Los autores desean agradecer el apoyo financiero del Consejo Nacional de Salud y Consejo de Investigación Médica (NHMRC) de Australia (Proyecto de Donación ID: 632512). Louise Dunn fue apoyado por una beca de Carrera Temprana NHMRC y Christina Bursill por una beca de la Fundación Nacional de Desarrollo de la Carrera del Corazón.

1. Preparación de férulas y vendajes oclusivos <ol><li> Esquema 10 círculos mm en 0,5 mm de espesor y láminas de silicona con unas tijeras o un punzón de biopsia para crear discos de silicona. </li><li> Centro de un punzón de biopsia de 5 mm en el centro del círculo de 10 mm y presione firmemente para crear un agujero para formar una &quot;rosquilla&quot;-como disco que se utilizará como una férula. </li><li> Esquema 10 mm círculos sobre un apósito oclusivo transparente como Opsite y tijeras utilizar para crear vendajes circulares. </li></ol> 2. Animales de experimentación <ol><li> Obtener la aprobación del Comité de Ética Animal de todos los experimentos que se realizarán. </li><li> Utilice 8 semanas de edad (22 a 26 gramos) ratones C57BL/6J macho de un criador comercial (por ejemplo, los Laboratorios Jackson). </li><li> Mantenga los ratones en condiciones estándar de 21 º C y una HR del ciclo 12 de luz-oscuridad con acceso libre a comida y agua. </li><li> (Opcional): La diabetes puede ser inducida en 6 a 7 semanas de edad ratones por un bolo intraperitoneal Injección de 165 mg / kg de estreptozotocina (en tampón citrato, pH 4,5), con hiperglucemia confirmada por control de la glucemia normal (accuchek glucómetro). Los ratones diabéticos pueden tener poliuria y así pueden necesitar su ropa de cama que cambiar con mayor frecuencia para eliminar la humedad y los pesos deben ser estrechamente monitorizados. </li></ol> 3. Anestesia y preparación Operativo <ol><li> Inducir la anestesia general utilizando 5% de isoflurano en oxígeno al 100% (tasa de flujo de 1 l / min) y mantener la anestesia utilizando 1-3% de isoflurano. </li><li> Asegúrese de que los reflejos profundos del pedal del ratón se suprimen y coloque el puntero del ratón en la posición prona. </li><li> Preparar la región operativa mediante la eliminación de piel con una maquinilla de la base del cuello a 3 cm más abajo en la parte posterior y entre las dos láminas de hombro. </li><li> Una ligera aplicación de crema depilatoria puede aplicarse por no más de 2 min. Hisopos de gasa húmeda se pueden usar para asegurar que todos crema y se retira de piel restante. </li><li> Limpiar la piel con unaalgodón con alcohol y dos aplicaciones de 10% de povidona yodada (Betadine) y cubren el ratón. </li></ol> 4. La escisión y el entablillado de la herida <ol><li> Representación esquemática del modelo de curación de heridas murino se presenta en la Figura 1. </li><li> Utilice un estéril 4 mm biopsia en sacabocados para delinear dos patrones circulares para la herida a cada lado de la línea media del ratón en el nivel de los hombros (véanse las figuras 2a-2b). </li><li> El uso de fórceps dentadas para levantar la piel en el medio del esquema y tijeras iris para crear una herida de espesor total que se extiende a través del tejido subcutáneo (Figuras 2c-2d), incluyendo el panículo carnoso (Figura 2e), y la pieza circular especiales de tejido. </li><li> Repetir el proceso para la herida en el otro lado de la línea media (Figura 2f). </li><li> Eliminar capa protectora de plástico de cada lado de la férula de silicona. </li><li> Aplicar cyanoacrylaTE adhesivo (por ejemplo, Super Glue o Krazy Glue) a un lado de una férula de silicona. </li><li> Centro de la férula sobre la herida (Figura 2g) y el ancla de la férula con interrumpidas 6-0 suturas de nylon para asegurar el posicionamiento (figura 2h). </li><li> Repetir el proceso de colocación de una férula en la otra herida. </li><li> Si es necesario en este punto de tiempo, aplicar el compuesto terapéutico a ensayar (de hasta 100 l) a una herida (figura 2i), y el control del vehículo a la otra. </li><li> Cubra la herida con un apósito oclusivo transparente (por ejemplo, OpSite) (figura 2j-2k). </li><li> Un gobernante se coloca por debajo de las férulas y una microfotografía tomada (Figura 2l). </li></ol> 6. Tratamiento postoperatorio <ol><li> Carprofeno (5 mg / kg) se administra una vez al día mediante inyección sub-cutánea para el alivio del dolor post-operatorio. </li><li> Después de la cirugía los animales están enjaulados y se mantienen a fuego m individualats hasta que esté completamente recuperado. </li><li> Supervisar los animales dos veces al día durante las manifestaciones de dolor y pérdida de peso. No se observaron muestras de comportamiento graves de dolor o pérdida de peso. </li></ol> 7. Medición de la herida y Tratamiento <ol><li> La herida puede medirse todos los días. </li><li> Inducir la anestesia general utilizando 5% de gas isoflurano (tasa de flujo de 1 l / min), y a continuación, asegurar los reflejos profundos sensoriales del ratón se suprimen utilizando 1-3% de isoflurano. </li><li> Suavemente jale el vendaje oclusivo con fórceps. </li><li> Use pinzas quirúrgicas para medir el diámetro de la herida. Tomamos el promedio de tres mediciones a lo largo de los ejes X, Y y Z (Figuras 2m-2o), así como una microfotografía para referencia futura (figura 2p). </li><li> Opcional: En este momento el animal se puede evaluar la perfusión arterial mediante un Doppler impresora láser (Figura 2q). </li><li> Re-aplicación del compuesto terapéutico y el control del vehículo puede ser realizadosmed en este punto. </li><li> Un vendaje oclusivo transparente limpia se vuelve a aplicar y los animales mantiene caliente hasta que esté completamente recuperado. </li><li> Nota: si las férulas no se fijan correctamente la herida se contrae rápidamente (Figura 2r). </li></ol> 8. Análisis histológico <ol><li> La eutanasia a los ratones después de 10 días con una sobredosis de anestesia. </li><li> Con unas pinzas y un bisturí quitar las suturas y con cuidado retire la férula. </li><li> Con unas tijeras iris para crear una amplia, la escisión completa alrededor y debajo de la zona de la herida e incubar el tejido en 4% de paraformaldehído en tampón fosfato salino (PBS) a 4 ° C durante la noche. </li><li> Transferencia de tejido a 17% de sacarosa en solución de PBS durante una hora 24 más a 4 º C y después eliminar el exceso de solución frotando cuidadosamente sobre el tejido y el lugar en compuesto OCT y congelar a -80 ° C. </li><li> Tinción con hematoxilina y eosina se pueden utilizar para visualizar la estructura y la brecha de la herida epitelial. Neovascularisation puede ser evaluada por análisis inmunohistoquímico para determinar el número de capilares (utilizando el factor de 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>

Los autores no tienen nada que revelar.

Este es un modelo experimental murino de cicatrización de heridas cutáneas. Una característica importante de este modelo es el uso de férulas de silicona para prevenir la contracción de la herida por lo que pueden producirse de re-epitelización y formación de nuevo tejido, por lo que es similar al proceso que se produce en los seres humanos. Este modelo es muy versátil y se puede utilizar para evaluar la cicatrización de heridas en la configuración tanto fisiológicos y fisiopatológicos (por ejemplo,...

Cicatrización de las heridas es responsable de la morbilidad y mortalidad en todo el mundo significativo, lo que es particularmente cierto para los que sufren de diabetes mellitus 1,2. En los seres humanos, la cicatrización de heridas es un proceso continuo de procesos, en la que no es significativa superposición 3. Inmediatamente se inició a raíz de los procesos inflamatorios, heridas. Las células inflamatorias liberan factores que estimulan los procesos de proliferación celular, la migración y la angiogénesis. Después de re-epitelización y formación de nuevo tejido hay una fase de remodelación que implica tanto la apoptosis y de re-organización de proteínas de la matriz tales como colágeno. La complejidad de la cicatrización de heridas no puede actualmente ser replicado in vitro y esto requiere el uso de modelos animales. Hasta la fecha, los estudios de curación de heridas se han limitado a modelos animales grandes, tales como cerdos, para garantizar que los procesos de curación son equivalentes y comparables a los seres humanos. Sin embargo, el uso de grandes animals para este tipo de estudios pueden ser difíciles de casa y no siempre son prácticos 4. El ratón de laboratorio representa un modelo animal económica que puede ser fácilmente manipulado genéticamente para la investigación mecanicista 5-7. Sin embargo, las heridas se curan murinos de manera diferente a los humanos, principalmente debido al proceso de contracción 8. Esto es en parte, debido a una amplia capa de músculo estriado subcutánea llamado el panículo carnoso que es en gran medida ausente en los seres humanos. En los ratones, esta capa muscular permite que la piel se mueva independientemente de los músculos más profundos y es responsable de la rápida contracción de la piel siguiente heridas. Para superar esta limitación, la curación de heridas murino se puede adaptar para replicar la curación de heridas humanos mediante el uso de una férula (Figura 1) 8,9. En este video se demuestra la herida modelo murino entablillado que elimina contracción de la herida y se aproxima más a los procesos humanos de re-epitelizaciónción y la formación de nuevos tejidos. En este modelo de dos escisiones de espesor completo que incluyen el panículo carnoso se crean en el dorso, uno a cada lado de la línea media del ratón. Una férula de silicona se coloca alrededor de la herida con la ayuda de adhesivo y la férula entonces asegurado con suturas interrumpidas. Cada ratón actúa como su propio control, con una herida de recibir tratamiento y otro de control del vehículo, lo que reduce el número de animales. Después de la aplicación tópica, se aplica un vendaje oclusivo transparente. El apósito se puede quitar cuando sea necesario para otras aplicaciones tópicas y / o de medición del área de la herida 10,11. En la realización de experimentos, cierre de la herida, la arquitectura morfológica y grado de neovascularización se puede evaluar mediante técnicas de inmunohistoquímica. Este económico y fácil de realizar modelo también se puede utilizar para evaluar la cicatrización de heridas en el contexto de la diabetes mellitus o otros patofisiología.

<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 curación y la reparación de heridas son los procesos biológicos más complejos que se producen en la vida humana. Después de la lesión, múltiples vías biológicas se activan. Cicatrización de las heridas, que se produce en los pacientes diabéticos, por ejemplo, puede conducir a resultados desfavorables graves como la amputación. No es, por lo tanto, un impulso creciente para desarrollar nuevos agentes que promueven la reparación de la herida. Las pruebas de estos se ha limitado a modelos animales grandes tales como cerdos, que son a menudo poco práctico. Los ratones representan el modelo preclínico ideal, ya que son económicos y susceptibles de manipulación genética, que posibilite la investigación mecanicista. Sin embargo, la curación de heridas en un ratón es fundamentalmente diferente a la de los seres humanos, ya que se produce principalmente a través de la contracción. Nuestro modelo murino supera este mediante la incorporación de una férula alrededor de la herida. Por entablillado la herida, el proceso de reparación es entonces dependiente epitelización, proliferación celular y la angiogénesis, que reflejan de cerca laprocesos biológicos de cicatrización de la herida humana. Mientras que requieren consistencia y la atención, este modelo murino no implica técnicas quirúrgicas complicadas y permite la prueba robusta de agentes prometedores que pueden, por ejemplo, promover la angiogénesis o inhibir la inflamación. Además, cada ratón actúa como su propio control como se preparan dos heridas, lo que permite la aplicación tanto del compuesto de ensayo y el control del vehículo en el mismo animal. En conclusión, se demuestra un modelo práctico, fácil de aprender, y robusto de cicatrización de la herida, que es comparable a la de los humanos.

Una curva de cierre de la herida se determina calculando el diámetro medio de la herida y expresando los resultados como un porcentaje, es decir, 100 - (Día 0 / diámetro X Día). En este experimento se aplicó un compuesto terapéutico (o vehículo de control) al día a la herida. El compuesto terapéutico de cierre acelerado en gran medida la herida (Figura 3). Es importante señalar que las férulas deben mantenerse durante la duración del experimento, como la eliminación de las férulas...

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

Murine Model of Wound Healing

Un modelo murino de cicatrización de heridas cutáneas que se puede utilizar para evaluar los compuestos terapéuticos en los entornos fisiológicos y fisiopatológicos.

Modelo murino de Curación de Heridas

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

63.6K vistas.  The Heart Research Institute. Un modelo murino de cicatrización de heridas cutáneas que se puede utilizar para evaluar los compuestos terapéuticos en los entornos fisiológicos y fisiopatológicos.

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 para<em&gt; En Vivo</em&gt; Healing Multimodal Imaging de Carcinogénesis y Evaluación de la herida y la inflamación intestinal

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

Investigación

Medicina

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Un ratón fetal Modelo de la piel de la reparación de heridas sin cicatrices

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

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.

Demostración de la rata isquémica de la piel herida Modelo

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.

Multimodal Fase cuantitativa de imágenes con Digital holográfica Microscopía evaluación exacta de la inflamación intestinal Curación de heridas y epitelial

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.

Creación y el transplante de una hoja de células madre derivadas de adiposo (ASC) en un modelo de curación de herida diabética

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.

Desarrollo de un Modelo de recubrimiento pulpar directo para la Evaluación de la Pulpa Curación de Heridas y formación de dentina reparadora en ratones

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.

Métodos basados en microscopía para la evaluación de la migración de células epiteliales durante la cicatrización de la herida In Vitro

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.

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

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.

Cicatrización de heridas de abrasión epitelial corneal con rebabas Ocular como un modelo de córnea

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.

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