Name: a microbiomechanical system for studying varicosity formation and recovery in central neuron axons
Uploaded: 2018-04-30T13:00:00.000+00:00
Duration: 9 min 58 s
Description: Watch this Scientific Journal Video about A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons at JoVE.com

Todos los experimentos con animales se ha realizado según los institutos nacionales de Salud Animal uso directrices. Este trabajo fue financiado en parte por las subvenciones de los institutos nacionales de salud (R01NS093073 y R21AA024873) a C. Gu.

<ol>
	<li>Niki&#263;, I., 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=A+reversible+form+of+axon+damage+in+experimental+autoimmune+encephalomyelitis+and+multiple+sclerosis.">A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis.</a> Nat. Med. 17, 495-499 (2011).</li><li>Yang, J., 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=Regulation+of+axon+degeneration+after+injury+and+in+development+by+the+endogenous+calpain+inhibitor+calpastatin.">Regulation of axon degeneration after injury and in development by the endogenous calpain inhibitor calpastatin.</a> Neuron. 80 (5), 1175-1189 (2013).</li><li>Debanne, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Information+processing+in+the+axon.">Information processing in the axon.</a> Nat. Rev. Neurosci. 5, 304-316 (2004).</li><li>Gu, Y., Jukkola, P., Wang, Q., Esparza, T., Zhao, Y., Brody, D., Gu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+of+varicosity+initiation+in+central+neuron+mechanosensation.">Polarity of varicosity initiation in central neuron mechanosensation.</a> J Cell Biol. 216 (7), 2179-2199 (2017).</li><li>Chung, R. S., 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=Mild+axonal+stretch+injury+in+vitro+induces+a+progressive+series+of+neurofilament+alterations+ultimately+leading+to+delayed+axotomy.">Mild axonal stretch injury in vitro induces a progressive series of neurofilament alterations ultimately leading to delayed axotomy.</a> J. Neurotrauma. 22 (10), 1081-1091 (2005).</li><li>Staal, J. A., Dickson, T. C., Chung, R. S., Vickers, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclosporin-A+treatment+attenuates+delayed+cytoskeletal+alterations+and+secondary+axotomy+following+mild+axonal+stretch+injury.">Cyclosporin-A treatment attenuates delayed cytoskeletal alterations and secondary axotomy following mild axonal stretch injury.</a> Dev. Neurobiol. 67 (14), 1831-1842 (2007).</li><li>Tang-Schomer, M. D., Johnson, V. E., Baas, P. W., Stewart, W., Smith, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+interruption+of+axonal+transport+due+to+microtubule+breakage+accounts+for+the+formation+of+periodic+varicosities+after+traumatic+axonal+injury.">Partial interruption of axonal transport due to microtubule breakage accounts for the formation of periodic varicosities after traumatic axonal injury.</a> Exp. Neurol. 233 (1), 364-372 (2012).</li><li>Donkin, J. J., Vink, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cerebral+edema+in+traumatic+brain+injury:+therapeutic+developments.">Mechanisms of cerebral edema in traumatic brain injury: therapeutic developments.</a> Curr Opin Neurol. 23 (3), 293-299 (2010).</li><li>Gardner, A., Jukkola, P., Gu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+of+rodent+hippocampal+neurons+in+culture.">Myelination of rodent hippocampal neurons in culture.</a> Nat Protoc. 7 (10), 1774-1782 (2012).</li><li>Wang, Q., Zhang, X., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromechanical+stimulator+for+localized+cell+loading:+fabrication+and+strain+analysis.">Micromechanical stimulator for localized cell loading: fabrication and strain analysis.</a> J. Micromech. Microeng. 23, 015002 (2013).</li><li>Lu, Y. B., 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=Viscoelastic+properties+of+individual+glial+cells+and+neurons+in+the+CNS.">Viscoelastic properties of individual glial cells and neurons in the CNS.</a> Proc. Natl. Acad. Sci. USA. 103 (47), 17759-17764 (2006).</li><li>Elkin, B. S., Azeloglu, E. U., Costa, K. D., Morrison, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+heterogeneity+of+the+rat+hippocampus+measured+by+atomic+force+microscope+indentation.">Mechanical heterogeneity of the rat hippocampus measured by atomic force microscope indentation.</a> J. Neurotrauma. 24 (5), 812-822 (2007).</li><li>Franze, K., 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=Neurite+branch+retraction+is+caused+by+a+threshold-dependent+mechanical+impact.">Neurite branch retraction is caused by a threshold-dependent mechanical impact.</a> Biophys. J. 97 (7), 1883-1890 (2009).</li><li>Camp&#224;s, O., 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=Quantifying+cell-generated+mechanical+forces+within+living+embryonic+tissues.">Quantifying cell-generated mechanical forces within living embryonic tissues.</a> Nat. Methods. 11 (2), 183-189 (2014).</li></ol>

Los autores no tienen nada que revelar.

El procedimiento de este ensayo microbiomechanical es hacia adelante. Va a producir resultados confiables, si se realizan cuidadosamente todos sus pasos. Hay varios pasos fundamentales que, si se realiza incorrectamente, dificultará la recopilación de datos exitosa. Los pasos críticos comienzan aguas arriba de la aplicación real del estímulo que sopla. Disección cuidadosa, cultivo y cuidado de la cultura de la neurona primaria son primordiales. Si las neuronas cultivadas no son saludables, no reaccionarán, ya que ...

Formación de várices o inflamación o abalorios, a lo largo de los axones, es una característica prominente de la neurodegeneración observada en muchos trastornos o lesiones del sistema nervioso central, incluyendo esclerosis múltiple, enfermedad de Alzheimer, enfermedad de Parkinson y traumática cerebro lesiones1,2. A pesar de los significativos impactos fisiológicos de várices axonales en la propagación del potencial de acción y transmisión sináptica3, cómo se generan las várices se desconoce. Recientemente, usando un análisis microbiomechanical de reciente creación en neuronas hippocampal cultivadas de los roedores, encontramos que los estímulos mecánicos pueden inducir várices en estas neuronas con muy interesantes características. En primer lugar, la inducción de várices es rápida (< 10 s) y este proceso es reversible de forma inesperada. En segundo lugar, iniciación de varices depende de la fuerza que sopla a presión: cuanto mayor sea la presión, más rápido la iniciación. En tercer lugar, iniciación de varices depende de edad neuronal. Los axones de las neuronas más jóvenes aparecen más sensibles a la tensión mecánica, en comparación con los de las neuronas más viejas. En cuarto lugar, la forma de várices a lo largo de los axones de las neuronas hipocampales, dendritas y segmentos inicial del axón de estas neuronas no muestran ningún cambio bajo la misma condición que sopla. Por lo tanto, nuestro estudio reveló una característica novedosa de polaridad neuronal. Estos resultados con el sistema in vitro son fisiológicamente relevantes. Usando un modelo en vivo para lesiones traumáticas leves del cerebro (mTBI), demostramos que várices axonales desarrollaron de una manera multi-focal en la corteza somatosensorial de los ratones inmediatamente después del impacto de cierre-cráneo, consistente con nuestro en vitro resultados4. Es importante tener en cuenta que la tinción y proyección de imagen de los ratones de la LCT sólo proporcionan un tiro rápido de cambios morfológicos neuronales, desde realizar en vivo Time-lapse la proyección de imagen de morfología neuronal durante un impacto mecánico es todavía no es factible.
Este sistema de líquido soplar nos permitió no sólo para capturar características únicas asociadas con la formación de várices inducida por estrés mecánico, sino también para determinar el mecanismo subyacente. Probando diferentes soluciones extracelulares, los bloqueadores y abridores de diferentes candidatos de mechanosensitive canales del ion y electrofisiología celular, identificamos que el catión potencial transitorio del receptor canal de miembros de la subfamilia V 4 (TRPV4) canal que es permeable al Ca2 + y Na+ y activado por soplar es principalmente encargada de detectar el estrés mecánico inicial durante la formación de várices axonal4. Esto fue confirmado más con un enfoque de nocaut de siRNA. Tomados en conjunto, este nuevo sistema de análisis que hemos desarrollado con cultura de neurona del hipocampo, es muy valiosa para el estudio de las propiedades de la micromecánica de las neuronas centrales, especialmente en combinación con otras técnicas.
Este sistema de micromechanical que hemos establecido es única y difiere de los sistemas previamente existentes en varios aspectos importantes. En primer lugar, en este sistema, las neuronas experimentan estrés mecánico fuera de plano en las formas de compresión y corte. Durante el impacto mecánico, procesos neuronales permanecen adheridos a la superficie del cubreobjetos y no se mueven. Esto difiere de otros sistemas que participan principalmente de flexión y estiramiento en el plano experimental (o tensión), por ejemplo, la desviación de axones incluidas como mover cuerdas5,6 o estiramiento de axones en micropatterned canales y membranas elástica7,8. Por otra parte, aunque las várices axonales pueden ser también inducidas en estos ensayos como en nuestro sistema que sopla el fluido, el proceso en estos ajustes toma mucho más tiempo (de 10 minutos a varias horas6,7,8) y aparece irreversible. Por último, nuestro sistema usando soplar líquido local permite el examen de las características espaciales de la formación de várices (e.g., dendritas, espinas dendríticas, soma, segmentos iniciales axonales, terminales axonales), además de sus características temporales. Mediante este sistema, descubrimos varias características inesperados y únicos de la formación de várices axonal, especialmente Inicio rápido lenta reversibilidad y polaridad de axón-dendrita.
El sistema que comentaremos en este artículo es compatible con muchas de las técnicas de molecular y biología de la célula. Por ejemplo, para estudiar los efectos del estrés mecánico sobre la morfología neuronal y función, puede ser utilizado junto con cocultivo de mielina, Time-lapse de imágenes de fluorescencia de reflexión interna total (TIRF) y de transferencia de energía de resonancia fluorescente (FRET) proyección de imagen de calcio y parche abrazadera grabación. En este artículo, nos centramos en los componentes del sistema. Cultura de neuronas hippocampal, líquido soplar configuración, alta resolución imágenes de Time-lapse para transporte axonal y proyección de imagen de calcio se ilustran paso a paso a continuación.

<table><tbody><tr><td>12 mm coverslips&amp;nbsp;</td><td>Warner Instruments&amp;nbsp;</td><td>64-0702</td><td>for 24-well plate&amp;nbsp;</td></tr><tr><td>25 mm coverslips&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>12-545-102&amp;nbsp;</td><td>for 6-well plate&amp;nbsp;</td></tr><tr><td>Acetic acid</td><td>Fisher Scientific&amp;nbsp;</td><td>A38-212</td><td></td></tr><tr><td>Poly-D-lysine</td><td>Sigma&amp;nbsp;</td><td>P6407</td><td></td></tr><tr><td>Rat tail collagen&amp;nbsp;</td><td>Roche&amp;nbsp;</td><td>11 179 179 001&amp;nbsp;</td><td></td></tr><tr><td>10X PBS&amp;nbsp;</td><td>National Diagnostics&amp;nbsp;</td><td>CL-253</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific&amp;nbsp;</td><td>S373-500</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific&amp;nbsp;</td><td>P304-500</td><td></td></tr><tr><td>HEPES&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>BP410-500</td><td></td></tr><tr><td>D-glucose&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>D16-500</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific&amp;nbsp;</td><td>BP214-500</td><td></td></tr><tr><td>NaOH</td><td>Fisher Scientific&amp;nbsp;</td><td>SS255-1</td><td></td></tr><tr><td>Protease enzyme&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>P4032</td><td></td></tr><tr><td>FBS</td><td>Gibco&amp;nbsp;</td><td>26140</td><td></td></tr><tr><td>Sodium pyruvate&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>11360-070</td><td></td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>25030081</td><td></td></tr><tr><td>Penicillin/Streptomycin 100x (P/S)</td><td>Gibco&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>MEM Earle&#039;s Salts&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>11090</td><td></td></tr><tr><td>B27 supplement&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>17504-044</td><td></td></tr><tr><td>Neurobasal&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>21103-049</td><td></td></tr><tr><td>Arabinosylcytosine (Ara-C)</td><td>Sigma&amp;nbsp;</td><td>147-94-4</td><td></td></tr><tr><td>Opti-MEM media&amp;nbsp;</td><td>Gibco&amp;nbsp;</td><td>31985-070</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Invitrogen&amp;nbsp;</td><td>1854313</td><td>transfection reagent&amp;nbsp;</td></tr><tr><td>Borosilicate rods&amp;nbsp;</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>PG52151-4</td><td>for puffing pipette&amp;nbsp;</td></tr><tr><td>rubber tubing&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>14-169-1A</td><td></td></tr><tr><td>10cc plastic syringe and plunger</td><td>Becton Dickinson&amp;nbsp;</td><td></td><td></td></tr><tr><td>micromanipulator&amp;nbsp;</td><td>Sutter Instruments&amp;nbsp;</td><td></td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>S640-3</td><td></td></tr><tr><td>KCl&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>BP366-500</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific&amp;nbsp;</td><td>C70-500</td><td></td></tr><tr><td>Cell culture dish (35 mm x 10 mm)</td><td>Corning&amp;nbsp;</td><td>3294</td><td></td></tr><tr><td>Fluo-4 AM</td><td>Molecular Probes</td><td>F14201</td><td>for calcium imaging&amp;nbsp;</td></tr><tr><td>Mito-YFP construct&amp;nbsp;</td><td>Takara Bio Inc.&amp;nbsp;</td><td></td><td>for cell transfection&amp;nbsp;</td></tr><tr><td>YFP-N1 construct&amp;nbsp;</td><td>Takara Bio Inc.&amp;nbsp;</td><td></td><td>for cell transfection&amp;nbsp;</td></tr><tr><td>Model P-1000 Flaming/Brown Micropipette puller&amp;nbsp;</td><td>Sutter Instruments&amp;nbsp;</td><td></td><td></td></tr><tr><td>Eclipse TE2000-U Mcroscope&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr><tr><td>Plan Fluor ELWD 20x lens&amp;nbsp;&amp;nbsp;</td><td>Nikon</td><td>062933</td><td>objective&amp;nbsp;</td></tr><tr><td>Apo TIRF 100x/1.49 oil lens&amp;nbsp;</td><td>Nikon</td><td>MRD01991</td><td>objective&amp;nbsp;</td></tr></tbody></table>

a microbiomechanical system for studying varicosity formation and recovery in central neuron axons

Várices axonales son estructuras ampliadas a lo largo de los ejes de axones con un alto grado de heterogeneidad. Están presentes no sólo en los cerebros con lesiones o enfermedades neurodegenerativas, sino también en el cerebro normal. Aquí, describimos un sistema de micromechanical recién establecida rápidamente, confiable y reversible inducir várices axonales, lo que nos permite entender los mecanismos que regulan la formación de várices y de composición heterogénea. Este sistema representa un nuevo medio para evaluar los efectos del estrés de compresión y cizalla en diferentes compartimentos subcelulares de las neuronas, diferentes de otros sistemas en vitro que se centran principalmente en el efecto de estiramiento. Importante, debido a las características únicas de nuestro sistema, recientemente hizo un nuevo descubrimiento que demuestra que la aplicación de fluido a presión puede inducir várices axonales a través de la activación de un canal potencial de receptor transitorio rápido y reversible. Nuestro sistema biomecánico puede utilizarse convenientemente en combinación con perfusión de drogas, proyección de imagen de células vivas, proyección de imagen de calcio y parche abrazadera grabación. Por lo tanto, este método puede ser adoptado para el estudio de canales del ion mechanosensitive, regulación de transporte axonal, dinámica del citoesqueleto axonal, señalización de calcio y cambios morfológicos relacionados con traumatismo craneoencefálico.

Antes de soplar, axones normalmente muestran poca formación de várices. Siguientes soplar con la presión estándar (190 mmH2O altura), el comienzo de axones a desarrollar várices grano-como muchos. La formación de várices es parcialmente reversible, como se muestra por regiones del axón regresan a su estado pre-inflado después de un período de recuperación de 10 minutos (figura 2A-B). Después de un período de recuperaci...

Watch this Scientific Journal Video about A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons at JoVE.com

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Este protocolo describe un acercamiento fluido fisiológicamente relevante, presurizado para la inducción rápida y reversible de las várices en las neuronas.

Un sistema de Microbiomechanical para estudiar la formación de várices y recuperación en axones de la neurona Central

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

a-microbiomechanical-system-for-studying-varicosity-formation

Research

JoVE Journal

Neuroscience

6.2K vistas.  The Ohio State University. Este protocolo describe un acercamiento fluido fisiológicamente relevante, presurizado para la inducción rápida y reversible de las várices en las neuronas.

Video: A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Mechanical Manipulation of Neurons to Control Axonal Development

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the 10-1000 &mu;dyne range1,2. Force measurements are obtained through observation of the Hookean bending of the glass needles, which are calibrated by a direct and empirical method3. Equipment requirements and procedures for fabricating, calibrating, treating, and using the needles on cells are fully described. The force regimes previously used and different cell types to which these techniques have been applied demonstrate the flexibility of the methodology and are given as examples for future investigation4-6. The technical advantages are the continuous 'visualization' of the forces produced by the manipulations and the ability to directly intervene in a variety of cellular events. These include direct stimulation and regulation of axonal growth and retraction7; as well as detachment and mechanical measurements on any type of cultured cell8.

Application and direct measurements of forces on neurons in the 2-1000 microdyne range are achieved with high precision using calibrated glass needles. This methodology can be used to control and measure several aspects of axonal development, including axonal initiation, axonal tension, velocity of axonal elongation, and force vectors.

La manipulación mecánica de las neuronas para controlar el desarrollo axonal

Cell manipulations and extension of neuronal axons can be accomplished with calibrated glass micro-fibers capable of measuring and applying forces in the ...

Investigación

Neurociencias

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Laser Nanocirugía del cerebelo Los axones<em&gt; En Vivo</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Una<em&gt; Ex Vivo</em&gt; Médula espinal inducida por láser Lesiones modelo para evaluar los mecanismos de degeneración axonal en tiempo real

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.

Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury.

Imaging intravital de axonales Interacciones con macrófagos y microglia en un ratón Dorsal Crush Columna Lesión

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Rewiring Neuronal Circuits: Un Nuevo Método Para La Extensión Neurítica Rápida Y La Conexión Neuronal Funcional

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases. More specifically the neuromuscular junction (NMJ) in Drosophila has proven to be an effective system to study neuromuscular diseases because of the ability to analyze the structural connections between the neuron and the muscle. Here, we report on an in vivo motor neuron injury assay in Drosophila, which reproducibly induces neurodegeneration at the NMJ by 24 h. Using this methodology, we have described a temporal sequence of cellular events resulting in motor neuron degeneration. The injury method has diverse applications and has also been utilized to identify specific genes required for neurodegeneration and to dissect transcriptional responses to neuronal injury.

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Here we describe a simple and widely accessible method to injure segmental nerves in Drosophila larvae to visualize and quantify neurodegeneration of motor neurons at the neuromuscular junction (NMJ) of third instar larvae.

Una Metodología Neuronal Simple de Lesiones Mecánicas para Estudiar<em&gt; Drosophila</em&gt; Degeneración de Neuronas Motoras

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

Un modelo de lesión de Drosophila en Vivo para el estudio de Neurorregeneración en el periférico y sistema nervioso Central

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Medicina

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

In vivo Evaluación de la dinámica y orientación de los microtúbulos en las neuronas caenorhabditis elegans

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

Ampliación del kit de herramientas para imágenes in vivo del transporte axonal

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

Inducing Axonal Varicosities with a Micromechanical Stimulus on Neurons

Source: Servello, D., et al. <a href="https://app.jove.com/v/57202">A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons.</a> J. Vis. Exp. (2018).
This video demonstrates the induction of axonal varicosities by applying micromechanical stimulus on neurons. The system comprises a micropipette attached to a syringe containing buffer via tubing. Using a micromanipulator, the micropipette is positioned above the neurons. Fluid pressure is then applied to induce varicosities in axons.

Un sistema de Microbiomechanical para estudiar la formación de várices y recuperación en axones de la neurona Central

Resumen

Explorar más videos

Capítulos en este video

La manipulación mecánica de las neuronas para controlar el desarrollo axonal

Laser Nanocirugía del cerebelo Los axones

Una

Imaging intravital de axonales Interacciones con macrófagos y microglia en un ratón Dorsal Crush Columna Lesión

Rewiring Neuronal Circuits: Un Nuevo Método Para La Extensión Neurítica Rápida Y La Conexión Neuronal Funcional

Una Metodología Neuronal Simple de Lesiones Mecánicas para Estudiar

Un modelo de lesión de Drosophila en Vivo para el estudio de Neurorregeneración en el periférico y sistema nervioso Central

In vivo Evaluación de la dinámica y orientación de los microtúbulos en las neuronas caenorhabditis elegans

Ampliación del kit de herramientas para imágenes in vivo del transporte axonal

Inducción de varices axonales con un estímulo micromecánico en las neuronas