Name: live imaging of dorsal root axons after rhizotomy
Uploaded: 2011-09-01T12:00:00
Description: Watch this Scientific Journal Video about Live Imaging of Dorsal Root Axons after Rhizotomy at JoVE.com

Мы благодарим д-р Алан Тесслер для комментариев и редакционных помощь. Эта работа была поддержана NIH NS062320.

1. Микроскоп настройки и подготовки изображений <ol><li> Наши изображения созданы состоит из Leica MZ16 флуоресцентного стереомикроскопа с быстрым затвором и охлаждением ПЗС-камера управляется программой Метаморф. </li><li> Подготовка термостатом грелку и настроить выход до 32,5 ° С для поддержан...

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	<li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+the+neuromuscular+junction.">Watching the neuromuscular junction.</a> J Neurocytol. 32, 767-775 (2003).</li><li>Bishop, D. L., Misgeld, T., Walsh, M. K., Gan, W. B., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+branch+removal+at+developing+synapses+by+axosome+shedding.">Axon branch removal at developing synapses by axosome shedding.</a> Neuron. 44, 651-661 (2004).</li><li>Balice-Gordon, R. J., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+visualization+of+the+growth+of+pre-+and+postsynaptic+elements+of+neuromuscular+junctions+in+the+mouse.">in vivo visualization of the growth of pre- and postsynaptic elements of neuromuscular junctions in the mouse.</a> J Neurosci. 10, 894-908 (1990).</li><li>Trachtenberg, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+imaging+of+experience-dependent+synaptic+plasticity+in+adult+cortex.">Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex.</a> Nature. 420, 788-794 (2002).</li><li>Pan, F., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+dendritic+spine+development+in+the+mouse+cortex.">Two-photon imaging of dendritic spine development in the mouse cortex.</a> Dev Neurobiol. 68, 771-778 (2008).</li><li>Grutzendler, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+synaptic+plasticity+and+pathology+in+the+living+mouse+brain.">Two-photon imaging of synaptic plasticity and pathology in the living mouse brain.</a> NeuroRx. 3, 489-496 (2006).</li><li>Kerschensteiner, M., Schwab, M. E., Lichtman, J. W., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+axonal+degeneration+and+regeneration+in+the+injured+spinal+cord.">in vivo imaging of axonal degeneration and regeneration in the injured spinal cord.</a> Nat Med. 11, 572-577 (2005).</li><li>Misgeld, T., Nikic, I., Kerschensteiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+single+axons+in+the+mouse+spinal+cord.">in vivo imaging of single axons in the mouse spinal cord.</a> Nat Protoc. 2, 263-268 (2007).</li><li>Davalos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+in+vivo+imaging+of+densely+populated+glia,+axons+and+blood+vessels+in+the+mouse+spinal+cord+using+two-photon+microscopy.">Stable in vivo imaging of densely populated glia, axons and blood vessels in the mouse spinal cord using two-photon microscopy.</a> J Neurosci Methods. 169, 1-7 (2008).</li><li>Maio, D. D. i. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+dorsal+root+regeneration:+Rapid+immobilization+and+presynaptic+differentiation+at+the+CNS/PNS+border.">in vivo imaging of dorsal root regeneration: Rapid immobilization and presynaptic differentiation at the CNS/PNS border.</a> Journal of Neuroscience. 31, 4569-4582 (2011).</li></ol>

Изображений DR регенерации непосредственно в живых мышей является особенно сложным, поскольку он требует существенных спинного ламинэктомии для контроля роста аксонов на большой площади следуют несколько инвазивных хирургических и обезболивающие процедуры в последующих сессиях из?...

<table border="1"><tbody><tr><th> Название реагента </th><th> Компания </th><th> Номер в каталоге </th><th> Комментарии </th></tr><tr><td> H-линия thy1 YFP (2-4 месяцев, либо секс) </td><td> Jackson Laboratory (Bar Harbor, ME) </td><td> 003782 </td><td></td></tr><tr><td> Ксилазин (AnaSed инъекции, стерильный раствор) </td><td> Ллойд Laboratories, (Шенандоа, LA) </td><td> 4811 </td><td> 8 мг / кг </td></tr><tr><td> Кетамин (инъекции кетамина гидрохлорид, USP) </td><td> Hospira, Inc (Лейк-Форест, Иллинойс) </td><td> 2051 </td><td> 120 мг / кг </td></tr><tr><td> Бупренорфин (Buprenex для инъекций) (0,05 мг / кг) </td><td> Reckitt Benckiser Pharmaceuticals Inc (Richmond, VA) </td><td> 7571 </td><td></td></tr><tr><td> Малый машинок для стрижки волос животных </td><td> Остер Professional, (McMinnville, TN) </td><td> 76059-030 </td><td></td></tr><tr><td> Лосьон Удаление волос </td><td> Церкви &amp; Dwight Co (Princeton, NJ) </td><td> НАИР с Детское масло </td><td></td></tr><tr><td> Марлевые губки </td><td> Fisher Scientific, (Питтсбург, Пенсильвания) </td><td> 22-362-173 </td><td></td></tr><tr><td> Хлопковые тампоны </td><td> Fisher Scientific, (Питтсбург, Пенсильвания) </td><td> 14-960-3-й квартал </td><td></td></tr><tr><td> 1 мл шприцев </td><td> Becton, Диксон и компании Франклин озер, NJ) </td><td> 309602 </td><td></td></tr><tr><td> Подкожный (Sub-Q) иглы, 26ga. </td><td> Becton, Диксон и Компания (Франклин озер, NJ) </td><td> 305115 </td><td></td></tr><tr><td> Весна ножницы и щипцы </td><td> Инструменты изобразительных наук, (Foster City, CA) </td><td></td><td></td></tr><tr><td> 2,5-мм изогнутые rongeurs </td><td> Инструменты изобразительных наук, (Foster City, CA) </td><td> 16221-14 </td><td></td></tr><tr><td> Лактата Рингера инъекций USP </td><td> B. Braun медицинской Inc, (Ирвин, Калифорния) </td><td> BBR-L7502 </td><td></td></tr><tr><td> Стерильный физиологический раствор </td><td> APP Pharmaceuticals, (Шаумбург, Иллинойс) </td><td> 918610 </td><td></td></tr><tr><td> Тонкие синтетические мембраны матрицы (Biobrane) </td><td> Bertek Pharmaceuticals, (Morgantown, WV) </td><td> 62794-096-251 </td><td></td></tr><tr><td> Искусственные оболочки </td><td> Гор Предотвращение MVP Dura Substitute, и WL Gore Associates, (Флагстафф, Аризона) </td><td> 1MVP40 </td><td></td></tr><tr><td> 5-0 швов шелка </td><td> Ethicon, Inc (Сомервилл, Нью-Джерси) </td><td> K-580 </td><td></td></tr><tr><td> Рана клипы </td><td> Perfect - Ets Бруно, (Burnea, Франция) </td><td> A75 </td><td></td></tr><tr><td> Флуоресцентные стереомикроскопа </td><td> Leica Microsystems (Wetzlar, Германия) </td><td> MZ16 </td><td></td></tr><tr><td> ПЗС-камера </td><td> Хамамацу (Бриджуотер, Нью-Джерси) </td><td> ORCA-Rx2 </td><td></td></tr><tr><td> Регулятор температуры </td><td> Всемирный точных приборов (Сарасота, Флорида) </td><td> ATC 1000 </td><td></td></tr><tr><td> Метаморф программного обеспечения </td><td> Molecular Devices, (Саннивейл, Калифорния) </td><td></td><td></td></tr><tr><td> Photoshop </td><td> Adobe Systems, San Jose, CA </td><td></td><td></td></tr></tbody></table>

live imaging of dorsal root axons after rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

Watch this Scientific Journal Video about Live Imaging of Dorsal Root Axons after Rhizotomy at JoVE.com

Live Imaging of Dorsal Root Axons after Rhizotomy

<em&gt; В естественных условиях</em&gt; Изображение протокол для мониторинга первичных сенсорных аксонов следующие спинной раздавить корень описано. Процедуры использования широкого поля флуоресцентной микроскопии и thy1-YFP трансгенных мышей, и разрешить повторяющиеся изображения регенерации аксонов более 4 см в ПНС и аксонов взаимодействия с интерфейсом ЦНС.

Живая съемка Спинной Аксоны Корневая после ризотомия

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

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Neuroscience

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Хроматин Иммунопреципитация из ткани спинного Ganglia Корневая следующие аксональное повреждение

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

Генетическое изучение регенерации аксонов с искусственного взрослых нейронов ганглиев

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Живая съемка никотина индуцированной сигнализации кальция и высвобождение нейромедиаторов Наряду вентральной гиппокампа Аксоны

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Спинальный ганглий Injection и дорсальных корешков размозжение как модель для регенерации Сенсорное Axon

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Спинной корень ганглиев изоляции и основной культуры для изучения нейромедиатора релиз

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Спектральная Рефлектометрические микроскопия на Миелинизированные аксоны в Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Следственный млекопитающих Axon регенерации: В Vivo электропорации взрослых мыши Спинной корень ганглия

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

Быстрая изоляция дорсал Корень Ganglion Макрофаги

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

Миелинизация периферических аксонов in vitro в кокультуре ганглиозных эксплантатов дорсальных корешков крыс и шванновских клеток

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

Обогащение культур нейронов ганглиев дорсальных корешков взрослых мышей методом иммунопаннирования

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as ...