Gli autori desiderano ringraziare il sostegno finanziamento dal National Health and Medical Research Council (NHMRC) dell&#39;Australia (Progetto di Grant ID: 632512). Louise Dunn è stato sostenuto da un NHMRC Early Career Fellowship e Christina Bursill dal National Heart Foundation Fellowship Career Development.

1. Preparazione di stecche e medicazioni occlusive <ol><li> Delineare 10 mm su cerchi 0,5 millimetri teli di silicone spessa e uso forbici o una biopsia per creare i dischi di silicone. </li><li> Centro un punzone 5 mm biopsia nel mezzo del cerchio 10 mm pressione al fine di generare un foro per formare una &quot;ciambella&quot;-come disco che verrà utilizzato come stecco. </li><li> Delineare 10 mm su cerchi una medicazione occlusiva trasparente come opsiti e usare le forbici per creare condimenti circolari. </li></ol> 2. Animali da esperimento <ol><li> Ottenere Animal approvazione Comitato Etico per tutti gli esperimenti che verranno eseguite. </li><li> Utilizzare 8 settimane di età (22-26 grammi) topi maschi C57BL/6J da un allevatore commerciale (ad esempio The Jackson Laboratories). </li><li> Tenere i topi in condizioni standard di 21 ° C e un ciclo di 12 ore luce-buio con libero accesso a cibo e acqua. </li><li> (Opzionale): Il diabete può essere indotta in 6-7 settimane di età i topi da un bolo intraperitoneale injection di 165 mg / kg streptozotocin (in tampone citrato, pH 4,5), con iperglicemia confermato dal monitoraggio della glicemia normale (accuchek glucometro). Topi diabetici possono avere poliuria e quindi la loro biancheria da letto possono avere bisogno di essere cambiato più frequentemente per eliminare l&#39;umidità e il loro peso deve essere attentamente monitorati. </li></ol> 3. Anestesia e preparazione Operativo <ol><li> Indurre l&#39;anestesia generale usando 5% isoflurano in ossigeno al 100% (portata 1 L / min) e mantenere l&#39;anestesia con isoflurano 1-3%. </li><li> Assicurarsi che i profondi riflessi pedale del mouse vengono soppressi e posizionare il mouse in posizione prona. </li><li> Preparare la regione operativa rimuovendo pelliccia con tagliaunghie dalla base del collo a 3 cm più in basso la schiena e tra le due scapole. </li><li> Una leggera applicazione di crema depilatoria può essere applicata per non più di 2 min. Tamponi di garza umida può essere utilizzato per garantire tutti crema e rimanendo pelo viene rimosso. </li><li> Asciugare la pelle con untampone imbevuto di alcool e due applicazioni di 10% di povidone-iodio (Betadine) ed attaccare il mouse. </li></ol> 4. Escissione e splintaggio Ferita <ol><li> Rappresentazione schematica del modello murino di guarigione è presentato nella figura 1. </li><li> Utilizzare una sterile 4 millimetri biopsia per delineare due schemi circolari per la ferita ai lati della linea mediana del topo a livello delle spalle (vedi figure 2a-2b). </li><li> Utilizzare seghettate pinze per sollevare la pelle nel mezzo del contorno iride e forbici per creare una ferita a tutto spessore che si estende attraverso il tessuto sottocutaneo (Figure 2c-2d), compreso il pannicolo carnosus (figura 2e), ed asportare il pezzo circolare di tessuto. </li><li> Ripetere il processo per la ferita sull&#39;altro lato della linea mediana (Figura 2f). </li><li> Rimuovere rivestimento protettivo plastico da ogni lato della stecca silicone. </li><li> Applicare cyanoacrylate collante (ad esempio colla eccellente o Krazy Glue) ad un lato di una stecca di silicone. </li><li> Centro la stecca sopra la ferita (Figura 2g) e ancora la stecca con interrotte 6-0 punti di sutura in nylon per garantire il posizionamento (Figura 2 ore). </li><li> Ripetere il processo di immobilizzazione, dall&#39;altro ferita. </li><li> Se richiesto a questo punto temporale, applicare il composto terapeutico da testare (fino a 100 microlitri) ad uno ferita (Figura 2i), e il controllo del veicolo all&#39;altro. </li><li> Coprire la ferita con una medicazione occlusiva trasparente (per esempio, nell&#39;Opsite) (Figura 2j-2k). </li><li> Un righello è posto sotto le stecche e una microfotografia presa (Figura 2l). </li></ol> 6. Gestione postoperatoria <ol><li> Carprofene (5 mg / kg) è somministrato una volta al giorno tramite iniezione sottocutanea per il sollievo del dolore post-operatorio. </li><li> Dopo gli animali di chirurgia sono in gabbia e mantenuto sui calore m individualmenteats fino pienamente recuperato. </li><li> Monitorare gli animali due volte al giorno per le manifestazioni di dolore e di perdita di peso. Non abbiamo osservato display comportamentali lordo di dolore o perdita di peso. </li></ol> 7. Ferita di misurazione e trattamento <ol><li> La ferita può essere misurato quotidianamente. </li><li> Indurre l&#39;anestesia generale, usando gas isoflurano 5% (tasso di flusso di 1 L / min), e quindi garantire i riflessi sensoriali profonde del topo sono soppresse utilizzando 1-3% isoflurano. </li><li> Delicatamente staccare la medicazione occlusiva con una pinza. </li><li> Utilizzare pinze chirurgiche per misurare il diametro ferita. Prendiamo la media di tre misurazioni lungo la X, Y e Z (Figure 2m-2o), nonché una microfotografia riferimento futuro (Figura 2p). </li><li> Opzionale: In questo momento l&#39;animale può essere valutata per la perfusione sanguigna utilizzando un laser Doppler Imager (Figura 2q). </li><li> Riapplicazione del composto terapeutico ed il controllo del veicolo può essere effetmed a questo punto. </li><li> Una medicazione occlusiva trasparente pulita viene poi riapplicata e gli animali tenuto caldo fino alla completa guarigione. </li><li> Nota: se stecche non sono protetti adeguatamente la ferita facilmente contrarre (Figura 2r). </li></ol> 8. Analisi istologica <ol><li> Euthanize i topi dopo 10 giorni con una dose eccessiva di anestesia. </li><li> Utilizzando pinze e una lama da bisturi rimuovere i punti di sutura e con attenzione staccarsi la stecca. </li><li> Usare le forbici iris per creare un&#39;ampia, completa escissione intorno e sotto l&#39;area della ferita e incubare il tessuto in 4% paraformaldeide in tampone fosfato salino (PBS) a 4 ° C durante la notte. </li><li> Trasferimento di tessuto al 17% di saccarosio in soluzione PBS per un ulteriore 24 ore a 4 ° C e rimuovere la soluzione in eccesso tamponando accuratamente sul tessuto e posto in OCT e congelare a -80 ° C. </li><li> Ematossilina ed eosina possono essere utilizzati per visualizzare la struttura ferita e gap epiteliale. Neovascularisation può essere valutata mediante analisi immunoistochimica per determinare il numero di capillari (utilizzando 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>

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Questo è un modello murino sperimentale di guarigione della ferita cutanea. Una caratteristica significativa di questo modello è l&#39;uso di stecche di silicone per prevenire la contrazione della ferita in modo che possono verificarsi riepitelizzazione e formazione del nuovo tessuto, rendendolo simile al processo che si verifica negli esseri umani. Questo modello è versatile e può essere utilizzato per valutare la guarigione delle ferite in ambienti sia fisiologici e fisiopatologici (diabete mellito). Il m...

La guarigione della ferita è responsabile per una significativa morbidità e mortalità in tutto il mondo, questo è particolarmente vero per chi soffre di diabete mellito di 1,2. Negli esseri umani, la guarigione della ferita è un continuum di processi, in cui vi sia una significativa sovrapposizione 3. Subito sono iniziati seguenti, processi infiammatori ferimento. Cellule infiammatorie rilasciano fattori che favoriscono i processi di proliferazione cellulare, la migrazione e l&#39;angiogenesi. Dopo riepitelizzazione e formazione del nuovo tessuto vi è una fase di rimodellamento che comporta sia apoptosi e riorganizzazione di proteine ​​della matrice come il collagene. La complessità di guarigione della ferita attualmente non può essere replicata in vitro e ciò richiede l&#39;uso di modelli animali. Ad oggi, gli studi cicatrizzanti sono stati limitati a grandi modelli animali, come maiali, per garantire che i processi di guarigione sono equivalenti e comparabili per l&#39;uomo. Tuttavia, utilizzando gran animals per tali studi possono essere difficili da casa e non sono sempre pratici 4. Il topo di laboratorio rappresenta un modello animale economico che può essere facilmente manipolato geneticamente per indagini meccanicistica 5-7. Tuttavia, le ferite guariscono murini diverso ai umana di, principalmente a causa del processo di contrazione 8. Questo è in parte dovuta ad un esteso strato muscolare striato sottocutaneo chiamato panniculus carnosus che risulta praticamente assente nell&#39;uomo. Nei topi, questo strato muscolare permette alla pelle di muoversi indipendentemente dei muscoli più profondi ed è responsabile della rapida contrazione della pelle seguente ferimento. Per superare questa limitazione, cicatrizzante murino può essere adattato per replicare guarigione della ferita umana mediante l&#39;uso di una stecca (Figura 1) 8,9. In questo video dimostriamo il modello murino ferita steccata che elimina la contrazione della ferita e più strettamente approssima i processi umani di ri-epithelializazione e la formazione di nuovo tessuto. In questo modello due escissioni tutto spessore che includono il pannicolo carnosus vengono creati sul dorso, uno su ciascun lato della linea mediana del mouse. Una stecca silicone è disposto intorno alla ferita con l&#39;assistenza di adesivo e la stecca poi fissato con punti staccati. Ogni mouse funge come proprio controllo, con una ferita in trattamento e l&#39;altro controllo del veicolo, riducendo così il numero degli animali. A seguito di applicazioni topiche, viene applicata una medicazione occlusiva trasparente. La medicazione può essere rimossa quando richiesto per ulteriori applicazioni topiche e / o misurazione della superficie della ferita 10,11. Al completamento di esperimenti, chiusura della ferita, architettura morfologica e grado di neovascolarizzazione può essere valutata mediante immunoistochimica. Questo economico e facile da eseguire modello può anche essere utilizzato per valutare la guarigione delle ferite nel contesto del diabete mellito o altre 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

Guarigione delle ferite e la riparazione sono i processi biologici più complessi che si verificano nella vita umana. Dopo l&#39;infortunio, diverse vie biologiche si attivano. La guarigione della ferita, che si verifica in pazienti diabetici, ad esempio, può portare ad esiti sfavorevoli gravi come l&#39;amputazione. Esiste, pertanto, una crescente impulso per sviluppare nuovi agenti che promuovono la riparazione della ferita. La sperimentazione di questi è stato limitato a grandi modelli animali quali maiali, che sono spesso impraticabile. Topi rappresentano il modello preclinico ideale, in quanto sono economici e suscettibili di manipolazione genetica, che consente l&#39;indagine meccanicistica. Tuttavia, la guarigione della ferita in un mouse è fondamentalmente diversa da quella degli esseri umani, come si verifica principalmente attraverso la contrazione. Il nostro modello murino supera questo incorporando una stecca intorno alla ferita. Con splintaggio la ferita, il processo di riparazione è quindi dipendente da riepitelizzazione, la proliferazione cellulare e l&#39;angiogenesi, che rispecchiano da vicino laprocessi biologici di guarigione della ferita umana. Pur richiedendo coerenza e cura, questo modello murino non comporta tecniche chirurgiche complicate e consente la prova robusta di agenti promettenti che possono, per esempio, promuovere o inibire l&#39;angiogenesi infiammazione. Inoltre, ogni mouse funge come proprio controllo come due ferite sono preparati, consentendo l&#39;applicazione sia del composto in esame e il controllo del veicolo sullo stesso animale. In conclusione, abbiamo dimostrato una, facile da apprendere, e robusto modello pratico di guarigione delle ferite, che è paragonabile a quello umano.

Una curva chiusura della ferita è determinato calcolando il diametro medio della ferita ed esprimere i risultati in percentuale, cioè 100 - (giorno 0 diametro / diametro X Giorno). In questo esperimento un composto terapeutico (o controllo veicolo) è stato applicato giornalmente sulla ferita. Il composto terapeutico chiusura della ferita notevolmente accelerato (Figura 3). È importante notare che le stecche devono essere mantenuti per tutta la durata dell&#39;esperimento, come la rimozione ...

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

Murine Model of Wound Healing

Un modello murino di guarigione della ferita cutanea che può essere utilizzato per valutare composti terapeutici in contesti fisiologici e fisiopatologici.

Modello murino di guarigione delle ferite

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

63.1K Views.  The Heart Research Institute. Un modello murino di guarigione della ferita cutanea che può essere utilizzato per valutare composti terapeutici in contesti fisiologici e fisiopatologici.

Video: 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.

Murino Endoscopia per<em&gt; In Vivo</em&gt; Guarigione Imaging Multimodale di Cancerogenesi e valutazione della ferita intestinale e infiammazione

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

Ricerca

Medicina

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.

Dimostrazione del Topo ischemica pelle ferita Modello

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

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.

Creazione e il trapianto di un foglio di cellule staminali di derivazione adiposa (ASC) in un modello diabetico di guarigione

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.

Sviluppo di un modello diretto Pulp-capping di valutazione dei polpa dentale guarigione delle ferite e riparativa dentina formazione nei topi

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.

La microscopia basato su metodi per la valutazione della migrazione delle cellule epiteliali durante In Vitro la guarigione arrotolata

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

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

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

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.

Guarigione delle ferite di abrasione epiteliale corneale con oculare Burr come un modello per Cornea

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 modello di cultura di organo corneale per la guarigione delle ferite studi

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.

Targeting grigio Rami Communicantes nello Sympathectomy lombare chimico selettivo

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 modello di abrasione epiteliale per lo studio della guarigione delle ferite corneali

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 ...