我们想感谢帮助安德烈特德斯奇设置的最初在实验室和Ricco林德纳芯片的实验，他的贡献，以微调芯片的条件。这项工作是由Hertie基金会的支持;财富补助金，图宾根大学，和东风集团的DI 1497/1-1补助（授予西蒙娜迪乔万尼）。

1。坐骨神经和背柱神经损伤<ol><li>动物被放置在一个手术巾，它下面的一个thermopad是目前整个过程中鼠标体温保持在37 ° C。所有动物都进行手术麻醉，用一个连续的异氟醚/ O 2的管理。手术器械灭菌前的程序。 </li><li>坐骨神经损伤，这两个躯仔细剃，脱毛是与一般的头发卸妆Betadyne 3倍的酒精清洁皮肤前完成。 </li><li>坐骨神经是由4毫米的切口后，通过皮肤在大腿中部股骨平行和蔓延除了用细镊子股二头肌肌肉暴露。 </li><li>暴露坐骨神经，然后受伤，皮肤与两个缝合剪辑封闭。 </li></ol> 注:同一切口暴露坐骨神经，但封闭的伤口缝合神经假伤的完好。 <ol start="5"><li>背柱损伤，鼠标的背面是经过精心剃光，脱毛是与一般的头发卸妆清洁皮肤用酒精喷/刷前完成。 </li><li>脊髓是暴露一个2.5厘米的切口约T7 T13。皮肤是分开，是沿着从T9 T11椎体删除和结缔组织。 </li><li>一个椎板切除术是在T10的水平</li></ol> 注:椎板切除术对于大多数实验，被认为是深水伤害。 <ol start="8"><li> Xylocain几滴组织麻醉线，硬脑膜被删除，注意不要触摸脊髓。 </li><li>受伤和肌肉缝合关闭，再次注意不要触摸脊髓背列。最后，皮肤缝合与缝合剪辑关闭。丁丙诺啡（0.1毫克/公斤体重）应每天两次48小时或直到不再需要。 </li></ol> 2。交联<ol><li>剖析受伤后euthanization小鼠L4和L5背根神经节。收集到冰冷的HBSS +蛋白酶抑制剂的鸡尾酒（2人受伤，并从每个动物深水DRG的共4个动物）共16 DRG的。 </li><li>短暂离心背根神经节，然后小心地取下的HBSS，加入500μL1％甲醛的PBS +蛋白酶抑制剂的鸡尾酒，并在37℃孵育30分钟的样品° C。 </li></ol> 关键的一步。使用新鲜，分子生物学级甲醛。 <ol start="3"><li>添加甘氨酸125毫米，停止为5分钟，在室温下的固定和孵化。 </li><li>短暂离心，吸出的缓冲区，并组织两次用500μL冰冷的PBS +蛋白酶抑制剂的鸡尾酒。 </li></ol> 3。核准备和染色质剪切<ol><li>吸过PBS中，加400μLSDS裂解液，转移到预冷的离心管，并破坏组织约30招与micropestle。 </li></ol> 关键的一步。裂解和破坏组织的交联的染色质好产量至关重要。 <ol start="2"><li>超声与8个脉冲，10秒的70％输出（Bandelin，Sonoplus GM70）每个样品。 </li></ol> 谨慎。剪切染色质必须为您的特定超声优化设置和适当的染色质分散在运行实际的IP实验之前，应检查（见第3节）。 注意碎裂的染色，也可以由微球菌核酸（MNase）消化。作为一个经验法则，通过超声分散是首选固定的组织，而MNase消化本地组织染色质免疫沉淀的青睐。剪切染色质交联，可存储于-80 ° C至2个月，但避免重复冻结/解冻循环。 4。染色质消化分析（推荐） <ol><li>取出10μL剪切染色质样品进行分析。添加氯化钠200毫米的样品和2H孵育65 ° C的反向交叉链接。 </li><li>净化DNA（见第8节），并运行一个1％的琼脂糖凝胶。 DNA应该是支离破碎，长度约200-1000 bp（图1）。 </li></ol> 谨慎。染色质的碎片可能导致信号下降，同时不完整的碎片会导致降低分辨率和增加背景。 5。免疫沉淀<ol><li>确定免疫反应（见下文附注），并平均分配到新管的样品。使每卷500片内缓冲+蛋白酶抑制剂的鸡尾酒液。 </li><li>取出5μL您的稀释后的样品，并转移到新管。这是1％输入采样，它会被存储在-20 ° C，直到需要（第7条）。 </li><li>对于每个免疫，添加相应的抗体或正常的IgG对照，在4 ° C旋转过夜孵育。 </li></ol> 注意:当确定的免疫，阳性对照（如组蛋白H3抗体）和阴性对照（普通IgG抗体）应予以考虑。数量为每个IP使用不同的抗体和抗体之间必须凭经验确定。但是，它通常是2-10范围内克/免疫。 <ol start="4"><li>为免疫沉淀抗体/蛋白质/ DNA复合物，加入30μl和芯片级的G蛋白的磁珠孵育2 h后，在4 °旋转彗星。 </li></ol> 6。洗涤<ol><li>下拉约束染色珠复杂，放在磁架管。等待，直到解决方案是明确的，然后小心地取下您上清。 </li><li>加入1毫升（片内缓冲）低盐洗珠和孵化3-5分钟旋转4 ° C，然后下拉珠和吸洗步骤6.1。重复这一步共3洗。 </li><li>添加高盐缓冲液洗1毫升（片内缓冲+氯化钠350毫米）的珠子和孵化为3-5分钟旋转，然后下拉珠和吸洗步骤6.1。 </li></ol> 7。洗脱和交联逆转<ol><li>把你的输入样本在-20 ° C，加入150μl和芯片洗脱缓冲液在室温下放在一旁，直到步骤7.5。 </li><li>每个步骤6.3 IP样品添加的1X芯片洗脱缓冲液150μL。 </li><li>放置在thermomixer管和孵化在65℃30分钟温和的震荡样品从珠洗脱染色质。 </li><li>下拉珠磁机架上，并小心地转移到新管洗脱染色（上清）。 </li><li>为了所有的管子，包括您的输入样本，2小时添加NaCl和40微克/蛋白酶K孵育反应65 ° C 200MM。 </li></ol> 注意。洗脱步骤，可以在室温下进行，太多，但它可能是效率不高。 8。 DNA回收<ol><li> 1体积酚/氯仿添加到您的样品从步骤7.5和30秒的旋涡。 </li><li>离心机以最大速度在室温离心5分钟。 </li><li>小心地恢复到新的管水（上）阶段，1体​​积的氯仿添加到水相中，和30秒的旋涡。 </li><li>离心机以最大速度在室温离心5分钟。 </li><li>仔细恢复到新的管水（上）阶段，再加入NaOAc 300MM，冰冷的100％的乙醇20微克/肝糖原的反应，和2.5册。 </li></ol>需要谨慎。载体，此外，如糖 ​​原，提高降水相对小型的DNA片段。 <ol start="6"><li>在-80 ° C孵育2小时，以沉淀DNA。 </li></ol>降水步骤也可以在-20 ° C过夜注意。 <ol start="7"><li>离心机中离心20分钟最高速度的样品在4 ° C。 </li><li>小心取出上清液，用300μL冰冷的70％乙醇沉淀DNA颗粒。 </li><li>离心机最高速度的样品离心10分钟，在4 ° C。 </li><li>小心取出多上清尽可能让颗粒风干。 </li><li>悬浮于20μL无菌H 2 O。样品现已准备好进行PCR或DNA可以储存在-20 ° C </li></ol> 9。代表性的成果一个代表在坐骨神经病变的病种付费的芯片实验结果显示（图1，图2）。首先，我们支离破碎的DNA长度约200-1000 bp（图1）。第二，我们展示了从相关蛋白43（GAP - 43）只有在坐骨神经损伤（泳道4，图2，5&#39;）的增长近端启动子的PCR信号以下芯片。无PCR信号是当动物只收到一个假伤（3巷），以及IP（巷5和6）当使用正常的抗体血清。要测试的特异性抗体，我们相同的DNA样本进行分析，但用于控制的引物，它可以检测在我们没有入住，预计感兴趣的基因3&#39; - UTR一个地区。 PCR信号缺席所有车道（车道3-6，图2，3&#39;），代表作为标准的PCR控制非IP DNA样本，除输入信号，（1-2车道，Figu再2）。这种芯片的程序，可以执行以下任一坐骨神经或脊髓后柱损伤，在原理图（图3）汇总。 <img src="/files/ftp_upload/2803/2803fig1.jpg" alt="图1" /> 图1。超声已被剪切片段长度适当的范围内，扭转了交联的DNA琼脂糖凝胶电泳。 <img src="/files/ftp_upload/2803/2803fig2.jpg" alt="图2" /> 图2。背根神经节组织的半定量PCR结果表明p53的乙酰化结合坐骨神经损伤后48小时内GAP - 43的近端启动子区域的坐骨神经病变。控制PCR方法从同一DRG组织表明，乙酰化P53不绑定到一个位于3&#39; -端非编码区的GAP - 43的基因（S =深水，我=受伤）（UTR）的DNA控制区。 <img src="/files/ftp_upload/2803/2803fig3.jpg" alt="图3" /> 图3。一个图显示坐骨神经病灶部位的位置，和背柱。

<ol>
	<li>Beirowski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+qualitative+analysis+of+Wallerian+degeneration+using+restricted+axonal+labelling+in+YFP-H+mice.">Quantitative and qualitative analysis of Wallerian degeneration using restricted axonal labelling in YFP-H mice.</a> Journal of Neuroscience Methods. 134, 23-35 (2004).</li><li>Carey, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromatin immunoprecipitation (ChIP). , (2009).</li><li>Dahl, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+micro+chromatin+immunoprecipitation+assay+(&amp;mu;ChIP.">A rapid micro chromatin immunoprecipitation assay (&amp;mu;ChIP.</a> Nat. Protoc. 3, 1032-1045 (2008).</li><li>Haring, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation:+optimization,+quantitative+analysis+and+data+normalization.+Plant+Methods.">Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods.</a> 11, 3-11 (2007).</li><li>Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neuronal+chromatin+from+brain+tissue.">Isolation of neuronal chromatin from brain tissue.</a> BMC Neurosci. 9, 42-42 (2008).</li><li>Lee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neural+stem+cells+from+the+postnatal+cerebellum.">Isolation of neural stem cells from the postnatal cerebellum.</a> Nat Neurosci. 8, 723-729 (2005).</li><li>Makwana, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+in+successful+peripheral+regeneration.">Molecular mechanisms in successful peripheral regeneration.</a> Febs J. 272, 2628-2638 (2005).</li><li>Tedeschi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+p53-CBP/p300+transcription+module+is+required+for+GAP-43+expression,+axon+outgrowth,+and+regeneration.">A p53-CBP/p300 transcription module is required for GAP-43 expression, axon outgrowth, and regeneration.</a> Cell Death and Differentiation. 16, 543-554 (2009).</li><li>Teng, F. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+regeneration+in+adult+CNS+neurons--signaling+molecules+and+pathways.">Axonal regeneration in adult CNS neurons--signaling molecules and pathways.</a> J Neurochem. 96, 1501-1508 (2006).</li></ol>

该协议提供了一种方法，直接在成年神经系统轴突再生性轴索损伤后在染色质环境要求。它采用了染色质免疫沉淀的背根神经节损伤模型探测后要么PNS或中枢神经系统损伤的转录和表观环境。这是调查谁愿意来描述假定为自己喜爱的转录因子结合位点，而这些网站的占用，以确定是否发生损伤反应特别有用。表观遗传修饰的组蛋白和DNA在这些网站也可以同时监控。以下的面部神经病变，面神经核?...

<table border="1"><tbody><tr><th>试剂</th><th>公司</th><th>目录编号</th></tr><tr><td> 10倍片内缓冲</td><td>细胞信号转导</td><td> 7008 </td></tr><tr><td> 2X的ChIP洗脱缓冲液</td><td>细胞信号转导</td><td> 7009 </td></tr><tr><td>芯片级G蛋白的磁珠</td><td>细胞信号转导</td><td> 9006 </td></tr><tr><td>麦格纳握架（8孔） </td><td> Millipore公司</td><td> 20-400 </td></tr><tr><td>氯仿</td><td>默克</td><td>联合国1888年</td></tr><tr><td> 37％的甲醛</td><td>罗仕</td><td> CP10.1 </td></tr><tr><td> 10X甘氨酸解决方案</td><td>细胞信号转导</td><td> 7005 </td></tr><tr><td>糖原</td><td>西格玛</td><td> G1767 </td></tr><tr><td> 10倍的HBSS </td><td> Gibco公司</td><td> 14185 </td></tr><tr><td>组蛋白H3抗体（兔） </td><td>细胞信号转导</td><td> 2650 </td></tr><tr><td>正常兔IgG </td><td>细胞信号转导</td><td> 2729 </td></tr><tr><td>酚/氯仿/异戊醇</td><td>罗仕</td><td> A156.1 </td></tr><tr><td>蛋白酶抑制剂鸡尾酒片</td><td>罗氏公司</td><td> 04 693 116 001 </td></tr><tr><td>蛋白酶K（20毫克/毫升） </td><td>细胞信号转导</td><td> 10012 </td></tr><tr><td> SDS裂解液</td><td>上州</td><td> 20-163 </td></tr></tbody></table><table border="1"><tbody><tr><th>所需设备</th></tr><tr><td> Sonicator </td></tr><tr><td> Micropestle </td></tr><tr><td>离心</td></tr><tr><td> Thermomixer </td></tr></tbody></table>

chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury

在中枢神经系统（CNS）的轴突无法再生，而在周围神经系统（PNS），再生伤后有限的范围内（登等人，2006年）。它是公认的，突起和轴突的生长至关重要的转录程序损伤在PNS（Makwana等人，2005年）后重新启动。然而，可用的工具来分析体内神经元的基因调控是有限的，往往具有挑战性。 背根神经节（DRG）提供了一个很好的损伤模型系统，因为无论是CNS和PNS来自相同SOMA的分叉轴突支配。神经节代表一个离散集合所有转录事件发生的细胞体，从而提供明确界定的区域，可以很容易地和重复地从动物中删除的转录活性。三七总皂苷（如坐骨神经），轴突再生发生，神经纤维损伤应揭示的是那些在中枢神经系统的响应类似的伤害，其中再生不会发生（如脊髓不同的转录方案）。转录因子结合，组蛋白和DNA修饰造成的伤害无论是PNS或CNS的网站都可以使用染色质免疫沉淀（ChIP）的特点。 在这里，我们描述了一个芯片使用固定的性轴索损伤后的小鼠背根神经节组织的协议。这种强大的组合提供了一个表征亲再生染色质环境，促进轴突再生的必要手段。

Watch this Scientific Journal Video about Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury at JoVE.com

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

我们目前从背根神经节组织中的染色质免疫沉淀性轴索损伤的方法。这种方法可以用来识别特定的转录因子结合位点，同时在周边及中枢神经系统受伤的轴突再生的表观遗传修饰的组蛋白与DNA的重要。

染色质免疫沉淀从以下轴索损伤的背根神经节组织

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 ...

chromatin-immunoprecipitation-from-dorsal-root-ganglia-tissue

Research

JoVE Journal

Neuroscience

16.1K 次观看.  University of Tuebingen. 我们目前从背根神经节组织中的染色质免疫沉淀性轴索损伤的方法。这种方法可以用来识别特定的转录因子结合位点，同时在周边及中枢神经系统受伤的轴突再生的表观遗传修饰的组蛋白与DNA的重要。

视频: Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

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.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

实时成像背根轴突后根切断术

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

科研

神经科学

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

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

培养成年的背根神经节神经元轴突再生的遗传研究

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

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.

轴突的相互作用与小胶质细胞和巨噬细胞在小鼠背柱破碎损伤活体成像

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

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

背根神经节神经元和区分脂肪干细胞:一种<em&gt;在体外</em&gt;共培养模型来研究周围神经再生

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

背根神经节的啮齿动物的脊髓和隔离的液压挤压

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

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

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

背根神经节内注射和背根挤压伤为感官轴突再生模型

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

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Laminotomy 对猪腰背根神经节通路和注射的治疗

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

背根神经节分离与原代培养对神经递质释放的研究

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

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

多尾根结鱼巨噬细胞的快速隔离

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