es ähnlich zu dem Verfahren, das in Menschen auftritt. Dieses Modell ist vielseitig und kann verwendet werden, um die Wundheilung bei sowohl physiologische und pathophysiologische (zB Diabetes mellitus) Einstellungen zu bewerten. Das Modell kann auch verwendet werden, um...

Wundheilungsstörungen ist verantwortlich für erheblichen Morbidität und Mortalität weltweit, dies gilt vor allem für Patienten mit Diabetes mellitus 1,2. Beim Menschen ist die Wundheilung ein Kontinuum von Prozessen, in denen es erhebliche Überschneidungen 3. Unmittelbar nach der Verwundung, entzündliche Prozesse initiiert werden. Entzündungszellen freizugeben Faktoren, die die Prozesse der Zellproliferation, Migration und Angiogenese zu fördern. Nach Reepithelisierung und neuem Gewebe gibt es eine Phase der Umgestaltung, die sowohl Apoptose und Neuorganisation der Matrixproteine ​​wie Kollagen bringt. Die Komplexität der Wundheilung können derzeit nicht in vitro repliziert werden, und dies erfordert die Verwendung von Tiermodellen. Bisher wurden Wundheilung Studien Großtiermodellen, wie Schweine beschränkt, um sicherzustellen, dass die Heilungsprozesse gleichwertig und vergleichbar Menschen. Allerdings mit großen animals für solche Studien kann schwierig sein, zu Haus und sind nicht immer praktisch 4. Das Labor Maus stellt eine kostengünstige Tiermodell die leicht genetisch für mechanistische Untersuchungen 5-7 manipuliert werden. Allerdings murinen Wunden heilen anders für die menschliche, vor allem auf den Prozess der Kontraktion 8. Dies ist zum Teil aufgrund einer umfangreichen subkutane quergestreiften Muskulatur Schicht genannt Panniculus carnosus, die weitgehend abwesend in den Menschen ist. Bei Mäusen, ermöglicht diese Muskelschicht der Haut, sich unabhängig von den tieferen Muskeln bewegen und ist verantwortlich für die schnelle Kontraktion der Haut folgende Verwundung. Um diese Einschränkung zu überwinden, können murine Wundheilung geeignet ist, menschliche Wundheilung durch Verwendung einer Schiene (1) 8,9 replizieren. In diesem Video zeigen wir die Wunde splinted murinen Modell, das Wundkontraktion eliminiert und mehr annähert die menschlichen Prozesse der Re-epithelialization und neuem Gewebe. In diesem Modell zwei voller Dicke Resektionen, die die panniculus carnosus enthalten, werden auf dem Rücken, einen auf jeder Seite der Mittellinie der Maus erzeugt. Ein Silikon-Schiene wird um die Wunde mit Hilfe von Klebstoff angebracht und die Schiene dann mit unterbrochenen Nähten befestigt. Jede Maus dient als seine eigene Kontrolle, mit einer Wunde, die Behandlung und die andere Fahrzeug-Steuerung, wodurch der Anzahl der Tiere. Nach topischer Anwendung wird eine transparente Okklusivverband aufgebracht. Der Verband kann entfernt werden, wenn weitere topische Anwendungen und / oder Messung der Wundfläche 10,11 erforderlich. Bei Beendigung von Experimenten Wundverschluss morphologischen Architektur und der Grad der Gefäßneubildung durch Immunhistochemie untersucht werden. Diese kostengünstige und einfach zu Modell ausführen, können auch verwendet werden, um die Wundheilung im Rahmen von Diabetes mellitus oder anderen pathophysiologies beurteilen.

<table border="0">
<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Press-to-seal silicone sheeting 0.5 mm thick</td>
 <td>Invitrogen</td>
 <td>P18178</td>
 <td>Cut into "donuts" with external diameter of 1cm external, 0.5 cm internal diameter</td>
 </tr>
 <tr>
 <td>Biopsy punch 5 mm</td>
 <td>Steifel</td>
 <td>BC-B1-0500</td>
 <td>To outline wound area to be excised</td>
 </tr>
 <tr>
 <td>Vannas scissors 8.5 cm curved</td>
 <td>World Precision Instruments </td>
 <td>501232</td>
 <td>For wound incision and excision</td>
 </tr>
 <tr>
 <td>Dumonte #7b forceps, 11 cm</td>
 <td>World Precision Instruments</td>
 <td>501302</td>
 <td>To grip skin when creating incision and excising skin </td>
 </tr>
 <tr>
 <td>Graefe forceps, serrated 10cm</td>
 <td>World Precision Instruments</td>
 <td>14142</td>
 <td>To help attach silicone splint to skin</td>
 </tr>
 <tr>
 <td>Needle holder, smooth jaws, curved, 12.5 cm</td>
 <td>World Precision Instruments</td>
 <td>14132</td>
 <td></td>
 </tr>
 <tr>
 <td>Malis forceps, smooth, straight, 12 cm</td>
 <td>Codman and Shurtleff, Inc (J&amp;J)</td>
 <td>80-1500</td>
 <td>To suture the silicon rings to the skin</td>
 </tr>
 <tr>
 <td>Ruler, 0.5 mm gradation</td>
 <td>n/a</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Calipers 0.25 mm gradation</td>
 <td>Duckworth and Kent</td>
 <td>9-653</td>
 <td>To measure wound area</td>
 </tr>
 <tr>
 <td>Opsite FlexiFix transparent adhesive film. 10 cm x 1 m</td>
 <td>Smith &amp; Nephew</td>
 <td>66030570</td>
 <td></td>
 </tr>
 <tr>
 <td>Rimadyl (Carprofen)</td>
 <td>Pfizer</td>
 <td>462986</td>
 <td></td>
 </tr>
 </tbody>
</table>

murine model of wound healing

Wundheilung und Reparatur sind die komplexesten biologischen Prozesse, die im menschlichen Leben auftreten. Nach einer Verletzung, werden mehrere biologische Pfade aktiviert. Wundheilungsstörungen, die bei Diabetes-Patienten zum Beispiel auftritt, kann zu schweren ungünstigen Ergebnisse wie Amputation führen. Es besteht daher ein zunehmender Impulse neuen Substanzen, die die Wundheilung fördern, zu entwickeln. Die Prüfung dieser hat Großtiermodellen wie Schweine, die oft unpraktisch beschränkt. Mäuse sind die ideale präklinischen Modell, da sie wirtschaftlich und zugänglich genetische Manipulation, die für mechanistische Untersuchung erlaubt sind. Allerdings Wundheilung in einer Maus unterscheidet sich grundlegend von dem des Menschen, wie es geschieht primär durch Kontraktion. Unsere Mausmodell überwindet diese durch den Einbau einer Schiene um die Wunde. Durch Schienung die Wunde, ist die Reparatur dann abhängig von Epithelisierung, Zellproliferation und Angiogenese, die eng spiegeln diebiologische Prozesse der menschlichen Wundheilung. Während erfordern Konsistenz und Pflege, bedeutet das Mausmodell nicht mit komplizierten chirurgischen Techniken und erlaubt die robuste Prüfung vielversprechende Wirkstoffe, zum Beispiel die Angiogenese fördern oder hemmen Entzündungen. Weiterhin wirkt jede Maus als seine eigene Kontrolle als zwei Wunden bereit sind, so dass die Anwendung sowohl der Testverbindung und der Fahrzeugsteuerung am selben Tier. Abschließend zeigen wir eine praktische, einfach zu erlernen und robustes Modell der Wundheilung, die vergleichbar mit der des Menschen ist.

(Tag 0 Durchmesser / Tag x Durchmesser) - Wundverschlusssystem Kurve durch Berechnung des durchschnittlichen Durchmessers der gewickelten und Exprimieren der Ergebnisse in Prozent, also 100 bestimmt. In diesem Experiment wurde eine therapeutische Verbindung (oder MFK) wurde täglich auf die Wunde aufgetragen. Die therapeutische Verbindung stark beschleunigte Wundheilung (Abbildung 3). Es ist wichtig zu beachten, dass die Schienen für die Dauer des Experiments muss beibehalten werden, das Entfe...

Watch this Scientific Journal Video about Murine Model of Wound Healing at JoVE.com

Murine Model of Wound Healing

Ein Mausmodell von kutanen Wundheilung, die zur therapeutischen Verbindungen in physiologischen und pathophysiologischen Einstellungen beurteilen kann.

Mausmodell der Wundheilung

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Research

JoVE Journal

Medicine

63.3K Aufrufe.  The Heart Research Institute. Ein Mausmodell von kutanen Wundheilung, die zur therapeutischen Verbindungen in physiologischen und pathophysiologischen Einstellungen beurteilen kann.

Serie: Murine Model of Wound Healing

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Murine Endoskopie für<em&gt; In Vivo</em&gt; Multimodale Bildgebung der Krebsentstehung und Bewertung der Darm Wundheilung und Entzündung

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Forschung

Medizin

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.

Demonstration der Ratte ischämische Hautwunde Modell

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and ...

Multimodal Quantitative Phase Imaging with Digital Holographic Microscopy Accurately Assesses Intestinal Inflammation and Epithelial Wound Healing

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The etiology of IBD remains unknown and current therapeutic strategies are based on the unspecific suppression of the immune system. The development of treatments that specifically target intestinal inflammation and epithelial wound healing could significantly improve management of IBD, however this requires accurate detection of inflammatory changes. Currently, potential drug candidates are usually evaluated using animal models in vivo or with cell culture based techniques in vitro. Histological examination usually requires the cells or tissues of interest to be stained, which may alter the sample characteristics and furthermore, the interpretation of findings can vary by investigator expertise. Digital holographic microscopy (DHM), based on the detection of optical path length delay, allows stain-free quantitative phase contrast imaging. This allows the results to be directly correlated with absolute biophysical parameters. We demonstrate how measurement of changes in tissue density with DHM, based on refractive index measurement, can quantify inflammatory alterations, without staining, in different layers of colonic tissue specimens from mice and humans with colitis. Additionally, we demonstrate continuous multimodal label-free monitoring of epithelial wound healing in vitro, possible using DHM through the simple automated determination of the wounded area and simultaneous determination of morphological parameters such as dry mass and layer thickness of migrating cells. In conclusion, DHM represents a valuable, novel and quantitative tool for the assessment of intestinal inflammation with absolute values for parameters possible, simplified quantification of epithelial wound healing in vitro and therefore has high potential for translational diagnostic use.

Accurate assessment of anti-inflammatory effects is of utmost importance for the evaluation of potential new drugs for the treatment of inflammatory bowel disease. Digital holographic microscopy provides assessment of inflammation in murine and human colonic tissue samples as well as automated multimodal evaluation of epithelial wound healing in vitro.

Multimodale Quantitative Phase Imaging mit digitalen holografischen Mikroskopie Beurteilt Exakt Darmentzündung und Epithelial Wundheilung

The incidence of inflammatory bowel disease, i.e., Crohn&#39;s disease and Ulcerative colitis, has significantly increased over the last decade. The ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

Schaffung und Transplantation von einem Fettgewebe-derived Stem Cell (ASC) Blatt in einem diabetischen Wundheilung Modell

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Development of a Direct Pulp-capping Model for the Evaluation of Pulpal Wound Healing and Reparative Dentin Formation in Mice

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is often capped with biocompatible materials in order to expedite pulpal wound healing. The ultimate goal is to regenerate reparative dentin, a physical barrier that functions as a "biological seal" and protects the underlying pulp tissue. Although this direct pulp-capping procedure has long been used in dentistry, the underlying molecular mechanism of pulpal wound healing and reparative dentin formation is still poorly understood. To induce reparative dentin, pulp capping has been performed experimentally in large animals, but less so in mice, presumably due to their small sizes and the ensuing technical difficulties. Here, we present a detailed, step-by-step method of performing a pulp-capping procedure in mice, including the preparation of a Class-I-like cavity, the placement of pulp-capping materials, and the restoration procedure using dental composite. Our pulp-capping mouse model will be instrumental in investigating the fundamental molecular mechanisms of pulpal wound healing in the context of reparative dentin in vivo by enabling the use of transgenic or knockout mice that are widely available in the research community.

We describe a step-by-step method of performing direct pulp capping on mice teeth for the evaluation of pulpal wound healing and reparative dentin formation in vivo.

Entwicklung eines Direkt Pulp-Kappungsmodell für die Beurteilung von Pulpal Wundheilung und Reparative Dentinbildung bei Mäusen

Dental pulp is a vital organ of a tooth fully protected by enamel and dentin. When the pulp is exposed due to cariogenic or iatrogenic injuries, it is ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

Mikroskopie-basierten Methoden für die Beurteilung der Epithelzelle Migration bei der In-vitro- Wundheilung

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

Epitheliale Korneaabnutzung mit okulärer Burr als Modell für die Hornhaut Wundheilung

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

Ex Vivo Wundheilung Hornhaut Orgel Kulturmodells für Studien

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Targeting Gray Rami Communicantes in Selective Chemical Lumbar Sympathectomy

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the lower extremities, hyperhidrosis, etc. It is commonly practiced to position the puncture needle tip in front of the anterior fascia of the psoas major muscle and inject the inactivating agent around the sympathetic trunk, which is defined as conventional CLS. Although relatively rare, ureteropelvic damage is the most frequently reported complication of conventional CLS and can cause serious harm to patients. We found that injecting the inactivating agent behind the anterior fascia, which only targets gray rami communicantes, helped achieve therapeutic efficacy in&#160;vasodilation, sweat reduction, and pain relief comparable to conventional CLS, and serious complications were largely reduced. We define this procedure as selective CLS. Here, we present a protocol of selective CLS. The precise needle tract and accurate evaluation of the spreading of the contrast agent are critical to ensure that the drug is injected behind the anterior fascia of the psoas major muscle. The needle tip is at approximately one-third the dividing line of the vertebral body in the lateral view of a lumbar X-ray. The contrast is mainly confined around the needle tip and spreads outward and downward along the psoas muscle fibers. In this way, the anterior fascia provides a natural barrier for the ureteropelvic area, and the psoas major muscle provides a natural barrier for the lumbar nerve root. There are several highlights of this article, including 1) a detailed description of the selective CLS procedures, 2) an explanation of the anatomical basis for the implementation of selective CLS, and 3) an explanation of the differences between selective and conventional CLS.

We present a protocol for selective chemical lumbar sympathectomy (CLS), which inactivates only gray rami communicantes and not the sympathetic trunk. Selective CLS can help achieve therapeutic efficacy in vasodilation, sweat reduction, and pain relief that are comparable to conventional CLS, and serious complications, particularly ureteropelvic damages, can be reduced.

Ausrichtung auf graue Rami Communicantes im selektiven chemischen lumbale Sympathektomie

Chemical lumbar sympathectomy (CLS) is a commonly used, minimally invasive procedure for the treatment of conditions including ischemic diseases of the ...

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

Ein epitheliales Abriebmodell zur Untersuchung der Hornhautwundheilung

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...