Este trabajo fue apoyado en parte por una subvención del NIH subvención R01 GM087609 (a HPL), un regalo de Ingrid Lai y Bill Shu en honor a Anthony Shu (a HPL), NIH U01 subvención HL099776 (a MTL), el Laboratorio de Hagey Pediátrica Medicina Regenerativa y La Fundación Oak (a MTL y HPL). GGW recibió el apoyo de la Escuela de Medicina de Stanford, el Programa de Capacitación de Stanford Medical Scientist, y GM07365 beca de formación NIGMS. MSH fue apoyada por el CIRM Clinical Fellow Formación Subvención TG2-01159. De WXH fue apoyado por fondos de la Fundación Cardiovascular Sarnoff.

NOTA: Todos los procedimientos descritos en este documento se realizan de acuerdo a los lineamientos establecidos por el Grupo Administrativo de Stanford en Laboratorio Animal Care (APLAC). 1. temporizadas Embarazos - Técnica Superovulación (Figura 1) NOTA: Precisamente temporizar la edad gestacional de embriones de ratón para la cirugía fetal en E16.5 y E18.5 es de importancia crítica. En esta sección detallamos nuestro protocolo de sincronización embarazos ratón utilizando suero embarazada yeguas (PMS) y gonadotropina coriónica humana (HCG) inyecciones para inducir superovulación. <ol><li> Inyectar ratones hembra (&lt;5 por jaula) por vía intraperitoneal (IP) con 3,0 a 5,0 unidades internacionales (UI) de PMS en un volumen de 100 l de PBS 1:00-15:00 para el día 1. </li><li> Entre las 12:00 y las 14:00 del día 3 (cuarenta y cinco a 47 horas después de la inyección PMS), inyectar ratones hembra IP con 3,0 a 5,0 UI de HCG en un volumen de 100 l de PBS. NOTA: Los induce inyección de HCG<html> ovulación, aproximadamente 12 horas después de la inyección. </li><li> Inmediatamente después de las inyecciones de HCG, aparearse con hembras varones de 8 a 16 semanas. NOTA: Por lo general colocamos dos hembras en una jaula de machos individuales. </li><li> Mujeres separadas de los hombres en la mañana del día 4 (06:00-10 a.m.) y registro como E0.5 edad fetal. NOTA: tapones vaginales pueden ser revisados ​​en este momento; sin embargo, la observación de un tapón vaginal no garantiza el embarazo y hembras puede quedar embarazada cuando se observa ningún enchufe. Dado que el embarazo es normalmente observable mediante inspección visual y / o palpación en edades E16.5 y E18.5 gestacional, la comprobación de tapones vaginales en la mañana del día 4 no es estrictamente necesario. En nuestra experiencia, y dependiendo de la cepa, aproximadamente el 30-50% de las mujeres súper-ovulado quedan embarazadas usando la técnica descrita aquí. </li></ol>pg &quot;/&gt; Figura 1. Esquema de Superovulación Técnica. Procedimiento de actuación Esquema de superovulación cronometrado embarazos en ratones. <a href="https://www.jove.com/files/ftp_upload/52297/52297fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> 2. Cirugía Fetal Murino (Dorsal hiriente) en E16.5 y E18.5 embriones (Figura 2) <ol><li> Antes del procedimiento, limpiar todas las superficies de la sala de operaciones y equipos con 70% de alcohol isopropílico. Además, esterilizar todos los suministros quirúrgicos e instrumentos que se utilizan en el procedimiento en autoclave ellos. Algunas instituciones pueden permitir el uso posterior de la esterilización del grano caliente. Por operación, use compresas estériles que incluyen gasas y los instrumentos quirúrgicos. </li><li> Inducir la anestesia en las madres embarazadas (E16.5 edad fetal o E18.5) bajo 2.5% mezcla de isoflurano / oxígeno a 2 l por min seguido de mantenimiento de la anestesia en 1 L por minuto. </li><li> Para confirma anestesia adecuada, asegúrese de que los reflejos de pedal profundas del ratón se suprimen y colocar el ratón en la posición prona. </li><li> Aplique un ungüento oftálmico veterinario como Puralube para evitar irritación de los ojos o la sequedad durante el procedimiento. </li><li> Preparar abdomen mediante la administración de una ligera aplicación de crema depilatoria durante no más de 30 segundos (Figura 2). </li><li> Preparar abdomen para la cirugía aséptica con povidona-yodo y alcohol (Figura 2B y 2C). </li><li> Realizar laparotomía media bajo el microscopio utilizando tijeras de microcirugía (Figura 2D y 2E). </li><li> Exponer suavemente útero y el feto seleccionado para la cirugía (Figura 2F). </li><li> Riegue campo quirúrgico con agua tibia (38 ° C) solución salina tamponada con fosfato (PBS) usando una aguja roma-tip NOTA: Uno puede hacerse doblando con cuidado la punta de una aguja de calibre grande. (Figura 2G y 2H). </li><li> Coloque el feto de una manera que permites acceso completo al dorso. </li><li> Pasar una puntada de hilo de bolsa utilizando sutura de nylon 7-0 a través del útero se sitúa el emplazamiento de la herida dorsal previsto (Figura 2I). Posición cordón de bolsa sobre una región de dorso hacia la izquierda o derecha de la médula espinal, y en una región de la pared uterina desprovista de vasos sanguíneos grandes. </li><li> Hacer una incisión de 3 mm a través de la pared uterina y el saco amniótico en el centro de la bolsa de tabaco (Figura 2J). </li><li> Riegue sitio de la incisión con agua tibia (38 ° C) PBS. </li><li> Con unas tijeras de microcirugía, cortar una sola herida excisional de espesor total, de aproximadamente 1 mm de longitud, en el dorso del feto. </li><li> Suavemente sitio de la incisión séquela con aplicador de punta de algodón. </li><li> Inyectar 3 l volumen de la India por vía subcutánea de tinta en la zona de la herida para marcar la posición de la herida (Figura 2C). </li><li> Irrigar con agua caliente (38 ° C) PBS para asegurarse de tinta ha sido retenido dentro de sitio de la herida. </li><li> Tener quirúrgica asistente inject caliente (38 ° C) a través de PBS con puntas romas 10 G jeringa en el saco amniótico como el cordón de bolsa está cerrada (Figura 2 l). Repliegue jeringa como cierre en bolsa de tabaco llega a su fin (Figura 2 M). </li><li> Volver suavemente útero en la cavidad abdominal (Figura 2 N). </li><li> Piel Evert y el peritoneo. </li><li> Han asistente quirúrgico irrigar la cavidad abdominal con agua caliente (38 ° C) PBS. </li><li> Cerrar abdomen rápidamente por grapado piel y peritoneo cerrado (Figura 2O). El cierre estándar se realiza en dos capas; peritoneo y músculo abdominal en una capa, tejido subcutáneo y de la piel en la segunda capa. Para el sacrificio inmediato y la cosecha del feto, nuestra demostración muestra el cierre en una capa. </li><li> Colocar el animal en observación en una incubadora caliente fijado en 37 ° C durante 30 min o hasta que el animal recupera la consciencia suficiente para mantener decúbito esternal. </li><li> No returna del animal a la compañía de otros animales hasta que se haya recuperado por completo de la intervención. </li><li> Al despertar de la anestesia y durante la posterior de 48 horas, administrar la inyección subcutánea de buprenorfina (0,05 mg / kg) cada 12 horas de analgesia según sea necesario, basado en la evaluación del dolor. Administrar carprofeno (5 mg / kg) por vía subcutánea para el alivio del dolor post-operatorio adicional según sea necesario. </li><li> Vuelta de los animales a la jaula y darles comida y agua ad libitum. </li><li> Seguir de cerca las manifestaciones de dolor. </li><li> 48 horas después de la cirugía, sacrifica madre embarazada con una sobredosis de isoflurano y la cosecha heridos feto. Con el fin de hacer esto, ajustar la concentración de isoflurano al 5% o superior y mantener la exposición de 1 min después de la cesación de la respiración. Confirme la eutanasia con dislocación cervical. Cosecha de un embrión no herido por el control de la misma edad. Finales de los embriones deben tener un método separado de la eutanasia consistente con las recomendaciones del IACUC, como decapitación, dislocación cervical, o de inyección de químicos. </li></ol><img alt="Figura 2" src="/files/ftp_upload/52297/52297fig2highres.jpg" /> Figura 2. Esquema de Cirugía murino fetal. Los pasos generales para dorsal hiriendo en E16.5 y E18.5 embriones de ratón. (A) de depilación abdomen del ratón. (B y C) Preparación de abdomen del ratón. (D) Microscopio para uso quirúrgico procedimiento. (E) de la línea media laparotomía. (F) La exposición del útero. (G) Creación de aguja roma-punta. (H) Riego del útero con una solución salina tibia. (I) Creación de sutura en bolsa. (J) a través de la incisión uterina pared y 1 mm generación herida por escisión de espesor total. (K) La inyección subcutánea de tinta china. (L y M) Clausura de sutura en bolsa. (N y O)Cierre del abdomen. <a href="https://www.jove.com/files/ftp_upload/52297/52297fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a>

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The authors have nothing to disclose.

El protocolo quirúrgico se presenta aquí se describe un modelo de escisión de la curación sin cicatrices fetal murino publicado por primera vez en 2006 por nuestro laboratorio 10. Además de otros modelos establecidos de la herida excisional 11, existen modelos de incisión de la curación sin cicatrices fetal murino, así 12,13. Las investigaciones de la cicatrización de heridas sin cicatrices fetal en el mono, cordero, conejo, tlacuache, y la rata se han reportado 14-17.</sup...

Heridas en la piel fetales curan rápidamente y scarlessly hasta finales de la gestación 1. La reparación de heridas sin cicatrices fetal se caracteriza por la regeneración de la arquitectura del tejido normal y la función. La transición de una cicatriz de fenotipo cicatrización se produce en el tercer trimestre del embarazo en los seres humanos y en todo el día embrionario 18 (E18) en ratones 2,3. En comparación con los adultos, la reparación de heridas fetal se caracteriza por una rápida epitelización, la deposición de tejido conectivo, y la migración de fibroblastos. Muchos estudios han ofrecido posibles explicaciones para el fenómeno de la cicatrización de heridas sin cicatrices durante el desarrollo fetal temprana. La inflamación es un componente fundamental de la reparación de heridas de adultos; Sin embargo, las heridas fetales se caracterizan por una falta de inflamación aguda 4. Si esto es una consecuencia de la inmadurez funcional del sistema inmune durante las etapas fetal permanece poco clara. Un estudio reciente sugiere que las diferencias en la abundancia, esteraureza, y la función de los mastocitos en E15 E18 vs. piel fetal pueden ser responsables de la transición de un fenotipo sin cicatrices, al menos en el ratón 3. Otros estudios postulan que las diferencias en las propiedades y la abundancia de macrófagos heridas fetales y adultos son responsables de la reforma de la matriz extracelular normal (ECM) para la reparación de la herida fetal al 5. Las diferencias en los factores ambientales durante el desarrollo fetal y adulto también pueden afectar a la reparación de heridas. Longaker y sus colegas mostraron que el fluido de la herida del feto posee altos niveles de actividad estimulante de ácido hialurónico en comparación con ninguno de heridas en adultos fluido 6. En consecuencia, los niveles más altos de ácido hialurónico, un glicosaminoglicano que promueve un microambiente favorable para la motilidad celular y la proliferación, en el entorno de la herida fetal puede ser responsable del fenotipo sin cicatrices visto durante el desarrollo fetal temprana. Otras líneas de evidencia apuntan al hecho de que el queso fetaambiente l herida es relativamente hipoxémica y sumergido en el líquido amniótico estéril rico en factores de crecimiento 7. Sin embargo, hay una respuesta definitiva se ha previsto un evento crítico o factor durante la embriogénesis que desencadena la transición de la regeneración de cicatrices de reparación fibrótica. La comprensión de los mecanismos responsables de la curación sin cicatrices en el feto requiere un modelo preciso y reproducible. A continuación te detallamos un modelo reproducible de fetal sin cicatrices cicatrización de heridas en el dorso de E16.5 (cicatrices) y embriones E18.5 (cicatrización) del ratón. Además, pequeñas variaciones de este modelo se pueden utilizar para realizar una serie de estudios adicionales, como el análisis de la expresión génica de las heridas fetales y 8,9 piel. Dado que los embarazos precisamente cronometrados son críticos para la recapitulación con éxito de este modelo de curación sin cicatrices de heridas fetal, también detalle nuestro protocolo de superovulación cronometrado embarazos.

<table border="0" cellpadding="0" cellspacing="0" width="665">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:219px;">Name of Material/Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:155px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7-O MONOSOF Suture</td>
			<td>eSuture</td>
			<td>SN-1647G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Forceps</td>
			<td>Kent Scientific</td>
			<td>INS650916</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro-scissors</td>
			<td>Kent Scientific</td>
			<td>INS600127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Autoclip 9mm</td>
			<td>Texas Scientific Instruments</td>
			<td>205060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin Syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-272-382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Black Pigment</td>
			<td>AIMS</td>
			<td>242</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BD Safety-Lok 3ml Syringe</td>
			<td>BD Biosciences</td>
			<td>309596</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OPMI-MD Surgical Microscope</td>
			<td>Carl Zeiss Surgical Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pregnant Mares Serum (PMS)</td>
			<td>Millipore</td>
			<td>367222</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Chorionic Gonadotropin (HCG)</td>
			<td>Sigma-Aldrich</td>
			<td>CG10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Povidone Iodine Prep Solution</td>
			<td>Dynarex</td>
			<td>1415</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nair (depilatory cream)</td>
			<td>Church and Dwight Co.</td>
			<td>22600267058</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Para el análisis histológico, heridas cutáneas en la piel dorsal de E16.5 y E18.5 embriones de ratón se deben cosechar 48 horas después de la herida, fijado en el 4% PFA, y embebidos en parafina. En modelos transgénicos fluorescentes, la criopreservación de octubre puede ser apropiado. Hay varias manchas que se pueden usar para visualizar la arquitectura del tejido celular y conectivo. Hematoxilina y eosina es una mancha de dos colores que tiñe los núcleos azules y estructuras de eosinófilos (es decir.,</e...

Watch this Scientific Journal Video about A Mouse Fetal Skin Model of Scarless Wound Repair at JoVE.com

A Mouse Fetal Skin Model of Scarless Wound Repair

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 ...

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Medicine

12.2K vistas.  Stanford University School of Medicine. 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.

Video: A Mouse Fetal Skin Model of Scarless Wound Repair

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

Un modelo de la contundente unilateral Lesión Medular Cervical Uso del Impactador Infinite Horizon

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

Investigación

Medicina

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

Una técnica simplificada para fabricar un modelo de lesión isquémica

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Evaluación de los cambios teratogénicos en un modelo de pez cebra de la exposición fetal al alcohol

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

Un nuevo enfoque quirúrgico para la administración intratraqueal de agentes bioactivos en un modelo de ratón fetal

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

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. ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Ecocardiografía Fetal y Ecografía Doppler de onda pulsada en un modelo de conejo de Restricción del crecimiento intrauterino

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Un modelo murino Nueva endovascular de aneurismas aórticos

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

Lesiones tejido isquémico en la Cámara de los pliegues cutáneos dorsal del ratón: Un modelo de la piel de la aleta para Investigar la isquemia aguda persistente

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

Un procedimiento quirúrgico para resecar la costilla del ratón: un modelo a gran escala Reparación huesos largos

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

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.