Name: functional and morphological assessment of diaphragm innervation by phrenic motor neurons
Uploaded: 2015-05-25T14:00:00
Description: Watch this Scientific Journal Video about Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons at JoVE.com

This work was supported by the NINDS (grant #1R01NS079702 to A.C.L.) and the SURP Program at Thomas Jefferson University (M.M.).

Los procedimientos experimentales fueron aprobados por el comité de la Universidad Thomas Jefferson cuidado de los animales institucional y el uso y llevaron a cabo en cumplimiento de la Directiva Comunidades Europeas (2010/63 / UE, 86/609 / CEE y 87 a 848 / CEE), la Guía del NIH para el cuidado y uso de animales de laboratorio, y la Sociedad para las Políticas de Neurociencia en el uso de animales en Investigación de Neurociencia. 1. Potenciales de Acción Compuesto musculares (CMAPs) </p...

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Como la función respiratoria se ve comprometida en tanto SCI traumático y la ELA, el desarrollo de terapias que se dirigen a la respiración y, específicamente, la inervación del diafragma son clínicamente relevantes 5,6. Con el fin de estudiar exhaustivamente la función respiratoria, se debe utilizar un método de enfoque combinado. CMAPs miden el grado de inervación funcional de la membrana a través de la estimulación del nervio frénico externa, pero el impulso respiratorio bulboespinal no endóge...

Esclerosis Lateral Amiotrófica (ELA) es una enfermedad de la motoneurona debilitante asociada con la pérdida de las neuronas motoras superiores e inferiores y la consiguiente parálisis muscular. Tras el diagnóstico, la supervivencia del paciente es en promedio de sólo 2.5 años 1. Pérdida frénico motoneurona (PhMN) es un componente crítico de la patogénesis de la ELA. Los pacientes mueren en última instancia debido a la pérdida de PhMN inervación del diafragma, el músculo principal de inspiración 2,3. Lesión medular traumática (SCI) es también un problema serio con dificultades respiratorias asociadas. Aproximadamente 12.000 nuevos casos de SCI se producen cada año 4 debido a daño traumático de la médula espinal. A pesar de la heterogeneidad de la enfermedad con respecto a la ubicación, el tipo y la gravedad, la mayoría de los casos de SCI implica trauma en la médula espinal cervical, que a menudo resulta en compromiso respiratorio debilitante y persistente. Además de ALS y SCI, otro sistema nervioso central (SNC) enfermedades se puede asociar wdisfunción respiratoria diafragmática ITH 5,6. El nervio frénico es un nervio motor eferente que inerva el hemi-diafragma ipsilateral y que se origina en los cuerpos celulares PhMN ubicados en los niveles de C3-C5 del mismo lado de la médula espinal cervical. Salida PhMN es controlado por descender de entrada bulboespinal del tronco cerebral en un área conocida como el grupo rostral ventral respiratoria (rVRG) 7. El circuito rVRG-PhMN-diafragma es central para el control de la respiración de inspiración, así como otros comportamientos de diafragma no ventilatorios. Diversas lesiones traumáticas y enfermedades neurodegenerativas que afectan a este circuito pueden conducir a una profunda disminución de la función respiratoria y la calidad de vida del paciente. Descendente de entrada para PhMNs de la supervivencia rVRG, PhMN, la integridad del nervio frénico y la inervación adecuada en la unión neuromuscular diafragma (NMJ) son todos necesarios para la función normal del diafragma. Por tanto, es importante emplear técnicas quepuede evaluar cuantitativamente este circuito in vivo en modelos de roedores de ALS, SCI y otras enfermedades del SNC. Con este protocolo, el objetivo es describir las herramientas experimentales para evaluar PhMN inervación del diafragma, tanto a nivel electrofisiológico y morfológicos. Los potenciales de acción muscular compuesto (CMAPs) se registran mediante la estimulación de todos los axones de la neurona motora eferente de un nervio motor dado y luego analizar la respuesta de despolarización elicitada de las miofibras de destino. Esta técnica se puede utilizar in vivo en ratas y ratones anestesiados para cuantificar la inervación funcional de la hemi-diafragma por PhMNs 8. Debido al hecho de que CMAPs representan la grabación simultánea de todos (o al menos muchos / la mayoría) miofibras del todo-hemi diafragma, es útil examinar también los fenotipos de los axones motores individuales y miofibras en la UNM diafragma con el fin de realizar un seguimiento de la enfermedad - y terapia relevante cambios morfológicos como parcial y compledenervación te, surgimiento de regeneración y reinervación. Esto se puede lograr a través de todo el montaje inmunohistoquímica (IHC) de la membrana, seguido por la evaluación morfológica detallada de NMJs individuales a lo largo del músculo 9. Combinando CMAPs y análisis UNM proporciona un enfoque poderoso para estudiar cuantitativamente inervación diafragmática en modelos de roedores de enfermedad del SNC y SNP.

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			<td height="21" style="height:21px;width:332px;">Paraformaldehyde</td>
			<td style="width:273px;">Fisher</td>
			<td style="width:131px;">T353-500</td>
			<td style="width:167px;">Make 10% solution first in de-ionized distilled water; make 4% with 1X PBS, adjust pH to 7.4</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:332px;">1X Phosphate Buffered Saline, pH 7.4</td>
			<td style="width:273px;">Invitrogen</td>
			<td style="width:131px;">10010049</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:332px;">2% Bovine serum albumin (2% BSA)</td>
			<td style="width:273px;">Sigma-Aldrich</td>
			<td style="width:131px;">A3059-100g</td>
			<td>Dissolve 2g BSA into 100mL of 1X PBS</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:332px;">0.2% Triton X100 in 2% BSA/PBS (Blocking Buffer)</td>
			<td style="width:273px;">Sigma-Aldrich</td>
			<td style="width:131px;">T9284-100mL</td>
			<td>Dissolve 0.2ml/100mL 2% BSA/PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">0.1M Glycine</td>
			<td style="width:273px;">Sigma-Aldrich</td>
			<td style="width:131px;">G-7126</td>
			<td>Add 0.185g to 25mL of 2% BSA/PBS</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:332px;">&#945;-bungarotoxin</td>
			<td style="width:273px;">Invitrogen</td>
			<td style="width:131px;">T1175</td>
			<td>Concentration 1:400</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:332px;">SMI-312&#160;</td>
			<td style="width:273px;">Sternberger Monoclonals</td>
			<td style="width:131px;">SMI312</td>
			<td>Concentration 1:1,000</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:332px;">SV2</td>
			<td style="width:273px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:131px;">SV2-Supernatant</td>
			<td>Concentration 1:10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">FITC goat anti-mouse IgG1</td>
			<td style="width:273px;">Roche</td>
			<td style="width:131px;">3117731001</td>
			<td>Concentration 1:100</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Silicone rubber</td>
			<td style="width:273px;">Sylgard, Dow Corning</td>
			<td style="width:131px;">Part # 184</td>
			<td>Follow instructions that come with kit: can use multiple sized culture dish (30mm, 60mm, 100mm) depending on needs</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Vectashield fluorescent mounting medium</td>
			<td style="width:273px;">Vector laboratories</td>
			<td style="width:131px;">H-1000</td>
			<td>This is not a hard-set medium. You will need to secure the cover slip with clear nail polish.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Small Spring Scissors</td>
			<td style="width:273px;">Fine Science Tools</td>
			<td style="width:131px;">15002-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Dissection forceps</td>
			<td style="width:273px;">Fine Science Tools</td>
			<td style="width:131px;">11295-51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Software for CMAP recordings</td>
			<td style="width:273px;">Scope 3.5.6; ADI</td>
			<td style="width:131px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Disk surface electrodes</td>
			<td style="width:273px;">Natus neurology</td>
			<td style="width:131px;">019-409000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Subdermal needle electrodes</td>
			<td style="width:273px;">Natus neurology</td>
			<td style="width:131px;">019-453100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Conductive gel</td>
			<td style="width:273px;">Aquasonic&#160;</td>
			<td style="width:131px;">122-73720</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:332px;">Stimulator/recording system for CMAP recordings</td>
			<td style="width:273px;">ADI Powerlab 8SP stimulator&#160;</td>
			<td style="width:131px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:332px;">Amplifier for CMAP recordings</td>
			<td style="width:273px;">BioAMP</td>
			<td style="width:131px;"></td>
			<td></td>
		</tr>
	</tbody>
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functional and morphological assessment of diaphragm innervation by phrenic motor neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Adultos ratas Sprague-Dawley recibieron o bien laminectomía solamente (control no lesionado) o unilateral hemi-contusión SCI a nivel de la médula espinal C4 10-12. A las 5 semanas después de la cirugía, el pico CMAP amplitud registrada de la hemi-diafragma ipsilateral al sitio laminectomía / lesión se redujo significativamente en ratas SCI (Figura 2C) en comparación con el control-laminectomía solamente (Figura 2B). Todos NMJs en el hemi-diafragma estaban completamen...

Watch this Scientific Journal Video about Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons at JoVE.com

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Evaluación funcional y morfológica de Diafragma Inervación por frénico neuronas motoras

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

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