这项研究获得资助 (授予 F Q。Z.) 由 NIH (R01NS064288, R01NS085176, R01GM111514, R01EY027347), 克雷格 h 尼尔森基金会和 BrightFocus 基金会。

所有动物实验都是按照约翰霍普金斯机构动物保育和使用委员会批准的动物协议进行的。
1. 材料和试剂
<ol><li>
动物
	<ol>
<li>用六周大的雌性 CF1 小鼠称量 30–35 g 进行实验。 注: 在单独通风灭菌笼中, 小鼠组内 (每笼5只小鼠), 1/4 "玉米芯寝具和 2" 方形 nestlets 用于筑巢。笼保持在一个控制的12小时的光暗循环和室温是介于19.4 摄氏度和25摄氏度之间。用全球啮齿动物饮食和自动供水系统喂养小鼠。</li>
	</ol>
</li>
	<li>
仪器
	<ol>
<li>使用下列外科器械有效地进行手术: 一次性注射器 (27 克, 1.0 毫升) 腹腔注射, 毛皮剪, 立体解剖显微镜, 显微手术钳, 显微手术剪刀, 小骨 rongeurs和灭菌的无纺布海绵。 注: 所有仪器和材料均在手术前蒸压, 或由生产者预先消毒。在手术过程中, 这些仪器用75% 酒精喷洒来维持灭菌。</li>
	</ol>
</li>
	<li>
麻醉解决方案
	<ol>
<li>使用麻醉液 #1 诱发麻醉, 并在根背根注射前使用麻醉液 #2 维持麻醉。<ol>
<li>为使麻醉溶液 #1, 稀释氯胺酮和甲苯噻嗪在无菌盐水中的最终浓度为10毫克/毫升和1.2 毫克/毫升。</li>
			<li>为使麻醉溶液 #2, 在20毫克/毫升的最终浓度下, 稀释 avertin 在无菌盐水中。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
DNA 质粒与 RNA 寡核苷酸
	<ol>
<li>根据制造商的协议, 使用商业工具包准备质粒。将无菌去离子水中的质粒在2.0 µg/µL 浓度上稀释。</li>
		<li>添加快速绿色染料溶液达到最终浓度的 0.005–0.01% (v/v), 以更好地可视化背节轮廓在注射过程中。</li>
		<li>在制造商提供的相应缓冲器中溶解 siRNA 或 microRNA 寡核苷酸, 以达到50µM 的最终浓度。</li>
	</ol>
</li>
	<li>
微注射吸管
	<ol>
<li>用微拉拔毛细管, 用显微外科剪刀剪下拉毛毛细管玻璃吸管的尖端, 产生一个大约50µm 外径的开口。然后在紫外线照射下将玻璃吸管消毒在干净的长凳上, 20–30分钟。</li>
		<li>将灭菌过的玻璃吸管安装在与细胞内微量注射分配系统相连的吸管支架上。将微注射系统的参数设置在压力的 30 psi 和8毫秒的时间内。</li>
	</ol>
</li>
	<li>
电穿孔系统
	<ol>
<li>将电极连接到方波电穿孔系统, 并设置以下参数:15 毫秒脉冲在 35 V, 950 毫秒间隔用于体内电穿孔。</li>
	</ol>
</li>
	<li>
灌注和固定液
	<ol>
<li>将多聚甲醛 (粉煤灰) 粉溶于 1x PBS 中, 浓度为 4% (瓦特/v)。将粉煤灰溶液贮存在摄氏4摄氏度。</li>
	</ol>
</li>
</ol>2. 实验程序
<ol><li>
L4 和 L5 DRGs 的手术暴露
	<ol>
<li>麻醉使用腹腔注射麻醉溶液 #1 前准备 (与氯胺酮100毫克/千克体重和甲苯噻嗪12毫克/千克体重)。</li>
		<li>用毛皮剪刀把手术部位剃掉。 注: 由于左坐骨神经粉碎还会有另一项手术, 左后大腿的皮毛可以同时剃光。</li>
		<li>将鼠标放在加热毯上 (35.0 °c), 用温度探头监视直肠温度。</li>
		<li>检查麻醉, 捏住老鼠的脚趾和尾巴, 观察行为反应。</li>
		<li>把老鼠的四只四肢贴在软木砖上。</li>
		<li>用碘伏溶液擦拭手术部位, 然后用75% 乙醇清除碘伏, 然后再切开皮肤。</li>
		<li>在麻醉时, 眼睛涂抹眼膏以防止干燥。</li>
		<li>切口前, 用细标记笔标记髂嵴两侧。画一条连接髂嵴两点的线, 以方便辨认 L5 DRGs 的位置。</li>
		<li>用微剪刀在低背中线上做一个3厘米的切口。此外, 分离的 paraspinous 肌肉, 如小 multiﬁdus 和 longissimus 肌, 从 L3 到 S1 棘突过程, 并暴露了 L4-5 和 L5-6 的小关节。</li>
		<li>使用微钳去除 L4-5 和 L5-6 的小关节。此外, 移除 L4 和 L5 的左神经弓, 以暴露 DRGs 的背侧。</li>
	</ol>
</li>
	<li>
根背根注射液
	<ol>
<li>注射麻醉液 #2 (用 avertin 200 毫克/千克体重) 腹腔维持麻醉前根节注射。</li>
		<li>将 DNA 质粒或 RNA 寡核苷酸 (每根背节的1µL) 加载到玻璃毛细管吸管中。</li>
		<li>小心地将毛细管玻璃吸管的尖端插入背节。</li>
		<li>用细胞内微量注射分配系统 (30 psi, 8 ms) 逐步注入1.0 µL 溶液的 DNA 质粒或 RNA 寡核苷酸进入背节。 注: 注射时间应持续不少于5分钟。</li>
	</ol>
</li>
	<li>
穿孔
	<ol>
<li>将 PBS 放在电极的尖端上。</li>
		<li>用消毒的方棉纱布清理出血。</li>
		<li>用电极轻轻捏住靶背节, 并将5平方电脉冲与电击系统结合使用。</li>
		<li>用5-0 尼龙缝线分别关闭肌肉和皮肤层。</li>
		<li>把鼠标放在加热的毯子上 (35.0 °c), 直到它完全恢复足够的意识来维持胸骨卧床。从麻醉中完全恢复后, 将鼠标返回到主笼子。 注: 在37°c 毯上, 鼠标从麻醉中完全恢复需要大约60分钟。将一丸 (200 毫克) 布洛芬溶入1000毫升水 (0.2 毫克/毫升), 用于每日喂养, 以减轻术后疼痛。</li>
	</ol>
</li>
	<li>
坐骨神经粉碎
	<ol>
<li>三天 (根据实验设计) 后背节电穿孔, 麻醉鼠标腹腔与麻醉解决方案 #2 (与 avertin 400 毫克/千克体重)。</li>
		<li>把老鼠的四只四肢贴在软木砖上。</li>
		<li>沿中线向左侧1厘米的切口0.5 厘米。切开肌肉, 如臀肌和梨肌, 纵向。揭示大坐骨神经孔与坐骨神经切口之间的坐骨神经段。</li>
		<li>十二年代用显微外科钳粉碎神经, 用10-0 尼龙 epineural 缝合标记粉碎部位。在硬脑膜膜上打个结来标记粉碎部位。</li>
		<li>用5-0 尼龙缝线闭合肌肉和皮肤层。</li>
		<li>把鼠标放在加热的毯子上 (35.0 °c), 直到它完全恢复足够的意识来维持胸骨卧床。从麻醉中完全恢复后, 将鼠标返回到主笼子。 注: 在37°c 毯上, 鼠标从麻醉中完全恢复需要大约60分钟。将一丸 (200 毫克) 布洛芬溶入1000毫升水 (0.2 毫克/毫升), 用于每日喂养, 以减轻术后疼痛。</li>
	</ol>
</li>
	<li>
小鼠灌注、根神经节和坐骨神经收获
	<ol>
<li>三天后坐骨神经粉碎 (根据实验设计), 麻醉鼠标腹腔与麻醉解决方案 #2。</li>
		<li>灌注小鼠 transcardially 与 pbs (ph 7.4) 其次是冰冷4% 多聚甲醛 (煤灰) (ph 7.5) 在 pbs。</li>
		<li>灌注后, 在解剖显微镜下用微剪刀和微钳将 DRGs 与神经根和坐骨神经在膝前仔细分离。将坐骨神经直接放置在4% 的粉煤灰中过夜4摄氏度。</li>
	</ol>
</li>
	<li>
荧光标记感觉神经轴突的成像与测量
	<ol>
<li>在解剖显微镜下, 用微剪刀和微钳剥去固定坐骨神经上的附着组织和膜。然后, 用 PBS 交换4% 的煤灰, 然后洗三次神经。</li>
		<li>把坐骨神经放在滑梯上, 保持笔直。在神经周围添加80µL antifade 溶液, 然后在它上放置一个盖玻片。压扁整个安装的组织。</li>
		<li>将ﬂattened 组织放在倒置 epiﬂuorescent 显微镜上, 配有用于马赛克采集和图像处理的附件。 注: 激发和发射长度为 488 nm 和 509 nm。还可以手动拍摄多个重叠图像, 并使用 ImageJ 与马赛克插件一起缝合图像。</li>
		<li>在测量再生轴突的长度时, 从粉碎部位 (标记为 10-0 epineural 缝合) 到远端轴突末端, 追踪坐骨神经中所有 identiﬁable ﬂuorescently 标记的轴突。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Saijilafu, , 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=PI3K-GSK3+signalling+regulates+mammalian+axon+regeneration+by+inducing+the+expression+of+Smad1.">PI3K-GSK3 signalling regulates mammalian axon regeneration by inducing the expression of Smad1.</a> Nature Communications. 4, 2690 (2013).</li><li>Zhang, B. Y., 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=Akt-independent+GSK3+inactivation+downstream+of+PI3K+signaling+regulates+mammalian+axon+regeneration.">Akt-independent GSK3 inactivation downstream of PI3K signaling regulates mammalian axon regeneration.</a> Biochemical and Biophysical Research Communications. 443 (2), 743-748 (2014).</li><li>Jiang, J. 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=MicroRNA-26a+supports+mammalian+axon+regeneration+in+vivo+by+suppressing+GSK3beta+expression.">MicroRNA-26a supports mammalian axon regeneration in vivo by suppressing GSK3beta expression.</a> Cell Death &amp; Disease. 6, 1865 (2015).</li><li>Krames, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+dorsal+root+ganglion+in+the+development+of+neuropathic+pain.">The role of the dorsal root ganglion in the development of neuropathic pain.</a> Pain Medicine. 15 (10), 1669-1685 (2014).</li><li>Salimzadeh, L., Jaberipour, M., Hosseini, A., Ghaderi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-viral+transfection+methods+optimized+for+gene+delivery+to+a+lung+cancer+cell+line.">Non-viral transfection methods optimized for gene delivery to a lung cancer cell line.</a> Avicenna Journal of Medical Biotechnology. 5 (2), 68-77 (2013).</li><li>Keeler, A. M., ElMallah, M. K., Flotte, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Therapy+2017:+Progress+and+Future+Directions.">Gene Therapy 2017: Progress and Future Directions.</a> Clinical and Translational Science. 10 (4), 242-248 (2017).</li><li>Neumann, E., Schaeferridder, M., Wang, Y., Hofschneider, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-Transfer+into+Mouse+Lyoma+Cells+by+Electroporation+in+High+Electric-Fields.">Gene-Transfer into Mouse Lyoma Cells by Electroporation in High Electric-Fields.</a> The EMBO Journal. 1 (7), 841-845 (1982).</li><li>Liu, C. M., 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=MicroRNA-138+and+SIRT1+form+a+mutual+negative+feedback+loop+to+regulate+mammalian+axon+regeneration.">MicroRNA-138 and SIRT1 form a mutual negative feedback loop to regulate mammalian axon regeneration.</a> Genes &amp; Development. 27 (13), 1473-1483 (2013).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Developmental Biology. 240 (1), 237-246 (2001).</li><li>Saijilafu, E. M., Hur, F. Q., Zhou, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+axon+regeneration+via+in+vivo+electroporation+of+adult+mouse+sensory+neurons.">Genetic dissection of axon regeneration via in vivo electroporation of adult mouse sensory neurons.</a> Nature Communications. 2, 543 (2011).</li><li>Pan, C., 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=Shrinkage-mediated+imaging+of+entire+organs+and+organisms+using+uDISCO.">Shrinkage-mediated imaging of entire organs and organisms using uDISCO.</a> Nature Methods. 13, 859 (2016).</li><li>Rao, R. D., Bagaria, V. B., Cooley, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterolateral+intertransverse+lumbar+fusion+in+a+mouse+model:+surgical+anatomy+and+operative+technique.">Posterolateral intertransverse lumbar fusion in a mouse model: surgical anatomy and operative technique.</a> Spine Journal. 7 (1), 61-67 (2007).</li><li>Dommisse, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood+supply+of+the+spinal+cord.+A+critical+vascular+zone+in+spinal+surgery.">The blood supply of the spinal cord. A critical vascular zone in spinal surgery.</a> The Journal of Bone and Joint Surgery. British Volume. 56 (2), 225-235 (1974).</li><li>Sorensen, D. R., Leirdal, M., Sioud, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Silencing+by+Systemic+Delivery+of+Synthetic+siRNAs+in+Adult+Mice.">Gene Silencing by Systemic Delivery of Synthetic siRNAs in Adult Mice.</a> Journal of Molecular Biology. 327 (4), 761-766 (2003).</li></ol>

作者没有什么可透露的。

一些手术步骤需要特别注意。L4 和 L5 DRGs (somas 的位置), 控制坐骨神经, 需要正确识别和注射的基因结构。否则, 在坐骨神经轴突中, GFP 标记将消失。髂嵴可以被视为有用的解剖标志, 以确定 L4 和 L5 DRGs。在大多数小鼠中, L5 和 L6 椎骨之间的关节与髂嵴12相近。另外, L3 根背根可以选择代替 L5, 特别是对免疫组化或西方印迹13的化验, 因为 L5 背节的手术暴露通常会遇?...

神经损伤或各种神经退行性疾病引起的神经系统损伤通常导致运动、感官和认知功能的缺陷。最近, 大量的努力已经致力于再生效能重建的成人神经元, 以恢复生理 functionsof 受损神经元1,2,3。神经节中的感觉神经元是一群神经细胞, 它传达不同的感官刺激, 如疼痛, 体温, 触觉, 或身体姿势, 对大脑。这些神经元都是伪单极, 包含一个单轴突, 分叉一个分支向周围延伸, 另一个分支朝向脊髓4。DRGs 中的成年感觉神经元是少数成熟的哺乳动物神经元, 在受伤后能积极地再生它们的轴突。因此, 感觉轴突损伤作为研究脑轴突再生机制的重要模型被广泛应用. 
在体内基因转染技术的建立和灵活性比使用转基因动物的时间要少, 在研究神经系统的基因功能和信号通路方面起着至关重要的作用。主要技术可以分为两种方法: 基于仪器的和基于病毒的5。以病毒为基础的成体神经元体内基因传递可以提供精确的时空操纵基因表达6。然而, 劳动密集型过程涉及基于病毒的方法, 如生产和纯化含有所需基因的病毒微粒。此外, 许多病毒载体可以激活宿主的免疫系统, 这可能会干扰数据的采集、数据分析, 并可能误导实验结果的解释。电穿孔是一种典型的以仪器为基础的转染方法, 使用电子脉冲增加细胞和核膜的通透性, 这有利于基因载体或小 RNA 寡核苷酸从细胞外的空间流入7.体外电穿孔被广泛认为是一种瞬态但高效的策略, 用于操纵多种细胞类型的靶向基因表达。虽然在体内电穿孔只会导致短暂的基因表达与病毒载体相比, 转染效率低, 它具有不同的病毒方法的优势。例如, 它可以应用到几乎所有的组织和细胞7,8,9。此外, 对某些转录蛋白编码的基因或小 RNA 寡核苷酸 (例如, siRNAs, microRNAs) 的质粒可以直接注入靶组织, 然后电脉冲, 这使得程序减少了劳动, 并耗时。此外, 转染多质粒和 RNA 寡核苷酸同时进行单电穿孔是可能的。
我们建立了体内电穿孔的方法来操纵成年小鼠感觉神经元中的基因表达, 并成功地应用和验证这种方法在许多先驱研究1,2,3 ,8,10。在这里, 我们提出了一个详细的协议, 以促进使用这种方法, 未来的研究哺乳动物轴突再生。

<table><tbody><tr><td>ECM 830 Square wave electroporation system</td><td>BTX Harvard Apparatus</td><td>45-0052</td><td>For &lt;em&gt;in vivo&lt;/em&gt; electroporation</td></tr><tr><td>Tweezertrodes electrodes</td><td>BTX Harvard Apparatus</td><td>45-0524</td><td>For &lt;em&gt;in vivo&lt;/em&gt; electroporation, 1 mm flat</td></tr><tr><td>Picospritzer III</td><td>Parker Instrumentation</td><td>1096</td><td>Intracellular Microinjection Dispense Systems</td></tr><tr><td>Glass Capillary Puller</td><td>NARISHIGE</td><td>PC-10</td><td></td></tr><tr><td>Borosilicate Glass Capillaries</td><td>World Precision Instruments, Inc.</td><td>1902328</td><td></td></tr><tr><td>Stereo Dissection Microscope</td><td>Leica</td><td>M80</td><td></td></tr><tr><td>Microsurgery Rongeur</td><td>F.S.T</td><td>16221-14</td><td></td></tr><tr><td>Microsurgery Forceps</td><td>FST by DUMONT, Switzerland</td><td>11255-20</td><td>Only for sciatic nerve crush</td></tr><tr><td>Glass Capillary</td><td>World Precision Instruments, Inc.</td><td>TW100-4</td><td>10 cm, standard wall</td></tr><tr><td>Tape</td><td>Fisherbrand</td><td>15-901-30</td><td>For fixing the mouse on the corkboard</td></tr><tr><td>2, 2, 2-Tribromoethanol (Avertin)</td><td>Sigma-Aldrich</td><td>T48402</td><td>Avertin stock solution</td></tr><tr><td>2-methyl-2-butanol</td><td>Sigma-Aldrich</td><td>152463</td><td>Avertin stock solution</td></tr><tr><td>siRNA Fluorescent Universal Negative Control #1</td><td>Sigma-Aldrich</td><td>SIC003</td><td>Non-target siRNA with fluorescence</td></tr><tr><td>microRNA Mimic Transfection Control with Dy547</td><td>Dharmacon</td><td>CP-004500-01-05</td><td>Non-target microRNA with fluorescence</td></tr><tr><td>Plasmids preparation kit</td><td>Invitrogen Purelink</td><td>K210016</td><td>GFP-coding plasmid preparation</td></tr><tr><td>Fast Green Dye</td><td>Millipore-Sigma</td><td>F7252</td><td>For better visualization of the DRG outline during injection</td></tr><tr><td>Ketamine</td><td>Putney, Inc</td><td>NDC 26637-731-51</td><td>Anesthesia induction</td></tr><tr><td>Xylazine</td><td>AnaSed</td><td>NDC 59399-110-20</td><td>Anesthesia induction</td></tr><tr><td>Acetaminophen</td><td>McNeil Consumer Healthcare</td><td>NDC 50580-449-36</td><td>Post-surgical pain relief</td></tr></tbody></table>

investigating mammalian axon regeneration: in vivo electroporation of adult mouse dorsal root ganglion

电穿孔是一种重要的非病毒基因转染方法, 将 DNA 质粒或小 RNA 分子引入细胞。背根神经节 (DRGs) 中的感觉神经元延伸一个单轴突, 两个分支。一个分支转到外围神经 (外围分支), 另一个分支进入脊髓通过背根 (中央分支)。神经损伤后, 外围分支恢复强劲, 而中心分支不再生。随着再生能力的提高, 感觉轴突再生已被广泛应用于研究哺乳动物轴突再生在周围神经系统 (三七) 和中枢神经系统 (CNS) 中的模型系统。在这里, 我们描述了一个以前建立的方法协议来操纵成熟的感官神经元在体内通过电穿孔的基因表达。在转染质粒或小 RNA 寡核苷酸 (siRNAs 或 microRNAs) 的基础上, 该方法既允许损失和增益功能的实验, 以研究基因利益或 microRNAs 的作用, 调节轴突再生在体内。此外,体内基因表达的操纵在相对较短的时间内可以在空间上和世俗地控制。该模型系统为研究哺乳动物轴突再生在体内调节的分子机制提供了一个独特的工具。

为了量化当前协议的细胞毒性, 并验证体内根节电穿孔的转染率是否足够高, 我们注射并转荧光标记的 microRNA 或 siRNA L4 和 L5 DRGs。分离 DRGs 经低温切片和免疫组化处理 (图 1A B)。在估计注射和电穿孔后的细胞存活率时, L4 和 L5 的完整 DRGs 通过冷冻切片和免疫组化进行了采集和处理。与完整 DRGs 的神经元密度相比, 神经元密度与 Tuj1 ?...

Watch this Scientific Journal Video about Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion at JoVE.com

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

电穿孔是一种有效的方法, 将兴趣基因传递给细胞。本文应用这种方法对成年小鼠背根神经节的神经元进行了研究, 并对其在体内的轴突再生模型进行了描述。

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 ...

investigating-mammalian-axon-regeneration-vivo-electroporation-adult

Research

JoVE Journal

Neuroscience

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

8.2K 次观看.  Johns Hopkins University School of Medicine. 电穿孔是一种有效的方法, 将兴趣基因传递给细胞。本文应用这种方法对成年小鼠背根神经节的神经元进行了研究, 并对其在体内的轴突再生模型进行了描述。

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

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 injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

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 ...

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. ...

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 ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

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 ...

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.

一种研究外周和中枢神经系统 Neuroregeneration 的果蝇体内损伤模型

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

医学

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

与嗅觉恩希廷格利亚合体培养的 Axotom 化大鼠视网膜甘利翁神经元，作为成人轴再生体外模型

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

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

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

大鼠背根神经节外植体和雪旺细胞共培养中外周轴突的体外髓鞘形成

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

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 well as various other cell types. Published culture protocols often refer to whole dissociated DRG cultures as being neuronal, despite the presence of fibroblasts, Schwann cells, macrophages, and lymphocytes. While these whole DRG cultures are sufficient for imaging applications where neurons can be discerned based on morphology or staining, protein or RNA homogenates collected from these cultures are not primarily neuronal in origin. Here, we describe an immunopanning sequence for cultured mouse DRGs. The goal of this method is to enrich DRG cultures for neurons by removing other cell types. Immunopanning refers to a method of removing cell types by adhering antibodies to cell culture dishes. Using these dishes, we can negatively select against and reduce the number of fibroblasts, immune cells, and Schwann cells in culture. This method allows us to increase the percentage of neurons in cultures.

This paper describes an immunopanning protocol for adult mouse dorsal root ganglia. By adhering antibodies to culture plates, we can negatively select and remove non-neuronal cells. We show that the cultures are enriched for neurons using this protocol, allowing for an in-depth study of neuronal responses to manipulation.

通过免疫淘选富集成年小鼠背根神经节神经元培养物

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 ...