Les auteurs tiennent à remercier le soutien financier du Conseil de recherches médicales (NHMRC) de l&#39;Australie (Project Grant ID: 632512) Santé nationale et du. Louise Dunn a été soutenue par un début de carrière NHMRC Fellowship et Christina Bursill par une Fondation carrière Bourse nationale de développement du coeur.

1. Préparation des attelles et des pansements occlusifs <ol><li> Décrivez 10 cercles mm sur 0,5 mm feuilles de silicone épais et utiliser des ciseaux ou un biopsie pour créer des disques de silicone. </li><li> Centre mm de biopsie 5 dans le milieu du cercle mm 10 et appuyez fermement pour créer un trou pour former un &quot;donut&quot;-comme disque qui sera utilisé comme une attelle. </li><li> Décrivez 10 cercles mm sur un pansement occlusif transparent comme Opsite et utiliser des ciseaux pour créer des pansements circulaires. </li></ol> 2. Animaux de laboratoire <ol><li> Obtenir l&#39;approbation du comité d&#39;éthique animale pour toutes les expériences qui seront effectuées. </li><li> Utilisez âgées de 8 semaines (22-26 grammes) C57BL/6J mâles chez un éleveur commercial (par exemple, les Jackson Laboratories). </li><li> Gardez souris dans des conditions standard de 21 ° C et un cycle lumière-obscurité de 12 heures avec un accès libre à la nourriture et à l&#39;eau. </li><li> (Facultatif): Le diabète peut être induite dans les 6 à 7 semaines souris par un bolus intra-péritonéale Injection de 165 mg / kg de streptozotocine (dans un tampon citrate, pH 4,5), l&#39;hyperglycémie confirmée par un contrôle de la glycémie normale (Accuchek glucomètre). Souris diabétiques peuvent avoir polyurie et ainsi de leur litière peuvent avoir besoin d&#39;être changé plus souvent pour éliminer l&#39;humidité et leur poids doivent être étroitement surveillés. </li></ol> 3. Anesthésie et préparation d&#39;Exploitation <ol><li> Induire une anesthésie générale en utilisant 5% d&#39;isoflurane dans 100% d&#39;oxygène (débit de 1 l / min) et maintenir une anesthésie avec 1-3% d&#39;isoflurane. </li><li> S&#39;assurer que les réflexes de pédale profondes de la souris sont supprimées et placer la souris dans la position couchée. </li><li> Préparer la zone opératoire en supprimant la fourrure avec une tondeuse à partir de la base du cou à 3 cm plus bas dans le dos et entre les deux omoplates. </li><li> Une légère application de crème dépilatoire peut être appliquée pendant une durée de 2 min. Des tampons de gaze humide peuvent être utilisés pour assurer toutes les crèmes et le reste fourrure est enlevée. </li><li> Essuyez la peau avec untampon imbibé d&#39;alcool et deux applications de 10% de povidone-iode (Betadine) et drapé de la souris. </li></ol> 4. L&#39;excision et la contention de la plaie <ol><li> Représentation schématique du modèle de cicatrisation murin est présentée dans la figure 1. </li><li> Utiliser un stérile mm biopsie 4 à esquisser deux motifs circulaires pour la blessure de chaque côté de la ligne médiane de la souris au niveau des épaules (voir figures 2a-2b). </li><li> Utilisez une pince dentelés pour soulever la peau au milieu de l&#39;esquisse et ciseaux à iris pour créer une plaie de pleine épaisseur qui s&#39;étend à travers le tissu sous-cutané (figures 2c-2d), y compris le panniculus carnosus (figure 2e), et de l&#39;accise de la pièce circulaire du tissu. </li><li> Répéter le processus pour la blessure de l&#39;autre côté de la ligne médiane (figure 2f). </li><li> Supprimer revêtement de protection en matière plastique à partir de chaque côté de l&#39;attelle de silicone. </li><li> Appliquer cyanoacrylate colle (par exemple Super Glue ou Krazy Glue) d&#39;un côté d&#39;une attelle silicone. </li><li> Centre de l&#39;attelle sur la plaie (figure 2g) et ancre l&#39;attelle avec interrompues 6-0 fils de nylon pour assurer un positionnement (figure 2h). </li><li> Répétez le processus contention d&#39;autre blessure. </li><li> En cas de besoin à ce point de temps, appliquer le composé thérapeutique à tester (jusqu&#39;à 100 ul) à une plaie (figure 2i), et le contrôle du véhicule à l&#39;autre. </li><li> Recouvrez la plaie avec un pansement occlusif transparent (par exemple, OpSite) (figure 2j-2k). </li><li> Une règle est placée sous les gouttières et une microphotographie prise (Figure 2l). </li></ol> 6. Gestion postopératoire <ol><li> Carprofène (5 mg / kg) est administré une fois par jour par injection sous-cutanée pour le soulagement de la douleur post-opératoire. </li><li> Après que les animaux de chirurgie sont en cage individuellement et maintenus à feu mats jusqu&#39;à ce que complètement rétabli. </li><li> De surveiller les animaux deux fois par jour pour les manifestations de douleur et de perte de poids. Nous n&#39;avons observé aucune affiche comportement brut de la douleur ou de la perte de poids. </li></ol> 7. Mesure et traitement des plaies <ol><li> La blessure peut être mesurée quotidiennement. </li><li> Induire une anesthésie générale en utilisant 5% de gaz isoflurane (débit de 1 L / min), puis s&#39;assurer que les réflexes sensoriels profonds de la souris sont supprimées en utilisant 1-3% d&#39;isoflurane. </li><li> Délicatement décoller le pansement occlusif avec une pince. </li><li> Utilisez étriers chirurgicales pour mesurer le diamètre de la plaie. Nous prenons la moyenne des trois mesures le long des axes X, Y et Z (Figures 2m-2o) ainsi que d&#39;une microphotographie pour référence future (figure 2p). </li><li> Facultatif: Pour le moment l&#39;animal peut être évalué pour la perfusion de sang en utilisant un appareil d&#39;imagerie laser Doppler (figure 2 po). </li><li> Ré-application du composé thérapeutique et la commande du véhicule peuvent être effectuées quemed à ce point. </li><li> Un pansement occlusif transparent propre est ensuite ré-appliquée et les animaux gardés au chaud jusqu&#39;à ce que complètement rétabli. </li><li> Note: si attelles ne sont pas sécurisés correctement la plaie va se contracter facilement (figure 2r). </li></ol> 8. L&#39;analyse histologique <ol><li> Euthanasier les souris après 10 jours avec une surdose d&#39;anesthésique. </li><li> En utilisant des pinces et d&#39;un scalpel enlever les sutures et décoller délicatement l&#39;attelle. </li><li> Utilisez des ciseaux iris pour créer une large excision complète autour et sous la surface de la plaie et incuber le tissu dans du paraformaldéhyde 4% dans du tampon phosphate salin (PBS) à 4 ° C pendant la nuit. </li><li> Transfert tissus à 17% de saccharose dans une solution de PBS pour une nouvelle heure 24 à 4 ° C puis retirez l&#39;excès de solution en tamponnant délicatement sur le tissu et le placer dans du composé OCT et congeler à -80 ° C. </li><li> Hématoxyline et éosine peuvent être utilisés pour visualiser la structure de la plaie et l&#39;écart de l&#39;épithélium. Neovascularisation peut être évaluée par des analyses d&#39;immunohistochimie pour déterminer le nombre de capillaires (à l&#39;aide du facteur Von Willebrand). </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>

Les auteurs n&#39;ont rien à révéler.

Il s&#39;agit d&#39;un modèle expérimental murin de la cicatrisation cutanée. Une caractéristique importante de ce modèle est l&#39;utilisation d&#39;attelles en silicone pour empêcher la contraction de la plaie afin que réépithélisation et la formation de nouveaux tissus peuvent se produire, ce qui en fait similaire au processus qui se produit chez l&#39;homme. Ce modèle est polyvalent et peut être utilisé pour évaluer la cicatrisation des plaies dans les milieux physiologiques et physiopathologiques (...

La cicatrisation des plaies est responsable de morbidité et de mortalité dans le monde entier significative, ce qui est particulièrement vrai pour les personnes souffrant de diabète sucré de 1,2. Chez l&#39;être humain, la cicatrisation des plaies est un continuum de processus, dans lequel il est important de chevauchement 3. Immédiatement processus inflammatoires, blessant suivantes sont initiées. Les cellules inflammatoires libèrent des facteurs qui favorisent les processus de prolifération cellulaire, la migration et l&#39;angiogenèse. Après réépithélisation et la formation de nouveaux tissus il ya une phase de rénovation qui implique à la fois l&#39;apoptose et la réorganisation des protéines de la matrice comme le collagène. La complexité de la cicatrisation ne peut actuellement pas être reproduit in vitro, ce qui nécessite l&#39;utilisation de modèles animaux. À ce jour, les études de cicatrisation ont été limitées aux grands modèles d&#39;origine animale, comme la viande porcine, afin de s&#39;assurer que le processus de guérison sont équivalentes et comparables pour les humains. Toutefois, l&#39;utilisation à grande animals pour de telles études peuvent être difficiles à la maison et ne sont pas toujours pratique 4. La souris de laboratoire représente un modèle animal économique qui peut être facilement manipulé génétiquement pour enquête mécaniste 5-7. Cependant, les blessures guérissent murins différemment à de l&#39;homme, principalement en raison du processus de contraction 8. Ceci est dû en partie à une vaste couche de muscle strié sous-cutanée appelée panniculus carnosus qui est largement absente chez les humains. Chez la souris, cette couche musculaire permet à la peau de se déplacer indépendamment des muscles plus profondes et est responsable de la contraction rapide des blessures de la peau suivant. Pour contourner cette limitation, la cicatrisation murin peut être adapté pour reproduire la cicatrisation des plaies de l&#39;homme par l&#39;utilisation d&#39;une attelle (Figure 1) 8,9. Dans cette vidéo, nous démontrons le modèle murin de la plaie attelle qui élimine contraction de la plaie et se rapproche plus étroitement les processus humains de re-epithelialization et la formation de nouveaux tissus. Dans ce modèle, deux excisions pleine épaisseur qui incluent le panniculus carnosus sont créés sur le dos, un de chaque côté de la ligne médiane de la souris. Une attelle silicone est placé autour de la plaie à l&#39;aide d&#39;un adhésif et l&#39;attelle ensuite fixé avec des sutures interrompues. Chaque souris agit comme son propre contrôle, avec une plaie de recevoir un traitement et l&#39;autre contrôle du véhicule, réduisant ainsi le nombre d&#39;animaux. Après applications topiques, un pansement occlusif transparent est appliqué. Le pansement peut être retiré en cas de besoin pour d&#39;autres applications topiques et / ou la mesure de la surface de la plaie 10,11. À l&#39;issue d&#39;expériences, la fermeture des plaies, l&#39;architecture morphologique et le degré de néovascularisation peut être évalué par immunohistochimie. Cette solution économique et facile à effectuer modèle peut également être utilisé pour évaluer la cicatrisation des plaies dans le cadre d&#39;un diabète ou d&#39;autres pathophysiologies.

<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

La cicatrisation des plaies et la réparation sont des processus biologiques les plus complexes qui se produisent dans la vie humaine. Après une blessure, de multiples voies biologiques s&#39;activent. La cicatrisation des plaies, qui se produit chez les patients diabétiques par exemple, peut conduire à des résultats défavorables sévères telles que l&#39;amputation. Il est, par conséquent, un élan croissant de développer de nouveaux agents qui favorisent la cicatrisation des plaies. Les essais de ces a été limitée aux grandes modèles animaux comme la viande porcine, qui sont souvent impraticables. Souris représentent le modèle préclinique idéal, car ils sont économiques et se prêtent à la manipulation génétique, ce qui autorise des investigations mécaniste. Cependant, la cicatrisation des plaies chez une souris est fondamentalement différente de celle de l&#39;homme comme cela se fait principalement par l&#39;intermédiaire de la contraction. Notre modèle murin surmonte ce en intégrant une attelle autour de la plaie. Moyen d&#39;une attelle la plaie, le processus de réparation est alors dépendante de l&#39;épithélialisation, la prolifération cellulaire et l&#39;angiogenèse, qui reflètent étroitement lales processus biologiques de cicatrisation des plaies de l&#39;homme. Bien que nécessitant cohérence et de soins, ce modèle murin ne comporte techniques chirurgicales complexes et permet de tester robuste des agents prometteurs qui peuvent, par exemple, favoriser l&#39;angiogenèse ou inhiber l&#39;inflammation. En outre, chaque souris agit comme son propre contrôle en deux plaies sont préparés, permettant l&#39;application à la fois le composé d&#39;essai et la commande du véhicule sur le même animal. En conclusion, nous démontrons un modèle pratique, facile à apprendre et robuste de cicatrisation de la plaie, ce qui est comparable à celle des humains.

Une courbe de fermeture de la plaie est déterminé en calculant le diamètre moyen de la plaie et d&#39;exprimer les résultats sous forme de pourcentage, soit 100 - (Jour 0 / diamètre diamètre du Jour de X). Dans cette expérience, un composé thérapeutique (ou le contrôle du véhicule) a été appliqué quotidiennement sur la plaie. Le composé thérapeutique de fermeture de plaies fortement accélérée (Figure 3). Il est important de noter que les attelles doivent être conservés pend...

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

Murine Model of Wound Healing

Un modèle murin de la cicatrisation cutanée qui peut être utilisé pour évaluer les composés thérapeutiques dans des contextes physiologiques et physiopathologiques.

Modèle murin de la cicatrisation

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

63.4K vues.  The Heart Research Institute. Un modèle murin de la cicatrisation cutanée qui peut être utilisé pour évaluer les composés thérapeutiques dans des contextes physiologiques et physiopathologiques.

Vidéo: 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.

Murin endoscopie pour<em&gt; In Vivo</em&gt; Guérison multimodale d&#39;imagerie de la cancérogenèse et de l&#39;évaluation de la plaie intestinale et l&#39;inflammation

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

Recherche

Médecine

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.

Démonstration de la peau plaies Modèle Rat ischémique

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.

Imagerie Multimodal quantitative phase avec Digital Holographic Microscopy Évalue avec précision guérison inflammation intestinale et épithéliales des plaies

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.

Création et la Transplantation d’une feuille adipeux dérivées de cellules souches (ASC) dans un modèle de cicatrisation diabétique

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.

Élaboration d&#39;un modèle de coiffage pulpaire direct pour l&#39;évaluation des pulpaire Wound Healing et Formation reconstructrice Dentin chez la souris

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.

Méthodes basées sur la microscopie pour l’évaluation de la Migration de cellules épithéliales lors In Vitro la cicatrisation des plaies

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.

Abrasion épithéliale cornéenne avec oculaire Burr comme modèle pour la cornée, la cicatrisation

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, modèle de Culture d’organes cornéenne pour la cicatrisation des études

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.

Ciblage gris Rami Communicantes dans une sympathectomie lombaire chimique sélective

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.

Un modèle d’abrasion épithéliale pour étudier la cicatrisation des plaies cornéennes

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 ...