这项研究得到了德意志 Forschungsgemeinschaft (DFG;SFB1118)。作者想感谢一周半跳 Erhardt 的大部分解剖和有用的传递这方面的知识, 并 Dr. 沃克 Eckstein 协助技术方面的流式细胞仪。

野生型 C57BL/6 小鼠 (雄性; 10-12 周) 被保存在标准的12小时光照/暗循环中, 并提供免费的标准膳食和水。所有动物实验都是按照当地动物保育和使用委员会的有关指导方针进行的, 并由德国卡尔斯鲁厄 (G216/10) 区域当局的当地动物保育和使用委员会批准。
1. 鼠标的灌注
<ol><li>牺牲鼠标 (C57BL/6;男性;10-12weeks) 使用 CO2或按照当地规定。 注: 使用 EDTA 涂敷的1.5 毫升管, 可进行逆行眶出血以获得控制白细胞。这种流血的方法只是在牺牲后立即有效。此外, 腹腔灌洗 (PCL) 可以获得控制巨噬细胞, 使用前加热 PBS 在37° c, 以避免冷凝集的血液, 这可能会妨碍灌注。</li>
	<li>用70% 伏/v 乙醇喷洒在腹部的皮毛, 用棉布纱布反复擦拭, 以消毒小鼠。使用钝剪刀, 切开腹部皮肤通过一个纵向切口。</li>
	<li>在胸腔内先用弯曲的钝剪刀将切口切开, 然后沿肋笼的整个长度延伸切口。在对侧做一个类似的切口, 直到胸骨可以被小心地抬走。</li>
	<li>用清晰的心和主要的血管, 插入一只蝴蝶23¾测量针, 连接到50毫升注射器充满冰冷的 PBS, 进入心脏的左室。</li>
	<li>用虹膜剪刀在心脏的右心房做一个切口, 尽可能大地创建一个插座, 而不会损害降主动脉, 然后轻轻地压低注射器, 使其通过大约30毫升的 PBS. 渗出液应该是清澈的。肝脏和/或肾脏的清除可以用来作为一个良好的灌注指标。</li>
</ol>2. 坐骨神经和脊神经节解剖
注意: 对于坐骨神经的解剖, 臀肌上方的皮肤被移除, 在伸展下肢的同时, 将老鼠置于腹侧向下。
<ol><li>双边解剖整个坐骨神经通过分离的肌肉组织从上背的大腿, 并沿着神经沿脊柱。切断神经, 它从脊柱的出口, 并立即在另一端的膝盖以上。 注意: 避免过度的神经处理, 如用钳子挤压或拉扯。</li>
	<li>使用镊子, 转移切除神经 (s), 约2厘米每神经, 1.5 毫升管含有1毫升冰冷 PBS 和储存在冰上。 注: 神经可以储存在冰上过夜。 注意: 对于背根解剖, 使用镊子和手术刀, 通过轻轻剥去残存的背部皮肤, 将皮肤从底层的软组织分离出来, 露出颈部、胸部和腰椎的区域。用钝的剪刀在头骨底部切开头部。</li>
	<li>在分离与脊柱前侧相连的内脏之前, 将肋骨与两侧的脊柱平行切开, 并将其靠近脊椎柱。切割股骨和删除脊柱的横向削减在水平的股骨。使用虹膜剪刀, 切断任何肌肉, 脂肪, 和其他软组织从脊柱。</li>
	<li>将脊柱与背侧朝上, 并打开它, 从尾端开始, 通过滑动剪刀的一个尖端到椎管内在三点位置和骨剪。在九点的位置重复步骤。 注意: 重要的是要准确地削减中心, 以避免损坏的背根。</li>
	<li>从侧面重复切割, 直到到达侧端, 脊髓完全暴露。使用解剖显微镜, 取出脊髓, 以揭露脊髓神经, 这可以用来定位在脊柱内的 DRGs。</li>
	<li>通过轻轻地拉动背部根部, 将背根拉入椎管内, 取出腰 DRGs, 然后用虹膜剪刀剪下神经和结缔组织, 去除 DRGs。 注意: 有可能限制收集到 L4-L6 背根, 这是助长坐骨神经。</li>
	<li>使用镊子, 转移 DRGs 到一个1.5 毫升管含有约1毫升冰冷 PBS, 并在冰上储存。 注: 神经可以储存在冰上过夜。</li>
</ol>3. 坐骨神经和脊神经节的消化
<ol><li>准备消化培养基 (DMEM, 10 毫米 HEPES, 5 毫克/毫升 BSA, 1.6 毫克/毫升胶原酶类型 4) 补充100µg/毫升 deoxyribonuclease, 并在冰上储存。 注: 每只老鼠所需的消化培养基量 (坐骨神经 + DRGs) 是1毫升。一旦准备好, 等分的消化介质可以储存在-20 ° c。</li>
	<li>将坐骨神经和 DRGs 从一只老鼠转移到单独的1.5 毫升管中, 其中含有500µL 消化介质。对于坐骨神经, 用弹簧剪刀将材料切成15-20 块, 然后在37° c 的摇动培养箱中孵育组织悬浮物, 20 分钟, 用柔和的搅动 (≤ 450 rpm)。</li>
	<li>反复研制细胞悬浮通过1000µL 吸管尖端机械离解任何不完全消化的组织。 注: 如果悬浮液难以滴定, 则用剪刀剪下吸管尖端以创建钝端。孵育组织悬浮在37° c 为20分钟以柔和的鼓动 (≤ 450 rpm)。</li>
	<li>重复研磨通过1000µL 吸管尖端和孵化为进一步10分钟在37° c 与温和的鼓动 (≤ 450 rpm)。</li>
	<li>准备的外地资产管制署缓冲 (PBS 补充 10% FCS 和1毫米 EDTA) 和储存在冰上。用 60 x 15 mm 的培养皿在冰上冲洗和浸泡一个网格大小为140µm 的不锈钢丝网。 注意: 应避免尼龙筛网过滤器, 因为它会导致有核细胞的产量显著下降。</li>
	<li>通过140µm 屏幕过滤器反复通过组织悬浮液, 因为它是在培养皿, 用钝钳, 在冰上举行。从培养皿中收集细胞悬浮液, 并将其转移到一个新的1.5 毫升的冰上储存。</li>
	<li>推动任何不完全消化的组织, 仍然在过滤通过重复研磨与1000µL 吸管尖端。冲洗屏幕过滤器两次与500µL 的外地资产管制系统缓冲区和收集结果细胞悬浮在培养皿结合与细胞悬浮从步骤3.6。 注: 如果使用的流式细胞仪对碎片敏感, 则步骤3.7 中的电池悬挂可通过70µm 滤网过滤;然而, 这可能导致有核细胞的产量的损失。</li>
	<li>离心的细胞悬浮在 300 x g 为5分钟在4° c。丢弃上清液, 在1毫升的重中进行细胞团的缓冲。重复这个洗涤步骤一次。</li>
	<li>重150-350 µL 的细胞团缓冲, 取决于所需的染色数量 (每染色面板150µL 的缓冲区)。</li>
</ol>4. 流式细胞术中荧光标记抗体对坐骨神经和背根的染色
<ol><li>将坐骨神经或背根细胞悬架的100µL 转移到一个96孔板上的 V 形井中。 注: 单染色细胞是必要的所有抗体, 因为他们将提供必要的控制, 以建立流式细胞仪关于光电倍增管 (PMT) 电压增益和补偿, 以及帮助区分特定的非特定绑定。由于来自坐骨神经和 DRGs 的有限的材料, 从 PCL (步骤 1.1-1.2) 的血白细胞和巨噬细胞可以作为单染色的控制, 其染色应与坐骨神经染色并行进行神经和 DRGs</li>
	<li>将剩余的细胞悬浮 (大约50µL 每样本) 从坐骨神经或根背根标本, 作为不控制。</li>
	<li>离心板在 400 x g 为 5 min 在4° c。通过把盘子翻转到垃圾箱上, 然后用手腕上的锋利的轻拍来丢弃上清, 然后轻轻地用纸巾擦干。</li>
	<li>对于表面染色, 重在20µL 中的每一个细胞球团都含有感兴趣的抗体 (CD45-A647, CD11b-PerCP/Cy5.5, 和 MHC II 类生物素) 和孵化, 保护免受光, 在冰上30分钟。 注: (1) 根据制造商的产品信息, 在使用稀释系列之前, 应确定最佳抗体稀释度;(2) 根据制造商的说明, 在这一步中, 通过 Fc 块孵化可以减少非特定的染色;和 (3) 同种控制染色通常是不需要使用的主要抗体直接与荧光和/或当抗原 (s) 的兴趣产生不同的人口。</li>
	<li>在每个井中加130µL, 并在4° c 下离心 400 x g 5 分钟。丢弃上清, 如前所述 (步骤 4.3), 并清洗细胞小球两次与150µL 的外地资产管制站缓冲区。 注: 如果使用游离主抗体或与生物素结合的主抗体, 则可在此时添加适当的二级试剂, 共轭于荧光, 并重复步骤 4.4-4.5。在本议定书的情况下, streptavidin-PE/Cy7 或亲和 PerCP 被用来检测 MHC 第二类与 CD45-A647/CD11b-PerCP/Cy5.5, 或结合 CD68-APC 和 F4/80-PE/Cy7, 分别。</li>
	<li>为固定, 在最后洗涤的步以后, 重细胞小球在100µL 4% 甲醛在 PBS (pH 7.4)。在冰上孵育板10分钟, 然后离心在 400 x g 5 分钟, 在4° c。丢弃上清, 如前所述 (步骤 4.3), 并清洗的细胞小球一次与150µL 的外地资产管制站缓冲区。 警告: 甲醛有毒;穿戴适当的保护例如手套和镜片,等。 注: 重150µL 中的细胞球团缓冲和存储保护免受光, 在 4 C 长达两天。如果不需要细胞内染色, 然后进行步骤4.11 后, 离心在 400 x g 5 分钟, 在4° c。</li>
	<li>对于细胞内染色, 重150µL 的细胞球团缓冲剂 + 0.5% 洗涤剂, 并在室温下孵育, 保护免受光15分钟. 离心板在 400 x g 为 5 min 在4° c。丢弃上清, 如前所述 (步骤 4.3) 和洗涤的细胞颗粒一次与150µL 的外地资产管制的缓冲 + 0.5% 洗涤剂。重20µL 的细胞球团缓冲剂 + 0.5% 洗涤剂含有的抗体的兴趣 (CD68-APC, CD206-PE) 和孵化, 保护免受光, 在室温下为30分钟。</li>
	<li>增加130µL 的外地资产管制的缓冲区 + 0.5% 洗涤剂每井和离心板在 400 x g 5 分钟, 在4° c。丢弃上清, 如前所述 (步骤 4.3) 和洗涤的细胞小球两次与150µL 的外地资产管制的缓冲区 + 0.5% 洗涤剂。 注: 如果使用游离主抗体或与生物素结合的主抗体, 则可在此时添加适当的二级试剂, 共轭至荧光, 并重复步骤 4.9-4.10。</li>
	<li>重150µL 的细胞球团缓冲 + 0.5% 洗涤剂和商店, 保护免受光, 在4° c 最多两天。</li>
	<li>重的细胞小球与150µL 的缓冲区 + 0.5% 洗涤剂补充5µg/毫升 DAPI 和孵化5分钟室温下, 保护免受光。离心板在 400 x g 为 5 min 在4° c。丢弃前述的上清液 (步骤 4.3)。</li>
	<li>重150µL PBS 中的细胞小球, 并转移到一个适当标记的外地资产管制管。储存在室温下, 免受光照, 直到准备好分析。 注意: 在 PBS 悬浮后, 细胞应在数小时内进行分析, 因为 DAPI 会慢慢脱离 DNA, 导致信号丢失。</li>
</ol>5. 流式细胞仪的设置和样品的运行
<ol><li>确保流式细胞仪配备适当的激光器和/或过滤器集, 以测量用于染色荧光的电池。这可以在系统的默认的细胞仪配置下确定。在本协议的情况下, 使用流式细胞仪进行分析, 配备三激光器 (405 nm、488 nm 和 633 nm), 允许检测≤5荧光。</li>
	<li>选择适当的渠道, 以检测荧光的利益。所有的荧光信号最好的检测与对数放大。</li>
	<li>为样本选择适当的流速。这个参量可能变化取决于使用的系统, 并且应该经验主义地被确定。在本协议的情况下, 一个流动率的 MED 到 HI (35-60 µL/分钟) 被用来防止堵塞的针。</li>
	<li>选择要在每个示例中收集的事件/单元格的总数。最小事件数应为100万。</li>
	<li>在全局工作表下创建散点图-a 与 FSC-a, 以及每个荧光的兴趣与 FCS a。创建一个统计视图, 它将显示所选荧光的事件数和平均荧光强度。</li>
	<li>设置 FSC 的电压-a 和 SSC-一个通道, 使最小的单元格符合散点图的左下10%。从坐骨神经或 DRGs 的不控制中确定背景荧光和最小样品荧光. 根据 DAPI 染色控制的散射图确定父 (P1) 门。 注意: 这不是一个集合门, 部分原因是, DAPI + 种群的异质性。</li>
	<li>使用 FSC 分散图为每个荧光的利益, 调整电压, 使不控制的细胞在其各自的散射地块的下四分之一。然后在这个区域之上创建门, 以定义那些对每个标记/荧光都是阳性的细胞。通过单染色控制 (步骤 4.1) 的运行, 确定了浇口的位置。 注: 重叠发射谱的补偿可以在这一点上进行, 或者追溯到适当的分析软件。</li>
	<li>一旦流式细胞仪已经建立, 如所述, 样品可以运行。</li>
	<li>在运行后, 剩余的样品材料可以被丢弃, 并可以导出 FCS 文件, 以便进一步分析。</li>
</ol>

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P., Fleming, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STZ+causes+depletion+of+immune+cells+in+sciatic+nerve+and+dorsal+root+ganglion+in+experimental+diabetes.">STZ causes depletion of immune cells in sciatic nerve and dorsal root ganglion in experimental diabetes.</a> J. Neuroimmunol. 306, 76-82 (2017).</li></ol>

作者在这项研究中没有利益冲突。

坐骨神经含有很大比例的脂质, 如胆固醇, 由于髓鞘周围的神经轴突的内容。由于油脂的性质随温度变化, 在不同的温度下可以得到不同的结果。为了保证细胞的保存, 消化后的所有步骤都在冰上进行。为了使结果重现性起见, 建议保持一致性, 但在室温下执行步骤3.6 和3.7 可以提高产量。如果3.8 步的神经没有被充分消化, 小块神经将在网格上可见。在不充分消化的情况下, 在网格上保留的材料可以通过转移到一个新的1.5 毫升管含有250µL 消化介质和重复步骤 3.2-3.6 的重新消化。如果产量很差, 那么在通过网筛 (步骤 3.6) 后的细胞悬浮样品, 应在一个光镜下观察台盼蓝。可行的, 非蓝色的, 细胞应该是可见的与杆状结构, 这是轴突的片段。如果没有可行的细胞存在, 那么消化条件和/或不正确的材料处理导致坏死。如果只有大的骨料是可见的, 那么建议收集的材料离心和重复消化 (步骤 3.2-3.6)。
小鼠坐骨神经和 DRGs 的流式细胞仪分析方法, 对于涉及全神经和多 DRGs 的问题是有用的. 最近有增加的兴趣在分析的总皂苷使用流化法,11,12.刘et al.已经开发出一种方法使用木瓜蛋白酶和机械解离坐骨神经和 DRGs 使用21G 和23G 专门分析1厘米的神经部分, 从而允许分析神经材料从特定地点的伤害11。这里提出的方法, 建议使用时, 对三七总皂苷的影响是系统性的, 如在毒和遗传模型神经病变或慢性疾病, 如糖尿病。由于从坐骨神经中获得的有核细胞的产量低, 因此不建议尝试从单个小鼠身上研究具有≤100门控事件的小型种群。然而, 从几只老鼠中收集材料将有助于研究神经中的这种小种群。然而, 这将是有兴趣的研究是否酶组织离解使用木瓜蛋白酶和21克/23 克针描述的刘et al.如本研究所述, 11可以与细胞核的流式细胞仪染色结合, 以提高单个细胞的收率。分析极少量组织的能力对于在单侧坐骨神经切断后进行信息学和对侧 DRGs 的比较至关重要。然而, 这超出了本研究的范围。有趣的是, 在小鼠的三七总皂苷中, 常见的用于识别巨噬细胞的标记物具有意想不到的异质表达。例如, CD11b 和 F4/80 都被发现在大比例的非造血, 非抗原呈递细胞在坐骨神经和 DRGs。此外, 在坐骨神经中, 并非所有的 CD68+巨噬细胞都表达了 CD11b 和 F4/80, 就像其他器官的研究所期望的那样。因此, 建议在使用这些标记来研究三七中的巨噬细胞时要谨慎, 并应研究其他标记物, 如 CD163。此外, 不同的标记对酶解离的敏感性也应建立, 因为这可能解释了在表达中观察到的差异, 如 CD11b 或 F4/80。
该协议以前被用来分析在毒性挑战后的三七总皂甙 (脲佐菌素)13的免疫细胞, 这种化学物质通常用于模型啮齿动物有机体中诱导糖尿病。结果表明, TRPV1 和 TRPA1 激活的佐菌素最初导致坐骨神经和 DRGs 的免疫细胞衰竭, 而不引起神经的任何炎症。虽然免疫细胞的数量在 DRGs 恢复, 免疫细胞, 特别是巨噬细胞仍然显着耗尽三周后毒性挑战。后来推测, 巨噬细胞吞噬神经碎片, 由离子通道兴奋造成的损伤可能导致了巨噬细胞的凋亡, 导致它们从一般细胞中耗尽。目前尚不清楚其他周围神经毒素如何影响神经驻留巨噬细胞。如果耗尽巨噬细胞是这种药物的共同结果, 这可能会影响治疗。
结果发现, 神经中只有5-10% 的有核细胞是造血的白细胞。其余的细胞可能是其他神经细胞类型的混合物, 即雪旺细胞、周、成纤维细胞和内皮细胞膜。由于流式细胞仪系统的能力, 以测量多个参数, 并提供了特定的标记和/或抗体可用, 它可以想象, 这些细胞类型也可使用此方法进行分析。该方法已被描述为小鼠材料。然而, 经过一些修改, 它也被用于分析猪和人的三七总皂苷的材料。预计这种方法在今后对比小鼠更大的种类的材料进行分析时将更有用。

从循环中进入三七总皂苷的免疫细胞, 如 CD45 和 CD11b 的表达, 有助于维持神经的完整性, 并在再生和变性方面发挥作用1。巨噬细胞 (由它们的 CD68 在老鼠中的表达) 可以偏向于炎症表型, 表达更多的 MHC 类 II 和 CD86 在他们的表面 (M1), 或对抗炎表型, 表达更多的胞内 CD206 (M2)2.巨噬细胞表型的倾斜是通过 Akt 信号3调节的动态过程, 反映了巨噬细胞在防御病原体 (M1) 和组织再生中的作用 (M2) 的不同任务。损伤的神经的再生首先要求吞噬髓鞘残骸由巨噬细胞在神经4,5和抗炎性 (CD68+CD206+) M2 巨噬细胞被证明促进轴突生长在6。减少招募或巨噬细胞的总皂苷, 或受损能力的吞噬可能导致受损的再生和维持神经完整性。炎症 M1 巨噬细胞, 表达 MHC 类 II, 是较少的能力吞噬比 M2 巨噬细胞, 和神经元炎症牵连的发病机制的几个神经退行性疾病7。
由于损伤引起的神经居民免疫系统的变化可能是定量的, 表现在失去 CD45 的+白细胞 (或增加渗透的 CD45+白细胞的情况下炎症), 或定性, 如巨噬细胞表型从 M2 到 M1 表型的变化。通过使用冰冻切片或石蜡嵌入材料8, 对三七总皂苷的免疫细胞进行了传统的分析。如果需要确定感兴趣的细胞的定位。然而, 定量使用如果经常依靠计数少量细胞在一个狭窄的部分组织, 使定量不可靠和易受选择偏倚。为了识别特定的免疫细胞子集, 同时检测细胞外和/或细胞内标记是必需的, 而巨噬细胞表型的确定至少需要两个标记, 特别是 CD206 和 MHC类 II。由于大多数常用的显微镜都被限制在至少两色的通道, 如荧光素异硫氰酸酯 (FITC) 和藻 (PE), 对免疫细胞的特定子集的表征, 如果可以是限制性和不完整的, 需要需要有多个幻灯片, 从相同的兴趣领域, 这是染色和分析并行。因此这费时的方面不必要借自己对大样品集合的分析。此外, 由于大部分的兴趣标记都是胞外的, 所以在组织中的检测, 或者被植入石蜡或 cryoconserved, 由于细胞膜完整性的破坏和表位的掩蔽, 以及失去由使用溶剂引起的抗原, 如丙酮和甲醇9。
相比之式细胞仪 , 它测量单细胞的光学和荧光特性的悬浮 , 因为他们通过一束光 , 提供了一个更实用和全面的方法来分析细胞的数量。流式细胞术, 而不是制作一个数字图像的组织, 提供了一个自动量化的设置参数, 其中包括一个细胞的相对大小和反射指数, 称为前散射 (FSC), 粒度/内部复杂度或侧散射 (SSC) 和相对荧光强度, 提供该细胞已被标记为适当的荧光, 如共轭抗体。典型的流式细胞仪由两个空冷激光器组成;氩激光在 488 nm 处产生蓝光, 氦氖激光器在 633 nm 产生光。这一组合允许检测和测量, 同时, 至少五目标, 无论是在表面上或在细胞内室。更先进的流 cytometers 可以由多个激光器组成, 它允许一次检测多达八种不同的荧光, 这就使得所选荧光的峰值发射波长不会有明显的重叠。
通过流式细胞仪分析, 首先要酶消化, 一般用胶原酶, 产生单细胞悬浮。小鼠坐骨神经的分析以前是困难的, 因为少量的组织从每只老鼠获得。此外, 高脂肪含量的髓鞘周围的轴突阻碍细胞的恢复和产生大量的碎片。本文对坐骨神经的制备和消化的方法进行了改编, 从发夹的雪旺细胞隔离协议的et al.7, 目的是从单个小鼠的神经中分离出足够的细胞进行流式细胞仪分析, 以减少小鼠之间的变异。在原始组织文摘中使用 DAPI 来识别单个细胞, 绕过需要去除轴突碎片, 这通常会导致细胞丢失。清洗几次用洗涤剂丰富的缓冲剂帮助释放细胞内的脂肪碎片, 从而提高产量。根据本协议, 从单个小鼠中消化两个全长坐骨神经, 产生≥30,000 单核事件, 至少3次从根背根中检索到这个数字。CD45 的+白细胞的比例约5% 的总细胞含量的坐骨神经消化和约5-10% 在根背根消化. 坐骨神经中的大多数 CD45+细胞表达了巨噬 CD68 和 CD206。

<table>
	<tbody>
		<tr>
			<td>C57BL/6 Mouse</td>
			<td>Charles River</td>
			<td>C57BL/6NCrl</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (+1g/L glucose, Glutamine, Pyruvate)</td>
			<td>Thermo</td>
			<td>31885023</td>
			<td>The source of this material is not important</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type 4</td>
			<td>Worthington Biochemical Corp., US</td>
			<td>LS004188</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease (DNase) I from bovine pancreas I</td>
			<td>Sigma-Aldrich</td>
			<td>DN25-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Calf Serum (FCS), heat inactivated</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody against CD11b-PerCP/Cy5.5</td>
			<td>Biolegend</td>
			<td>101228</td>
			<td>Clone M1/70</td>
		</tr>
		<tr>
			<td>Antibody against MHC class II, biotinylated</td>
			<td>Biolegend</td>
			<td>107603</td>
			<td>Clone M5/114.15.2</td>
		</tr>
		<tr>
			<td>Antibody against CD45-A647</td>
			<td>Biolegend</td>
			<td>107603</td>
			<td>Clone 30-F11</td>
		</tr>
		<tr>
			<td>Antibody against CD68-APC</td>
			<td>Biolegend</td>
			<td>137007</td>
			<td>Clone FA/11</td>
		</tr>
		<tr>
			<td>Antibody against CD206-PE</td>
			<td>Biolegend</td>
			<td>141706</td>
			<td>Clone C068C2</td>
		</tr>
		<tr>
			<td>Antibody against F4/80-PE/Cy7</td>
			<td>Biolegend</td>
			<td>123113</td>
			<td>Clone BM8</td>
		</tr>
		<tr>
			<td>Streptavidin-PE/Cy7</td>
			<td>Biolegend</td>
			<td>405206</td>
			<td>Used to detect MHCII-biotin with CD45-A647 and CDllb-PerCP/Cy5.5</td>
		</tr>
		<tr>
			<td>Streptavidin-PerCP</td>
			<td>Biolegend</td>
			<td>405213</td>
			<td>Used to detect MHCII-biotin with CD68-APC and F4/80-PE/Cy7</td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542-1MG</td>
			<td>Dilute in PBS to a 50x and store at 4 &#176;C in the dark</td>
		</tr>
		<tr>
			<td>Cell dissociation sieve - tissue grinder kit</td>
			<td>Sigma-Aldrich</td>
			<td>CD1</td>
			<td></td>
		</tr>
		<tr>
			<td>V-Shaped, 96-well plates</td>
			<td>Greiner/Sigma</td>
			<td>M8185</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml polystryene round-bottom tube, 12x75mm</td>
			<td>BD Biosciences</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr>
			<td>60x15mm petri dish</td>
			<td>Greiner/Sigma</td>
			<td>Z643084</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSR II Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of immune cells in single sciatic nerves and dorsal root ganglion from a single mouse using flow cytometry

神经驻留免疫细胞在周围神经系统 (三七) 是必不可少的保持神经元完整的健康神经。三七总皂苷的免疫细胞受损伤和疾病的影响, 影响神经功能和再生能力。神经免疫细胞通常通过免疫荧光 (如果) 进行分析。如果是确定神经的免疫细胞位置的必要条件, 如果只是半定量的, 并且方法仅限于可以同时分析的标记的数量和表面表达的程度。本研究采用流式细胞术对单个小鼠坐骨神经或背根节 (DRGs) 的白细胞浸润进行定量分析。用 DAPI 进行单细胞分析, 同时分析了几种蛋白质的表面或细胞内表达。根据本议定书处理的一只老鼠的坐骨神经都产生了≥3万单核事件。坐骨神经中白细胞的比例由 CD45 表达, 在坐骨神经中的总细胞含量约为 5%, 在根节中约5-10%。虽然本协议主要关注的是在三七总皂苷内的免疫细胞, 但流式细胞仪的灵活性, 同时测量一些标记, 意味着其他细胞存在于神经, 如雪旺细胞, 周、成纤维细胞和内皮细胞膜, 也可以用这种方法进行分析。因此, 该方法为研究三七总皂甙的系统性影响提供了新的途径, 如神经病变的毒和遗传模型, 以及糖尿病等慢性病。

根据该协议, 从六只健康的 C57BL/6 小鼠中制备了全长坐骨神经和所有主要根背根的细胞悬浮液, 并将其分为三等分染色。反染色与 DAPI, 其中着色 DNA, 允许检测一个单一的细胞数量 (图 1A-B, 上部面板)。冲洗的 DAPI, 在分析前流式细胞仪, 减少到一定程度上的非核碎片的荧光强度, 从而允许选择一个离散的人口在 FSC 轴的单核事件或汗衫。没有收集门的应用, 因为从汗衫的期票是分布在一个大范围的 FSC 和 SSC (图 1C)。在选择单线态填充 (图 1A-B) 时, 必须排除在 FSC 轴的高端上的材料, 其中包含未完全消化的组织。使用密度图 (下面板) 来显示结果可能会在排除大型碎片 (下面板) 时有所帮助。总的≥3万汗衫可以定期从两个完整的坐骨神经和≥200,000 汗衫可以获得从 DRGs (图 1D)。从坐骨神经, 大约5% 汗衫表达了 CD45 在他们的表面, 而大约5-10% 在 DRGs 被发现了, 从而定义了这些细胞作为造血的白血球起源 (图 2A)。
大多数 CD45+汗衫也以渐进方式 (图 2B) 表达了 MHC 类 II。在 DRGs 中, CD11b 可用于检测类似胶质的单元格 (CD45dim、MHCII+和 CD11b+) (图 2B, 上部面板)。然而, 这种小胶质细胞的人群没有从坐骨神经 (图 2B, 下部面板)。汗衫中表达 CD45 的大多数是 CD68+巨噬细胞。在坐骨神经和背节, 2-5% 的汗衫表达 CD68 (图 3A)。所有表达 CD68 的细胞也表达了 CD45 (未显示数据)。在 DRGs 中, CD68 的+巨噬细胞在 MHC II 类和 CD206 (M2 巨噬细胞) 的表达上比在坐骨神经 (图 3BD) 中更具异构性。在坐骨神经中, 大多数巨噬细胞共同表达的 MHC 类 II 和 CD206 (图 3B-D, 下部面板);通常, 大约60-80% 的 CD68+巨噬细胞表达了 CD206, 而在 DRGs (图 3B) 中, CD68+ co 表示 CD206 的比例较小。有趣的是, M1 巨噬细胞标记 CD86 没有产生任何阳性染色的巨噬细胞从神经和 PCL (数据没有显示)。巨噬细胞标记, F4/80, 显示了一个异构的表达在背根和坐骨神经 (图 3B-C), 染色比例的 CD68 和 MHCII 细胞. 此外, F4/80 表达式与 CD68 表达式没有完全重叠。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56538/56538fig1.jpg"> 图 1: 来自背根和坐骨神经 DAPI 染色的有核细胞的流式细胞仪分析的代表性数据.收集的所有事件, 显示为 scatterplots (上部面板) 或密度图 (下部面板), 从 (A) 背根或 (B) 坐骨神经 (SN) 的一个代表性的鼠标。下面板中的箭头表示门, 不包括顶部的物质。(C) 对 DAPI 门 (汗衫) 中收集的单元格进行后浇口, 在 SSC 与 FSC 散射图的面板 (A) 中显示。(D) 来自 SN 和所有主要背根的汗衫从六健康 C57BL/6 小鼠的数量。数据代表平均± SD.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56538/56538fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56538/56538fig2.jpg"> 图 2: 流式细胞仪分析 CD45、MHC II 类和 CD11b 对汗衫从背根和坐骨神经的表面表达.在图 1中所示的 DAPI 门 (汗衫) 中选择的事件进行了分析。(A) 在坐骨神经 (SN) 和六控制小鼠的背根中表达 CD45 的汗衫的比例。(B) 代表从背节 (上部面板) 和 SN (下部面板) 汗衫的散点图。表达 CD11b 的细胞用蓝色表示。数据代表平均± SD.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56538/56538fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56538/56538fig3.jpg"> 图 3: 流式细胞仪分析 CD68、MHC II 类和 CD206 对汗衫从背根和坐骨神经的表面表达.在图 1中所示的 DAPI 门 (汗衫) 中选择的事件进行了分析。(A) 汗衫表达 CD68 在坐骨神经 (SN) 和 DRGs 六健康 C57BL/6 小鼠中的比例。(B) CD68 的+巨噬细胞与六健康 C57BL/6 小鼠的 SN 和背根 CD206 的比例。(C) 具有代表性的汗衫从背节 (上面板) 和 SN (下部面板) 的散布图, 显示 CD68 和 MHC 类 II 表达。(D) 代表汗衫从背节 (上部面板) 和 SN (下部面板) 显示 CD68 和 CD206 表达式的散点图。表达 F4/80 的细胞用洋红色表示。数据代表平均± SD.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56538/56538fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry at JoVE.com

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

由于组织的缺乏, 小鼠坐骨神经内细胞含量的定量分析是困难的。本协议描述了一种组织消化和制备的方法, 它提供了足够的细胞, 用于对单个小鼠神经的免疫细胞进行流式细胞仪分析。

应用流式细胞仪分析单侧坐骨神经和背根神经节的免疫细胞

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

analysis-immune-cells-single-sciatic-nerves-dorsal-root-ganglion-from

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.4K 次观看.  University Hospital of Heidelberg. 由于组织的缺乏, 小鼠坐骨神经内细胞含量的定量分析是困难的。本协议描述了一种组织消化和制备的方法, 它提供了足够的细胞, 用于对单个小鼠神经的免疫细胞进行流式细胞仪分析。

视频: Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues presents a unique challenge because of the paucity of cells of interest and few transgenic reporter mouse models. Here, we describe a protocol using fluorescence-conjugated probes to selectively label LacZ-positive cells from the neonatal cochleae.
The most common underlying pathology of sensorineural hearing loss is the irreversible damage and loss of cochlear sensory hair cells, which are required to transduce sound waves to neural impulses. Recent evidence suggests that the murine auditory and vestibular organs harbor stem/progenitor cells that may have regenerative potential1,2. These findings warrant further investigation, including identifying specific cell types with stem/progenitor cell characteristics. The Wnt signaling pathway has been demonstrated to play a critical role in maintaining stem/progenitor cell populations in several organ systems3-7. We have recently identified Wnt-responsive Axin2-expressing cells in the neonatal cochlea, but their function is largely unknown8.
To better understand the behavior of these Wnt-responsive cells in vitro, we have developed a method of isolating Axin2-expressing cells from cochleae of Axin2-LacZ reporter mice9. Using flow cytometry to isolate Axin2-LacZ positive cells from the neonatal cochleae, we could in turn execute a variety of experiments on live cells to interrogate their behavior as stem/progenitor cells. Here, we describe in detail the steps for the microdissection of neonatal cochlea, dissociation of these tissues, labeling of the LacZ-positive cells using a fluorogenic substrate, and cell sorting. Techniques for dissociating cochleae into single cells and isolating cochlear cells via flow cytometry have been described2,10-12. We have made modifications to these techniques to establish a novel protocol to isolate LacZ-expressing cells from the neonatal cochlea.

Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of &#946;-galactosidase prior to separation via flow cytometry.

隔离LacZ基因表达的小鼠内耳组织用流式细胞仪检测细胞

Isolation of specific cell types allows one to analyze rare cell populations such as stem/progenitor cells. Such an approach to studying inner ear tissues ...

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 ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

单苍蝇检测方法的觅食行为的<em&gt;果蝇</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

在对小鼠坐骨神经体内的电测量

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

在癌神经侵袭的体外模型:背根神经节型号

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

一种方法来提高背根神经节的对齐和髓鞘

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

视网膜神经节细胞定义子集的单细胞RNA-Seq

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...

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 ...

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

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

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

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

哺乳动物轴突再生: 成年小鼠背根神经节体内电穿孔的研究

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