Este trabajo fue apoyado por becas de la Asociación de Investigación Neuromuscular CIHR (JNM - 90959, para Jaeb).

Antes de utilizar este modelo, los investigadores deben haber recibido la aprobación para el protocolo quirúrgico del uso de los animales de su institución órgano de gobierno. El modelo es aprobado por la Junta de Ética de la Investigación, Hamilton Health Sciences Corporation, la Universidad McMaster (AUP # 10/04/24) y se lleva a cabo en estricta conformidad con las recomendaciones formuladas por el Consejo Canadiense de los Animales. 1. Ratón Preparación <ol><li> Pesar el ratón. Inducir la anestesia con isoflurano al 5% o 2% de halotano. El circuito utilizado debe garantizar compactación adecuada de la anestesia para proteger el cirujano. Después de 2-3 minutos la respiración del animal se ralentizará. Asegúrese de que el reflejo de parpadeo está ausente y pellizcar los espacios interdigitales de la pata para confirmar la anestesia quirúrgica (es decir, no hay respuesta por el ratón). Aplicar lubricante oftálmica al ojo, para evitar el secado de la córnea durante la cirugía. </li><li> Shave la cara lateral del muslo y la nalga del ciáticoapuntarse a la rodilla y desinfectar con proviodine. Afeitar mantendrá el sitio de la incisión libre de pelo para asegurar una adecuada visualización del campo quirúrgico y para minimizar la interferencia con la disección del nervio y la transección. La escotadura ciática, que es superior y posterior en el fémur, se puede identificar mediante la palpación. </li></ol> 2. Procedimiento Operativo <ol><li> Reducir por inhalación isoflurano al 2% (halotano 1%) y colocar el ratón sobre un lado (el lado destinado a la cirugía hacia arriba), con un microscopio operativo o disección. Alternativamente, la cirugía se puede realizar con lupas quirúrgicas desde ampliación 3.5X es satisfactoria para un adulto (20-25 g) del ratón. </li><li> Ponte los guantes estériles. Identificar la escotadura ciática por palpación. El uso de un escalpelo, incisión de la piel de la cara lateral del muslo de la escotadura ciática a la rodilla (aproximadamente 1 cm). </li><li> Suavemente separa la piel. Identificar el músculo bíceps femoral, que es el músculo superficial plana de la cara lateral del musloinmediatamente debajo de la piel. Con unas tijeras finas, dividir el músculo bíceps femoral a lo largo de las fibras musculares y mantenga abierto con un retractor de primavera para exponer el nervio ciático y sus ramas. </li><li> Identificar el nervio ciático inmediatamente la profundidad del músculo bíceps femoral. Puede ser identificado por su color blanco brillante característico y es de aproximadamente 0,8 mm de diámetro. Se extiende desde la escotadura ciática a la rodilla, se ramifican en los nervios tibiales, peroneo y sural a nivel de la fosa poplítea. </li><li> Separar suavemente el tibial de las ramas del nervio peroneo y sural con fórceps ultrafinas y primavera tijeras microdissecting. El nervio tibial es la rama más grande y suele ser central. Es importante no aplastar el nervio mientras que separa las ramas. Sosteniendo el nervio sólo en la capa adventicial exterior con las pinzas ultrafinas, y mantener la holgura del nervio (no enseñados), evitará aplastamiento del nervio y la lesión por tracción. </li><li> Para obtener información completa y duradera denervatien, cortar el nervio tibial con microdissecting tijeras como distalmente como sea posible, evitando cuidadosamente los vasos poplíteos. Alternativamente, para la denervación temporal con reinervación completa esperada en 2-4 semanas, el nervio tibial puede simplemente ser aplastado con fórceps ultrafinas de 15 segundos en lugar de seccionado. (Nervios periféricos vuelven a crecer después de la lesión y se reinervar músculo deseado.) </li><li> Si se requiere la denervación completa, suturar el extremo del nervio tibial cortado transversalmente a la superficie anterior del músculo bíceps femoral con nylon 10-0 y volver a aproximar el bíceps femoral con Vicryl 5-0 para evitar aberrante de re-inervación de los gastrocnemio y músculos sóleo. </li><li> Cierre la piel con una sutura de Vicryl 5-0 correr. </li></ol> 3. Cuidado operativo del poste <ol><li> Apague la anestesia inhalatoria, pero mantener el flujo de oxígeno. Administrar buprinorphine (o sustituto) analgésico por vía subcutánea. </li><li> Transferir el ratón a una jaula limpia sinropa de cama, mientras que despierta de la anestesia. Mantener en una manta de calentamiento dentro de la jaula y bajo observación directa hasta deambular. </li><li> Transferencia y la casa en la jaula inferior suave (no cable) con amplia ropa de cama blanda. </li><li> Inspeccionar la extremidad operativa diaria para la condición de la herida quirúrgica y el pie para el desarrollo de las úlceras por decúbito talón o las pruebas de la masticación. Los problemas menores pueden ser tratados con antibióticos o antisépticos tópicos como proviodine. Indicadores de punto final que requieren la eutanasia de animales son la pérdida de peso, la evidencia de autocuidado deficiente (piel rizada) y postura encorvada. Además, los animales con mayor apertura de la herida o úlceras que no se curan en 1-2 semanas con antibióticos tópicos o parece tener dolor deberán ser sacrificados. </li></ol> 4. Gastrocnemius Denervado y cosecha del músculo sóleo <ol><li> En el punto de tiempo después de la operación deseada, pesar el ratón y el sacrificio con sobredosis de CO 2. </li><li> Shave el aspecto medial de tanto laoperativa y las piernas de control contralateral y limpio con alcohol. Coloque el ratón en un microscopio operativo o disección, o alternativamente usar lupas quirúrgicas para la ampliación de visualización. </li><li> En la extremidad operativa, una incisión en la piel pierna medial con un bisturí desde el tobillo hasta la rodilla y circunferencialmente alrededor del tobillo. Tire suavemente la piel con pinzas fuera del músculo y proximalmente hacia el muslo. Esto expone a todos los músculos de la pierna. Identificar el músculo gastrocnemio, que es el músculo de la pantorrilla que se extiende desde la rodilla hasta el tobillo en la cara posterior de la pierna y se encuentra inmediatamente debajo de la piel. Identificar la inserción distal del músculo bíceps femoral, proximal al músculo gastrocnemio en la cara medial de la rodilla. En su inserción distal del bíceps femoral aparece delgada y transparente y se superpone a la porción más proximal del gastrocnemio. Con unas tijeras y disección extremo romo, separar suavemente la inserción distal del bíceps femoral del gasmúsculo gastrocnemio. </li><li> En su inserción distal en el calcáneo, el músculo gastrocnemio se estrecha en el tendón de Aquiles. Identificar el tendón de Aquiles, que aparece blanco y musculoso. Sostenga el tendón de Aquiles con unas pinzas, teniendo cuidado de no agarrar ni triture los músculos gemelos, y dividir el tendón de Aquiles de la inserción del calcáneo con unas tijeras. </li><li> Sin soltar el tendón, levante suavemente el músculo gemelo (rojo claro) fuera el sóleo profundas (más rojo), a partir de la inserción distal a su origen en la rodilla (el sóleo se pueden cosechar por separado). </li><li> Diseccionar el gastrocnemio de la pierna dividiendo el origen de los gastrocnemio de los chondyles fémur medial y lateral con unas tijeras. Tracción muy suave en el músculo facilita este proceso. Tenga cuidado de no aplastar el músculo. </li><li> Los sóleo ahora serán claramente visibles, inmediatamente debajo del sitio de los gastrocnemio. Levante el sóleo de su inserción en el tendón de Aquiles de su oigin en la pantorrilla posterior. Si el sóleo se eleva inadvertidamente con el gastrocnemio, suavemente separarlo de la muestra cosechada. El gastrocnemio (rojo claro) y sóleo (rojo oscuro) siguen siendo fácilmente identificables en la muestra recolectada, debido a sus diferencias de color. </li><li> Pesar los músculos por separado en una balanza de precisión. </li><li> Dividir los músculos verticalmente, un medio para la congelación en nitrógeno líquido a presión (para su posterior proteínas y / o extracción de ARN), y un medio para la histología (es decir, evaluación morfométrica, inmunohistología) que se fijan ya sea en 10% de formalina, o la fijación congelados en isopentano enfriado con nitrógeno líquido , si lo deseas. </li><li> Repita desde el paso 4.3 en el control, no operado, de lado a cosechar gemelo de control y los músculos sóleo. </li></ol>

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No hay conflictos de interés se declaran.

El modelo de transección del nervio tibial de la atrofia del músculo esquelético denervación inducida es un modelo comúnmente empleado y bien validado en ratas. Hemos adaptado este modelo para su uso en los ratones, lo que permite al investigador para tomar ventaja de la existencia de ratones modificados genéticamente y estudiar el proceso de la atrofia muscular in vivo en ausencia de proteínas cruciales para la regulación de la masa muscular 7,8. Los músculos gemelos y sóleo, tanto desnerv...

Denervación del músculo esquelético, debido a una lesión traumática de nervios periféricos, la enfermedad o la intervención farmacológica, resulta en la pérdida inmediata de la función contráctil muscular voluntario. Muscle concomitantemente comienza a atrofiarse y esta atrofia es reversible si reinervación oportuna y de buena calidad produce 1,2. En la ausencia de reinervación, atrofia miofibrilar progresa, y cambios biológicos irreversibles en el músculo se produce con la fibrosis muscular y muerte miofibras. Aquí se demuestra el modelo de transección del nervio tibial, un modelo de la denervación inducida por la atrofia muscular y la fibrosis en los ratones. Este modelo permite a los científicos estudiar los mecanismos biológicos fisiológicos, celulares y moleculares que subyacen a la atrofia muscular in vivo en los músculos gastrocnemio y sóleo. Aunque se han utilizado históricamente predominante en ratas, más reciente aplicación de este modelo a nocaut y las líneas de ratones transgénicos en concreto, permite a los investigadores para evaluar el papel de la, las proteínas (s) de interés en la inducción, el desarrollo y el mantenimiento, o, alternativamente, la resolución de, la atrofia muscular y la fibrosis in vivo. El nervio tibial es un nervio periférico del motor-sensorial mixta en la extremidad posterior de roedores, y es una de las tres ramas terminales del nervio ciático. Transección del nervio tibial desnerva el gastrocnemio, sóleo y plantar (músculos y los tres músculos flexores profundos y pequeños de los pies, incluyendo tibial posterior, flexor largo de los dedos y flexor largo del dedo gordo hallicus), y es un modelo bien estandarizado y validado en ratas 3,4 . Los músculos gastrocnemio y sóleo pueden ser fácilmente diseccionados en puntos de tiempo de serie Publicación de transección del nervio tibial, fijaron y procesaron para la evaluación de la histología muscular y morfometría de la fibra muscular, o el flash congelado para la extracción de ARN y de proteínas musculares para el propósito de estudiar, por ejemplo, las redes de señalización celular que regulan la atrofia muscular. El gamuscular strocnemius es un tipo de fibra muscular mixta (de tipo I y de tipo II, aunque predominantemente de tipo II) y el músculo sóleo se compone de una gran proporción de fibras de tipo I, proporcionando de este modo contracción muscular a la vez rápido y lento para la evaluación de 5,6. El modelo de transección del nervio tibial es adecuado para estudiar el proceso de denervación inducida por la atrofia muscular, tanto a corto plazo (días) 7 y largo plazo (semanas a meses) 4,8. En contraste con el modelo de transección del nervio ciático (un segundo modelo de la denervación inducida por la atrofia muscular comúnmente utilizado en roedores), la transección del nervio tibial induce una menor morbilidad en el animal, por lo que es un modelo más atractivo. Transección del nervio ciático desnerva todos los músculos de la pierna (debajo de la rodilla) y el pie, mermando la capacidad del animal para caminar 2, mientras que la transección del nervio tibial deja las ramas del nervio peroneo y sural del nervio ciático intacto, conservando asísus músculos en cuestión y territorios sensoriales. El ratón es incapaz de flexión plantar o invertir el pie, pero es capaz de deambular fácilmente y el peso lleva igualmente en ambas extremidades traseras, disminuyendo así significativamente la morbilidad del modelo. Estudios de análisis de la marcha que evalúan los patrones de caminar se han realizado en ratas después de lesiones del nervio tibial y ciático y demostrar que la huella y la carga de peso se conserva mejor con una lesión tibial 9,10. Además, en el modelo de transección del nervio tibial, el nervio peroneo puede ser movilizado en un punto de tiempo más tarde y se transfiere como una fuente de reinervación retardada, si el diseño del estudio requiere 3. En contraste, la reinervación retardada en el modelo de transección del nervio ciático hace necesario el uso de un injerto de nervio al déficit nervio ciático, muy significativamente el aumento de la dificultad técnica del modelo y la limitación de su uso para cirujanos expertos. Mientras que el modelo de transección del nervio tibial requires familiaridad del operador con la técnica quirúrgica estéril en cirugía animal, tanto el nervio tibial y los músculos de la pantorrilla que inerva son fácilmente accesibles e identificables para la manipulación, de manera que las personas que no son cirujanos, o una amplia experiencia con la cirugía animal, pueden dominar fácilmente este modelo .

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents and Materials</td>
		</tr>
		<tr>
			<td>10-0 Nylon suture</td>
			<td>Ethicon</td>
			<td>2850G</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 Vicryl suture</td>
			<td>Ethicon</td>
			<td>J553G</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Spring microdissecting scissors</td>
			<td>Fine Surgical Tools</td>
			<td>15021-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra fine forceps</td>
			<td>Fine Surgical Tools</td>
			<td>11370-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Non locking micro needle holder (driver)</td>
			<td>Fine Surgical Tools</td>
			<td>12076-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring retractor</td>
			<td>Fine Surgical Tools</td>
			<td>17000-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

tibial nerve transection - a standardized model for denervation-induced skeletal muscle atrophy in mice

El modelo de transección del nervio tibial es un bien tolerado, validado, y reproducible modelo de atrofia del músculo esquelético inducida por desnervación en roedores. Aunque originalmente desarrollado y utilizado ampliamente en la rata, debido a su tamaño más grande, el nervio tibial en ratones es lo suficientemente grande que pueda ser manipulado fácilmente, ya sea con aplastamiento o transección, dejando las ramas del nervio peroneal y sural del nervio ciático intacta y preservando así sus músculos en cuestión. Por lo tanto, este modelo ofrece las ventajas de la inducción de una menor morbilidad y impedimento de la deambulación que el modelo de transección del nervio ciático y también permite a los investigadores estudiar los mecanismos biológicos fisiológicos, celulares y moleculares que regulan el proceso de la atrofia muscular en ratones diseñados genéticamente. El nervio tibial suministra el gastrocnemio, sóleo y plantar los músculos, por lo que su corte transversal permite el estudio de los músculos esqueléticos desnervado compuesto por fibras de contracción rápida de tipo II y / o de tipo I de contracción lentafibras. Aquí se demuestra el modelo de transección del nervio tibial en el C57Black6 ratón. Evaluamos la atrofia del músculo gastrocnemio, como representante de un músculo, en 1, 2, y 4 semanas después de la denervación mediante la medición de los pesos musculares y del tipo de área de la sección transversal de la fibra específica en parafina secciones histológicas immunostained para la miosina rápida contracción.

<html> Transección del nervio tibial desnerva el gastrocnemio, sóleo y plantar los músculos de la pantorrilla. Aquí se evalúa el desarrollo de la atrofia en los músculos gemelos, como un músculo representativo. Músculo gastrocnemio fue cosechada de 2-3 meses de edad C57Black 6 ratones (Jackson Laboratories) denervado para 1, 2, o 4 semanas. Pesos musculares disminuyen progresivamente (Figura 1), al igual que el área de sección transversal de las fibras de tipo II musculares de contracción ráp...

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Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

El modelo de transección del nervio tibial es un bien tolerado, validado, y reproducible modelo de atrofia del músculo esquelético. El modelo de protocolo quirúrgico se describe y se demuestra en C57Black6 ratones.

Transección del nervio tibial - Un modelo normalizado para la atrofia del músculo esquelético denervación inducida en ratones

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

tibial-nerve-transection-standardized-model-for-denervation-induced

Research

JoVE Journal

Medicine

24.3K vistas.  St Michaels Hospital.  El modelo de transección del nervio tibial es un bien tolerado, validado, y reproducible modelo de atrofia del músculo esquelético. El modelo de protocolo quirúrgico se describe y se demuestra en C57Black6 ratones. 

Video: Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

Myo-mechanical Analysis of Isolated Skeletal Muscle

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

Myo-mecánico Análisis de músculo esquelético aislado

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

Investigación

Medicina

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. 
The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn&#x2019;t require expert microsurgical skills.
The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. 
As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.

The Spared Nerve Injury SNI Model of Induced Mechanical Allodynia in Mice

The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

La lesión del nervio Salvados (SNI) Modelo de alodinia mecánica inducida en ratones

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared ...

Skeletal Muscle Gender Dimorphism from Proteomics

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

Músculo Esquelético dimorfismo de género de Proteómica

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

El nervio ciático esposar modelo de dolor neuropático en ratones

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

Procedimientos de biopsia de músculo esquelético humano utilizando la técnica de Bergström Modificado

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

A-crítico tamaño femoral Defecto sencillo modelo en ratones

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

Axotomía nervio facial en ratones: un modelo para estudiar las motoneuronas respuesta a la lesión

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Evaluación Ultrasonido base de la arteria coronaria de flujo y la reserva coronaria Utilizando la sobrecarga Modelo Presión en ratones

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis using the Weil-Blakesley conchotome.

Humano<em&gt; vasto externo</em&gt; Esquelético biopsia muscular utilizando el Weil-Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

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 ...