We would like to thank Alex Miller for manuscript revisions. This work was supported with the research grants awarded to M.G. from Cure CMD, Struggle Against Muscular Dystrophy (SAM) and Muscular Dystrophy Association (218938).

1. Tejido Cosecha y congelación de los tejidos musculares <ol><li> La eutanasia del ratón con una sobredosis de isoflurano (2-cloro-2- (difluorometoxi) -1,1,1-trifluoro-etano). Realice este paso en una campana de extracción y el uso de un método de contención secundaria, como un secador o una campana de vidrio que contiene un hisopo de algodón empapado en isoflurano. </li><li> Confirmar la muerte apretando firmemente la almohadilla plantar. Use un método secundario de la eutanasia como la dislocación cervical. </li><li> Humedezca la piel con alcohol al 70% para reducir la adherencia de los cabellos sueltos sobre los músculos. Pelar la piel de la extremidad utilizando unas pinzas finas como el # 5. </li><li> Separar cuidadosamente el músculo de interés (tibial anterior (TA) en este caso) de los huesos por debajo, a partir de tendón y quitar. </li><li> Extirpar el músculo, cubrirlo en octubre (compuesto temperatura óptima de corte), y déjelo sobre un molde de congelación desechables etiquetados. Asegúrese de que el músculo está en su orientación fisiológica normal (cabeza para tail) sin estirar. Alternativamente, congelar el músculo en pequeños cuadrados de espuma de poliestireno y el pin con alfileres de insectos para asegurar la longitud correcta de los músculos. </li><li> Chill 2-metilbutano (isopentano) en un vaso de precipitados de acero usando nitrógeno líquido. Esto toma en promedio 3-5 min. Mantener la agitación intermitente hasta precipitados blancos comienzan a formarse en la parte inferior y las paredes del vaso de precipitados. NOTA: Cuando esto ocurre, isopentano ha alcanzado la temperatura óptima de congelación (-150 ° C) </li><li> Sumerja los moldes que contienen músculos en el frío 2-metilbutano. Congele pequeños músculos como el diafragma y el extensor largo del dedo dedos (EDL) durante 6-12 segundos y músculos más grandes como el sóleo gastrocnemio (GS) y tríceps de 15 a 20 seg. No congelar los músculos durante demasiado tiempo ya que esto puede conducir a la rotura. </li><li> Transferir los tejidos congelados sobre hielo seco y dejar que se evapore isopentano (tenga en cuenta que este proceso toma en promedio alrededor de 15 a 20 min). Envuelva los tejidos en papel de aluminio y almacenarlos en ultra baja temperatura (-70 ° C a -80 ° C) hasta el corte. </li></ol> 2. Incorporación de los tejidos en octubre preparar Tissue Bloquear <ol><li> Tejidos Transporte en hielo seco para cyrostat para evitar la descongelación. Transferencia de tejido a criostato cámara (establecido a de -20 a -24 ° C para evitar la descongelación completa de los tejidos) y dejar que se equilibren durante 30 min. </li><li> Gire la bahía Peltier de enfriamiento en. Si el criostato no tiene este enfriamiento, utilice aerosol spray de refrigeración para congelar rápidamente la OCT. No deje que el deshielo del músculo en cualquier momento durante la incrustación ya que esto puede resultar en daños por congelación. </li><li> Añadir una fina capa uniforme de octubre en el disco muestra y dejar enfriar. Cuando la mayoría de la OCT se congela en el fondo mientras que todavía líquido en la parte superior, inserte el tejido en la orientación correcta (perpendicular a octubre para las secciones transversales y paralelas a octubre de secciones longitudinales). Utilice aerosol spray de refrigeración para congelar rápidamente octubre Toque la parte unembeded del tejido con el calor extractor y esperar 45 seg. </li><li> Añadir otra capa delgada de octubre alrededor del músculo, rociar con aerosol spray de enfriamiento y extraer el calor como en el paso anterior. </li><li> Mantenga la adición de octubre en pequeños incrementos y rápidamente congelar el octubre mediante una combinación de aspersión y extractor de calor de refrigeración aerosol hasta que el músculo está cubierto. </li><li> Ponga el extractor de calor en la parte superior del bloque de tejido y esperar 5 min. </li></ol> 3. Preparación de las secciones e identificación de las Secciones de Mid-vientre Región de los músculos <ol><li> Montar la muestra sobre la cabeza de la muestra. Apriete la perilla para asegurar el disco. Asegúrese de que el disco de la muestra se encuentra en buen contacto con la cabeza de la muestra. </li><li> Ajuste la temperatura de la cámara a entre -21 y -24 ° C y espere hasta que se alcanza la temperatura programada. </li><li> Ajuste los valores de espesor (7 micras); recortar el bloque mediante el avance o retroceso del bloque usando ajustes gruesos y finos. </li><li> Recoge las seccionesen pre-calentado portaobjetos cargados positivamente presionando la parte superior de la diapositiva en secciones, con la atracción entre cargas opuestas para facilitar la adherencia de las secciones cortadas a la diapositiva. </li><li> Identificar las secciones mediados de vientre mediante la medición de área de sección transversal (CSA) de las secciones. Para ello, el software de imágenes usando en el equipo que está conectado al microscopio. NOTA: Las imágenes de contraste de fase se toman y la circunferencia de todo el músculo se mide a través de una función de seguimiento del programa de software. Esto producirá tanto CSA así como hurón diámetro mínimo (medida de la fibra de tamaño de sección transversal que se expresa como la distancia más pequeña entre dos tangentes paralelas en lados opuestos de una partícula) mediciones. Nota: Cuando las mediciones CSA y diámetro mínimo hurón comienzan a estabilizarse, se ha llegado a mediados del vientre. </li><li> Inventario y archivar las diapositivas a -80 ° C para la tinción futuro. NOTA: octubre no tiene que ser eliminado antes de staininprocedimientos g. Una vez portaobjetos se cubrieron, que están listos para ser manchado inmediatamente. </li></ol> 4. La tinción NOTA: Si bien los procedimientos detallados paso a paso se pueden encontrar en la hoja de datos del producto y se deben seguir los procedimientos de tinción, la optimización del personal puede ser requerido para obtener la tinción óptima. <ol><li> Debido a que los medios de montaje (véase la tabla) no es miscible con agua, deshidratar diapositivas a través crecientes grados de alcohol (2 min cada uno en 70%, 95%, y 100% de etanol) y claro ellos con xileno (100% durante 5 min) para asegurar diapositivas no se vuelven opacas en el tiempo. </li><li> Cubreobjetos secciones y déjelos secar. Imagen con microscopio equipado con software de imágenes. </li></ol>

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History, clinical status, muscle strength, and biopsy findings.</a> AMA J Dis Child. 91, 326-338 (1956).</li><li>Platzer, A. C., Chase, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histologic+Alterations+in+Preclinical+Mouse+Muscular+Dystrophy.">Histologic Alterations in Preclinical Mouse Muscular Dystrophy.</a> The American journal of pathology. 44, 931-946 (1964).</li><li>Mercuri, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+magnetic+resonance+imaging+involvement+in+muscular+dystrophies+with+rigidity+of+the+spine.">Muscle magnetic resonance imaging involvement in muscular dystrophies with rigidity of the spine.</a> Ann Neurol. 67, 201-208 (2010).</li><li>Tasca, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+molecular+signatures+in+magnetic+resonance+imaging-staged+facioscapulohumeral+muscular+dystrophy+muscles.">Different molecular signatures in magnetic resonance imaging-staged facioscapulohumeral muscular dystrophy muscles.</a> PloS one. 7, e38779 (2012).</li><li>Girgenrath, M., Dominov, J. A., Kostek, C. A., Miller, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+apoptosis+improves+outcome+in+a+model+of+congenital+muscular+dystrophy.">Inhibition of apoptosis improves outcome in a model of congenital muscular dystrophy.</a> The Journal of clinical investigation. 114, 1635-1639 (2004).</li><li>Kumar, A., Yamauchi, J., Girgenrath, T., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle-specific+expression+of+insulin-like+growth+factor+1+improves+outcome+in+Lama2Dy-w+mice,+a+model+for+congenital+muscular+dystrophy+type+1A.">Muscle-specific expression of insulin-like growth factor 1 improves outcome in Lama2Dy-w mice, a model for congenital muscular dystrophy type 1A.</a> Human molecular genetics. 20, 2333-2343 (2011).</li><li>Mehuron, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+matricellular+proteins+is+an+early+signature+of+pathology+in+laminin-deficient+muscular+dystrophy.">Dysregulation of matricellular proteins is an early signature of pathology in laminin-deficient muscular dystrophy.</a> Skeletal muscle. 4, 14 (2014).</li><li>Yamauchi, J., Kumar, A., Duarte, L., Mehuron, T., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triggering+regeneration+and+tackling+apoptosis:+a+combinatorial+approach+to+treating+congenital+muscular+dystrophy+type+1+A.">Triggering regeneration and tackling apoptosis: a combinatorial approach to treating congenital muscular dystrophy type 1 A.</a> Human molecular genetics. 22, 4306-4317 (2013).</li><li>Sheriffs, I. N., Rampling, D., Smith, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+wax+embedded+muscle+is+suitable+for+the+diagnosis+of+muscular+dystrophy.">Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy.</a> Journal of clinical pathology. 54, 517-520 (2001).</li><li>Fox, C. H., Johnson, F. B., Whiting, J., Roller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+fixation.">Formaldehyde fixation.</a> Journal of Histochemistry &amp; Cytochemistry. 33, 845-853 (1985).</li><li>Maccarty, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Diagnostic+Reliability+of+Frozen+Sections.">The Diagnostic Reliability of Frozen Sections.</a> The American journal of pathology. 5, 377-380 (1929).</li><li>Shi, S. 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The authors have nothing to disclose.

Histología e inmunohistoquímica se han utilizado como herramientas clave en la comprensión de la manifestación y la progresión de la patología en diversos trastornos neuromusculares. Clásicamente, las biopsias musculares se fijaron por primera vez en formaldehído y luego incrustados con cera de parafina. Si bien la fijación de formaldehído conserva la arquitectura del tejido y permite el almacenamiento y transporte del tejido a temperatura ambiente, también inactiva las enzimas y los entrecruzamientos proteí...

El examen de histología muscular juega un papel crítico en la comprensión de los diferentes tipos de trastornos neuromusculares 1-4. Cuando se combina con otras técnicas, permite a los investigadores para obtener una mejor idea de la manifestación y la progresión de la patología y también pueden ser esenciales para evaluar la eficacia de una intervención terapéutica 5-10. Sin embargo, para ser precisos, los tejidos necesitan ser conservado en la manera apropiada a fin de preservar el estado fisiológico inmediatamente antes de la escisión. Esto requiere gran cuidado durante la extracción, preservación, seccionamiento y tinción. Los tejidos pueden prepararse de dos maneras diferentes para estudio microscópico de luz. El primer método, formol fija en parafina (FFPE) secciones, requiere tejidos que se fijaron en formaldehído al 10% tamponada naturales antes de ser integrado con cera de parafina 11,12. La etapa de fijación ayuda a preservar la morfología mejor, pero también vínculos transversales pr endógenaoteins necesitando por lo tanto los procedimientos de recuperación de antígeno intrincada de inmunotinción y eliminando la posibilidad de tinción enzimática en tales secciones 12. Las secciones congeladas, por otra parte, no tienen que ser pre-fijo o procesados; por lo tanto, las proteínas son capaces de retener conformaciones biológicos nativos 13,14. Además, las secciones congeladas también permiten el tiempo de respuesta rápida, que a veces es necesario, por ejemplo en el diagnóstico intraoperatorio de los sarcomas y otros biopsias quirúrgicas 15. Sin embargo, si no se hace correctamente, este método es propenso a los daños por congelación, que introduce cambios en la arquitectura muscular haciendo las secciones no aptos para el examen histológico. En este trabajo se expondrá el método más eficaz para preparar las biopsias musculares para lograr tinción óptimo para examen adecuado.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cryostat</td>
			<td>Leica CM 1850&#160;</td>
			<td>Leica Microsystems Inc.</td>
			<td>Buffalo Grove, IL 60089 United States, office phone 1-800 248 0123 
			Richard Allen Scientific</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon Eclipse 50i&#160;</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			Olympus</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Nikon Imaging Software Basic Research</td>
			<td>Nikon Instruments Inc.</td>
			<td>1300 Walt Whitman Road, Melville, N.Y. 11747-3064, U.S.A. 
			Phone: +1-631-547-8500 
			Image J, Metamorph</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>14043-220-05</td>
			<td>JD Medical Dist. Co., Inc.</td>
			<td>JD Medical Distrubuting Co., Inc 1923 West Peoria Avenue Phoenix, Arizona 85029</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>4583</td>
			<td>Sakura Inc.</td>
			<td>Sakrua Fintek, USA, Torrence, CA 90501, USA 
			Leica Microsystem</td>
		</tr>
		<tr>
			<td>Freeze It</td>
			<td>23-022524</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Disposable tissue mold</td>
			<td>22-363-554</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Colorfrost plus slides</td>
			<td>9991001</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>650501-4L</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>HC-1300-1GL</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>2-methyl butane</td>
			<td>M 32631-SL</td>
			<td>Sigma-Aldrich</td>
			<td>3050 Spruce Street, St.Louis MO, 63103 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>HC-7001GA</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Cytoseal 280</td>
			<td>8311-4</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Hematoxylin Gill #3</td>
			<td>CS402-1D</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>6766007</td>
			<td>Thermofisher</td>
			<td>790 Memorial Dr, Cambridge, MA, USA, 02139 
			VWR Scientific</td>
		</tr>
	</tbody>
</table>

do's and don'ts in the preparation of muscle cryosections for histological analysis

La evaluación histológica de las biopsias musculares ha servido como una herramienta indispensable en la comprensión del desarrollo y la progresión de la patología de los trastornos neuromusculares. Sin embargo, con el fin de hacerlo, el cuidado apropiado debe ser tomado cuando la escisión y la preservación de los tejidos para lograr tinción óptimo. Uno de los métodos de conservación de tejidos implica la fijación de tejidos en formaldehído y luego incrustarlos con cera de parafina. Este método preserva la morfología bien y permite el almacenamiento a largo plazo a temperatura ambiente pero es engorroso y requiere la manipulación de productos químicos tóxicos. Además, los resultados de fijación en formol antígeno entrecruzamiento, lo que requiere protocolos de recuperación de antígeno por inmunotinción eficaz. Por el contrario, seccionado congelado no requiere la fijación y por lo tanto retiene conformación antígeno biológica. Este método también proporciona una clara ventaja en vuelta rápida alrededor del tiempo, por lo que es especialmente útil en situaciones que requieren evaluación histológica rápida como intraoperaTIVE biopsias quirúrgicas. A continuación se describe el método más eficaz de preparar biopsias musculares para la visualización con diferentes tinciones histológicas e inmunológicas.

La puesta a punto para la cosecha y la congelación de los tejidos se puede ver en la Figura 1. Tejidos extirpados se deben almacenar en una gasa empapada en PBS para evitar el secado (Figura 1A). Cuando se seleccionan los tejidos a ser utilizado para el análisis histológico deben ser sumergidos en OCT y se presentarán en molde crio en la orientación adecuada y lo más cerca a la longitud fisiológica como sea posible para asegurar la morfología exacta (Figura 1B)....

Watch this Scientific Journal Video about Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis at JoVE.com

Do&#039;s and Don&#039;ts in the Preparation of Muscle Cryosections for Histological Analysis

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Qué hacer y qué no hacer en la preparación de Muscle criosecciones de Análisis histológico

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

do-s-don-ts-preparation-muscle-cryosections-for-histological

Research

JoVE Journal

Medicine

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

31.4K vistas.  Boston University. Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Video: Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

Ensayo in vivo del canino función muscular

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

Investigación

Medicina

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Ensayo de alto rendimiento basado en células citometría de flujo para detectar anticuerpos a la N-metil-D-aspartato receptor o dopamina-2 del receptor en suero humano

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

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 ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

Disección del páncreas de ratón para análisis histológico y perfiles metabólicos

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

Examen ecocardiográfico e histológico de la morfología cardiaca en el ratón

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

Evaluación in vivo de las propiedades mecánicas y viscoelásticas de la lengua de rata

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Muestreo y extracción in situ de tumores cerebrales para el análisis de metabolómica y lipidómica

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried out in large animals. Considering the complex anatomy of the heart, interspecies differences, and the types of compensatory and pathological reactions, consistent protocols are challenging to implement. The utilization of multiple dissection protocols is usually adapted to suit the prosector's experience, and personal preference continues to be a source of experimental and interobserver variability. Here, the aim is to present the main anatomical features and landmarks, dissection protocols, and histological sampling standards of the heart in some commonly used species (including dogs, pigs, ruminants, and cats) as models of cardiovascular diseases.
Two standard gross examination protocols are presented here. First, the inflow-outflow method, which follows the physiological blood flow direction through the heart and large vessels (frequently used in dogs, ruminants, and pigs), and second, the four-chamber dissection technique (exemplified in cats). Both techniques can be adapted to any species in certain experimental circumstances. The sampling protocols include all the areas of interest (sinoatrial node, ventricles, interventricular septum, atria, valves, and aorta), and if properly carried out, improve both the reproducibility and reliability of experimental studies.

Due to the complex anatomy, a consistently reproducible dissection and harvesting protocol for cardiac samples can be challenging to implement. This manuscript presents the key elements of some standard cardiac dissection protocols, highlighting both the gross examination approaches and the sampling sites commonly used for histopathologic examination.

Técnicas de disección y muestreo histológico del corazón en grandes modelos animales para enfermedades cardiovasculares

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried ...

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 &#181;m thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation.
Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

Preparación del tejido miocárdico humano para el cultivo a largo plazo

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...