We thank members of the Mariani lab for critical reading of the manuscript. Our funding sources were: the Baxter Medical Scholar Research Fellowship (to M.K.S.), USC undergraduate fellowships and the Provost, Dean Joan M. Schaeffer, and Rose Hills Fellowships, (to M.K.S.). We also acknowledge a CIRM BRIDGES fellowship through Pasadena City College (to T.T.T). and the James H. Zumberge Research and Innovation Fund, the USC Regenerative Medicine Initiative, and the NIAMS NIH under Award Number R21AR064462 (to F.V.M).

NOTA: Todos los procedimientos se ajustan con un animal protocolo aprobado por el Cuidado y Uso de Animales Comité Institucional (IACUC) de la Universidad del Sur de California. 1. Preparación para la Cirugía <ol><li> Coloque los instrumentos quirúrgicos y suministros necesarios (esterilizada, artículos 1 a 18 en la lista de materiales) y organizar un microscopio de disección y una fibra óptica de trans-iluminador en una campana o en una mesa de aspiración. </li><li> Coloque un ratón sexualmente maduros (20 - 30 g, 1 - 3 meses de edad) en una cámara de inducción con 4% de isoflurano para inducir la anestesia. </li><li> Confirme la anestesia con una pata y / o prueba del pellizco cola. </li><li> Aplique una pomada para los ojos del ratón para evitar la sequedad del ojo durante la cirugía. </li><li> Mueve el ratón a la platina del microscopio. Coloque el ratón en el cono de la nariz de la manguera de mantenimiento y posicionar lateralmente. Proporcionar una bolsa de calentamiento adyacente al animal para ayudar a mantener la temperatura corporal. NOE:. Posición del ratón depende de la preferencia y / o uso de las manos del cirujano personal, lateral izquierda o derecha está muy bien. </li><li> Ajuste isoflurano al 2-3% para el mantenimiento, y ver la frecuencia respiratoria. </li><li> Inyectar buprenorfina (0,05 mg / kg) por vía subcutánea (flanco lateral de la pierna) para el dolor después de la cirugía con una aguja 25 G. La pierna puede contracción que es una respuesta normal. </li></ol> Incisión 2. Apertura <ol><li> Detectar la costilla deseada por palpación, y limpiar la zona de pelo con una maquinilla de afeitar. NOTA: Costillas 8-10 se recomiendan desde las costillas 1-7 están más cerca de los pulmones y 11-13 son considerablemente más cortos. Contando las costillas debe comenzar con la costilla más rostral como número 1. </li><li> Prepare el área con la alternancia de povidona yodada y el isopropanol (70%) hisopado (3x se recomienda a menudo por los protocolos de uso animal). </li><li> Cortar una incisión de 2 cm a través de la piel, directamente arriba y paralela a la costilla deseado con tijeras de microcirugía de tamaño medio. Inc¿Estee a través de las capas de músculo y grasa subyacentes. </li><li> Coloque todos los 3 capas (piel, músculo, grasa) en un retractor para exponer el área quirúrgica, y reducir al mínimo el tamaño de la incisión. </li></ol> 3. Ablación la costilla <ol><li> Corte a través de los músculos intercostales suprayacentes la sección deseada de la costilla con un bisturí 5,0 mm. Posición de esta incisión alrededor de 5 mm proximal a la articulación condrocostal ya que es donde la costilla no es tan altamente curvada. Separar cuidadosamente el músculo del hueso con unas pinzas de punta fina. </li><li> Para crear una resección al tiempo que conserva el periostio en el animal, cortar a través del periostio, a lo largo de la longitud de la costilla con un bisturí 5,0 mm. Separar cuidadosamente el periostio del hueso subyacente lateralmente con unas pinzas de punta fina. Proceda con precaución, ya que el periostio es muy delicada y tiene una consistencia gelatinosa. </li><li> Siguiente hacer una sección transversal a través del hueso en un extremo con finas micro-tijeras. Si es necesario, medir la resección conuna retícula en el microscopio o un medidor de tipo regla. A continuación, levante con cuidado el hueso del periostio y cortar el otro extremo. NOTA: Proceda con precaución. Este es el paso más delicado, ya que tirando hacia arriba sin extirpar el nervio continuación se rompa la membrana pleural y dar lugar a un neumotórax. Si se rompe la membrana pleural este será fácilmente evidente como el interior de la cavidad torácica se hará visible. </li><li> Si el sangrado se produce como resultado de cortar el hueso, aplicar presión en el extremo de corte con un hisopo de algodón con punta de 4 - 5 segundos para detener el sangrado. NOTA: En las raras ocasiones en que el sangrado continúa, puede que sea necesario para detener y practicar la eutanasia del animal pérdida de sangre tan significativo comprometerá recuperación (sin reposición de líquidos, el volumen máximo de sangre que puede ser con seguridad se pierde es el 10% del volumen total de sangre o 7.7 -. 8 l / g Para un 25 g de ratón, esto es equivalente a alrededor de 180 a 200 l 9). </li><li> Coloque inmediatamente la costilla eliminado en4% PFA para el análisis futuro. NOTA: Si la resección de tanto el hueso y el periostio se llevará a cabo, omita la incisión periostio y la separación (paso 3.2). Desde el periostio está muy estrechamente unida a la membrana pleural proceder con cautela, las burlas la nervadura de distancia de la membrana pleural con pinzas para evitar que se rompa. </li></ol> 4. Cierre de la incisión <ol><li> Suturar los músculos intercostales sobre la parte superior de la manga perióstica restante con 9-0 suturas (2 suturas suelen ser suficientes). Coloque las suturas directamente sobre los extremos cortados de la costilla, para actuar como indicadores para la ubicación de la cirugía. </li><li> Retire el retractor. Suturar el músculo y la grasa que recubre con 9-0 suturas (3-4 suturas suelen ser suficientes). En ratones con capas particularmente gruesas de músculo y / o grasa, suturar cada capa por separado (por ejemplo, 1 capa de suturas para la capa muscular y 1 capa de suturas para la capa de grasa). </li><li> Cierre lapiel con 7-0 suturas (4-5 suturas suelen ser suficientes). </li><li> Asegure la incisión con pegamento de sutura, pellizcar los bordes juntos con grandes pinzas. </li><li> Lentamente destetar el ratón fuera isoflurano ajustando primero al 1% durante unos minutos y luego se apagará. </li><li> Coloque el ratón bajo una lámpara de calor y dejar hasta que se recupere la conciencia, 5-10 min. Después de la conciencia se recuperó y en todo el período de cicatrización, el ratón debe moverse y deambular normalmente y no muestran signos de sufrimiento. </li></ol> 5. Recuperación y análisis <ol><li> Proporcionar el manejo del dolor postoperatorio incluyendo la administración de buprenorfina en forma de gelatina oral a 0,5 mg / kg cada 12 horas durante 48 horas. La administración oral no requiere restringir el animal que puede provocar dolor a la región torácica. </li><li> Mantener el ratón en una jaula con libre acceso al alimento y agua durante el período de cicatrización. Después de la incisión es seco, las hembras pueden co-alojados mientras que el varóns debe permanecer aislado para evitar enfrentamientos. </li><li> Tras el período de cicatrización, siga el procedimiento aprobado por la eutanasia. Retire la caja torácica para la fijación y el análisis. </li></ol>

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The authors have nothing to disclose

La primera vez que el aprendizaje de este protocolo, determinar dónde ubicar la incisión inicial puede ser un reto. Sin embargo, la práctica en los ratones sacrificados ayuda al cirujano a aprender dónde colocar la incisión inicial y exponer el nervio deseado para ser resecado. Trabajando en cadáveres también mejora las habilidades motoras finas requeridas para eliminar la porción de la costilla con o sin el periostio. Además, alguien nuevo en este procedimiento podría encontrar la manipulación de las herrami...

Debilitante lesión esquelética, la osteoartritis crónica y los problemas graves asociados con la cirugía reconstructiva impactar la productividad económica, el bienestar de la familia, y la calidad de vida. Mientras que las pequeñas roturas y lesiones pueden sanar bastante bien, los seres humanos no son capaces de reparar defectos grandes y por lo tanto deben confiar en los procedimientos de reconstrucción para restaurar la estructura y función. Reconstrucción puede implicar injertos alogénicos o heterogeneic, hueso morcelado, andamios implantados, o osteogénesis de distracción. Desafortunadamente, no sólo hay factores de morbilidad persistentes asociados con estos tratamientos, pero la resistencia original del hueso reparado raramente se alcanza. Por lo tanto, se necesitan nuevos enfoques clínicos. De una vía para desarrollar métodos innovadores para el tratamiento de defectos segmentarios es estudiar situaciones en las que la reparación a gran escala se produce de forma natural. Anfibios famoso puede regenerar elementos esqueléticos, mientras que los mamíferos se consideran limitadas en ºes la capacidad. Sin embargo, desde principios del siglo 20, algunos informes de la regeneración de la costilla humana se han publicado lo que sugiere que los seres humanos no pueden ser tan limitados 1-4. Actualmente este fenómeno es el más conocido por los cirujanos plásticos que utilizan material de la costilla de la mandíbula, la cara y la oreja de reconstrucción, pero no es más ampliamente apreciado 5. Para estudiar esta reparación con más detalle, hemos desarrollado un modelo quirúrgico con el ratón. El uso de este protocolo, los investigadores pueden identificar los factores innatos involucrados y utilizar esta información para facilitar la curación esquelético en otros lugares. Hay muchas ventajas de utilizar las nervaduras como un modelo para estudiar la reparación esquelética. En primer lugar, las costillas que rodean proporcionan un fijador natural (en comparación con la resección del fémur 6,7). Esto disminuye el riesgo de morbilidad de fijadores internos y externos y simplifica el procedimiento quirúrgico. En segundo lugar, las capas musculares delgadas de los wa pecholl proporcionar un fácil acceso y una excelente visibilidad que hacen que el ensayo comparables a la comodidad de las resecciones de calota 8. En tercer lugar, en contraste con la calotas que forman por la osificación intramembranosa, la forma de las costillas por la osificación endocondral y crecer en longitud a través de la extensión en placas de crecimiento situados en cualquier extremo de una diáfisis central. Por lo tanto, la reparación de los nervios puede ser más comparable a la reparación de los huesos largos del esqueleto apendicular. Además, hemos encontrado que en comparación con el fémur, el periostio de la costilla es más gruesa y puede ser más fácilmente manipulada. Por lo tanto, los investigadores que desean ensayar la reparación del hueso con el propósito de estudiar el periostio o probar terapias celulares, agentes farmacológicos, y / o andamios de tejido pueden encontrar este modelo quirúrgica útil. En resumen, este modelo de resección de costilla proporciona un contexto en el que para estudiar la reparación ósea a gran escala natural en mamíferos como tal modelo de uso general en la actualidad existe.

<table border="0" cellpadding="0" cellspacing="0" width="896">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Name of Reagent/ Equipment</td>
			<td style="width:160px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:304px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">Medium sized micro-dissection scissors (Vannas-T&#252;bingen Spring Scissors 5 mm)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Fine micro-dissection scissors (Vannas Spring Scissors - 2mm Cutting Edge)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">15000-04</td>
			<td>curved tip is beneficial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Micro-scalpel 5.0 mm</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>10315-12</td>
			<td>other fine scalpels can be substituted</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Dumont 55 forceps</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">11295-51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Retractor&#160;</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>17004-05</td>
			<td>adjustability is convenient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Micro-needle holders</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">12060-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">9.0 nylon sutures (Ethilon), taper point best</td>
			<td style="width:160px;">Ethicon</td>
			<td>2819G or similar</td>
			<td>taper point best but reverse cutting is also good</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">7.0 prolene sutures (Prolene)</td>
			<td style="width:160px;">Ethicon</td>
			<td>8700H or similar</td>
			<td>6-0 can be used too, needle point can vary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Large forceps (Adson Forceps)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">11006-12</td>
			<td>other brands are fine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Lubricant Eye Ointment (Akwa Tears)</td>
			<td style="width:160px;">Akorn</td>
			<td style="width:157px;">17478-062-35</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Suture glue (GLUture Topical Tissue Adhesive)</td>
			<td style="width:160px;">Abbot</td>
			<td style="width:157px;">32046-01</td>
			<td>has excellent working time</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Shaver</td>
			<td style="width:160px;">Wahl</td>
			<td>9918-6171 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Clamp lamp</td>
			<td>Zoo Med</td>
			<td style="width:157px;">LF-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Infrared Bulb, 75W</td>
			<td style="width:160px;">Zoo Med</td>
			<td>RS-75</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:275px;">RC2 Rodent Anesthesia System</td>
			<td style="width:160px;">VetEquip&#160;</td>
			<td style="width:157px;">922100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">IsoFlo (Isoflurane)</td>
			<td style="width:160px;">Abbot</td>
			<td style="width:157px;">05260-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Buprenorphine (Buprenex)</td>
			<td style="width:160px;">Reckitt Benckiser</td>
			<td style="width:157px;">12496-0757-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Betadine</td>
			<td style="width:160px;">Purdue Frederick</td>
			<td>67618015017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Flavored Gelatin, raspberry</td>
			<td style="width:160px;">Jell-O</td>
			<td>B000E1FYL0</td>
			<td>made up firm, to the consistency of &#39;jigglers&#39;</td>
		</tr>
	</tbody>
</table>

a surgical procedure for resecting the mouse rib: a model for large-scale long bone repair

Este protocolo introduce investigadores a un nuevo modelo para la reparación ósea a gran escala utilizando el nervio de ratón. El procedimiento se detalla lo siguiente: preparación del animal para la cirugía, la apertura de la pared del cuerpo torácica, exponiendo la costilla deseado de los músculos intercostales circundantes, la escisión de la sección deseada de la costilla sin inducir un neumotórax, y el cierre de las incisiones. En comparación con los huesos del esqueleto apendicular, las costillas son muy accesibles. Además, no fijador interno o externo es necesario ya que las costillas adyacentes proporcionan una fijación natural. La cirugía utiliza comercialmente la oferta disponible, es fácil de aprender, y bien tolerado por el animal. El procedimiento puede llevarse a cabo con o sin retirar el periostio, y por lo tanto la contribución del periostio a la reparación se puede evaluar. Los resultados indican que si se mantiene el periostio, la reparación robusta se produce en 1 - 2 meses. Esperamos que el uso de este protocoloestimular la investigación en la reparación de costilla y que los hallazgos facilitarán el desarrollo de nuevas formas de estimular la reparación ósea en otros lugares del cuerpo.

Animales suelen recuperarse rápidamente de este procedimiento, la incisión cicatriza bien, y los animales presentan un comportamiento normal. El procedimiento utiliza los suministros disponibles comercialmente y después de la práctica, se puede completar en menos de 1 hr. Antes de utilizar este procedimiento para generar los datos experimentales, es importante analizar la resección en el punto de tiempo del día 0 para determinar si los fragmentos óseos grandes pueden permanecer dentro de la zona de resección. Un...

Watch this Scientific Journal Video about A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair at JoVE.com

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

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 ...

a-surgical-procedure-for-resecting-mouse-rib-model-for-large-scale

Research

JoVE Journal

Medicine

13.0K vistas.  Keck School of Medicine, University of Southern California. 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.

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

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 ...

Investigación

Medicina

Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs

This is a compact visual description of a combination of surgical technique and device for the delivery of (gene and cell) therapies into the spinal cord. While the technique is demonstrated in the animal, the procedure is FDA-approved and currently being used for stem cell transplantation into the spinal cords of patients with ALS. While the FDA has recognized proof-of-principle data on therapeutic efficacy in highly characterized rodent models, the use of large animals is considered critical for validating the combination of a surgical procedure, a device, and the safety of a final therapy for human use. The size, anatomy, and general vulnerability of the spine and spinal cord of the swine are recognized to better model the human. Moreover, the surgical process of exposing and manipulating the spinal cord as well as closing the wound in the pig is virtually indistinguishable from the human. We believe that the healthy pig model represents a critical first step in the study of procedural safety.

Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application.

Técnica quirúrgica para la entrega de la Médula Espinal de Terapias: Demostración de Procedimiento en Göttingen minipigs

This is a  compact visual description of a combination of surgical technique and device  for the  delivery of (gene and cell) therapies into the spinal ...

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 ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

El Enfoque del ratón Ronda-ventana para ototóxicos Agente de entrega: Una rápida y técnica fiable para la inducción de degeneración de las células cocleares

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

Un Modelo Murino novela de fístula arteriovenosa Fallo: El procedimiento quirúrgico en Detalle

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Angiogénesis quirúrgica en porcino Allotransplantation Tibial: un hueso de Animal grande nuevo vascularizado compuesto Allotransplantation modelo

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

Evaluación del procedimiento para realizar cistometría despierta en un modelo de ratón

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among ...

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus

We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane.

Un procedimiento quirúrgico para la administración de medicamentos para el oído interno en una no humanos primates común tití (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

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 ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

Evaluación histomorfométrica estandarizada de la osteoartritis en un modelo de ratón quirúrgico

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...