在本研究中，动物实验得到了加州大学旧金山分校机构动物护理和使用委员会的批准，并按照美国国立卫生研究院实验动物护理和使用指南进行。这里使用8-12周龄的成年C57BL / 6雄性和雌性小鼠（内部繁殖）。
1. DRG样品制备
<ol><li>用2.5%Avertin麻醉小鼠（参见材料表）。通过对疼痛刺激缺乏反应来确保充分麻醉。如前所述，用1x磷酸盐缓冲盐水（PBS）然后用4%甲醛经心灌注动物，如前所述14,15,16。</li>
	<li>从脊髓解剖 DRG 组织，如前所述14,15,16。</li>
	<li>在室温下将解剖的DRGs在4%甲醛中固定3小时。</li>
	<li>将DRGs置于30%蔗糖的PBS中，在4°C过夜。</li>
	<li>准备基础最佳切削温度 （OCT）<ol><li>加入OCT化合物（参见 材料表）以覆盖铝块的顶面，并在-20°C下冷冻5-10分钟（图1A）。</li>
		<li>使用低温恒温器（参见 材料表）切掉 OCT 的顶部，厚度为 30 μm，直到其表面平坦为基础 OCT（图 1B）。</li>
		<li>在将铝块从低温恒温器上取下之前，在底座 OCT 的底部（六点钟位置）做一个标记以标记其方向（图 1C）。</li>
		<li>在以下步骤中，将标记保持在六点钟位置以保持相同的方向，这可能会导致获得 DRG 样品的更多部分。 注意：如果失去方向并且以不同的角度切割 DRG 样品，则 DRG 样品无法从上到下完全切割，这将在基础 OCT 上留下一些样品并浪费，因为在尝试切割 DRG 样品时会遇到基础 OCT（图 1C）。</li>
	</ol></li>
	<li>执行 DRG 的 OCT 嵌入<ol><li>在将 DRG 组织放在 OCT 上之前将其擦干。<ol><li>在将 DRG 添加到模块上之前，确保去除样品周围的 30% 蔗糖/PBS 溶液。</li>
			<li>通过将DRG样品放在干燥的培养皿上干燥DRG样品（图2A），并用干镊子将其从一个位置移动到另一个位置两到三次（图2B）。</li>
			<li>在移动另一个DRG样品之前，用无绒组织擦干镊子（图2C）。</li>
		</ol></li>
		<li>将干燥的 DRG 放在底座 OCT 上。<ol><li>用干镊子将干燥的DRG放在12点钟位置的底座OCT上（图1D）。</li>
			<li>在 DRG 的上边缘和基础 OCT 的上边缘之间至少保持 5 毫米的距离。</li>
			<li>将DRG的背部放在12点钟位置，将腹侧放在6点钟位置（图1D）。</li>
		</ol></li>
		<li>用 OCT 包埋 DRG 组织。<ol><li>添加更多的OCT以圆形或椭圆形覆盖整个DRG样品，DRG位于中心（图1E）。</li>
			<li>确保 OCT 的上边缘覆盖基础 OCT 上边缘的 DRG（图 1F），因为这样可以更轻松地将截面转移到载玻片上（图 3A）。 注意：如果盖子 OCT 位于基础 OCT 的中间，则基础 OCT 的上边缘将阻挡载玻片以收集该部分（图 3B）。</li>
			<li>将覆盖的OCT和DRG样品在-20°C下再冷冻5分钟（图1G）。不要在 DRG 样品周围修剪 OCT。</li>
			<li>切掉 30 μm 厚的盖子 OCT 的顶部，直到 DRG 可见。</li>
		</ol></li>
	</ol></li>
</ol>2. DRG的冷冻切片
<ol><li>将低温恒温器截面厚度更改为12μm，将DRG截面切割到载玻片上（图1H）。</li>
	<li>用冷却至-20°C的小画笔将OCT部分保持在底部。 注意：重要的是不要完全切割整个部分，而是保留 1-3 毫米未切割（图 1I）。</li>
	<li>使用室温镊子的末端轻轻触摸该部分的底部（六点钟位置），使其粘在平台表面上（图 1J）。这样可以防止截面卷曲，从而难以在载玻片上收集截面。 注意：当截面的底部粘在平台表面上，并且截面的顶部仍然与盖子 OCT 连接时，该截面呈扁平形状（图 1K），这使得在载玻片上收集截面变得更加容易。</li>
	<li>取一个带电的载玻片（参见 材料表），然后慢慢地将载玻片放在切片上。一旦该部分开始粘在载玻片上，轻轻地向后拉载玻片（图1L）。</li>
	<li>按照相同的过程将更多的 DRG 部分放到载玻片上，而不会与这些部分重叠（图 1M）。确保新的 DRG 切片未放置在另一个 DRG 切片的 OCT 上（图 4），因为这样的切片不能很好地留在载玻片上，并且可以在免疫化学染色过程中被洗掉。</li>
</ol>3.载玻片上DRG部分的尼氏染色
<ol><li>在PBS和0.1%Triton X-10中洗涤切片10分钟。然后，用PBS洗涤切片两次。</li>
	<li>将 1：300 Nissl 染色剂（参见 材料表）涂在载玻片上，并在室温下孵育 20 分钟。</li>
	<li>用PBS加0.1%Triton X-100洗涤切片。然后，用PBS洗涤切片两次，每次5分钟，然后在室温下洗涤2小时。</li>
	<li>应用 4′，6-二脒基-2-苯基吲哚 （DAPI） fluoromount-G（参见 材料表）用盖玻片覆盖切片。</li>
</ol>

小鼠背根神经节冷冻切片

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作者没有什么可透露的。

该协议为小鼠DRG的低温恒温器切片提供了简单的分步程序，以可靠地在载玻片上获得高质量的DRG切片。该协议有四个关键步骤。首先，在将 DRG 样品放到基础 OCT 上之前，必须将 DRG 样品和镊子干燥。DRG 样品周围的任何液体都会在其周围形成冰壳，导致 DRG 部分与 OCT 分离并向上弯曲。其次，如果铝块没有标记，或者如果底座 OCT 覆盖了标记，则重要的是在底座 OCT 的底部（六点钟位置）做一个标记?...

背根神经节 （DRG） 包含初级感觉神经元、组织巨噬细胞和围绕初级感觉神经元的卫星细胞 1,2,3,4。它是处理无害和有害信号的关键解剖结构，在疼痛、瘙痒和各种周围神经疾病中起关键作用5、6、7、8、9、10、11、12、13。尽管已经开发了几种方法来解剖小鼠脊髓的 DRG 组织14,15,16，但由于 DRG 组织非常小，并且 DRG 样品的低温恒温器切片往往会弯曲成卷，因此很难将低温恒温器切片正确地转移到载玻片上。然而，DRG 组织的正确冷冻切片对于免疫组化研究和 DRG 感觉神经元的结构至关重要17、18、19、20、21、22、23。此外，由于单细胞RNA测序结果揭示了人类24和小鼠25中DRG感觉神经元的显着异质性，因此对DRG组织进行适当的冷冻切片对于研究不同DRG细胞在各种生理和病理条件下的功能作用至关重要。
尽管组织透明化技术已被应用于研究 DRG26 的 3D 重建，作为冷冻切除 DRG 的替代技术，但组织透明化技术既费时又费力。相比之下，DRG的冷冻切片快速且相对容易执行，因此它仍然是DRG和中枢神经系统其他区域的免疫组化和结构研究的关键技术。然而，在载玻片上获得高质量、完整和扁平的低温恒温器切片仍然是神经科学研究中的一个挑战，因为组织的样本量很小，如DRG和某些大脑区域，并且目前没有文章描述冷冻切片小尺寸组织样本的最佳方案，如小鼠DRG。
该协议为小鼠DRG的低温恒温器切片提供了一种简单的分步技术，以可靠地在载玻片上获得尽可能多的高质量DRG切片，用于后续的DRG研究。虽然该技术专为冷冻切除 DRG 样品而设计，但可用于冷冻切除样品量较小的各种其他组织。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Avertin</td>
			<td>Sigma-Aldrich</td>
			<td>T48402-25G</td>
			<td>Anesthetize animal</td>
		</tr>
		<tr>
			<td>Epredia&#160;Cryotome Cryostat Cryocassettes, 25 mm dia. Crosshatched</td>
			<td>Fisherbrand</td>
			<td>1910</td>
			<td>Hold the OCT section at the bottom&#160;</td>
		</tr>
		<tr>
			<td>Ergo Tweezers</td>
			<td>Fisherbrand</td>
			<td>S95310</td>
			<td>Using the end of a tweezer to gently touch the bottom (6 o&#8217;clock) of the section so that it sticks to the platform surface to prevent the section from curving back in a roll&#160;</td>
		</tr>
		<tr>
			<td>Fisherbrand&#160;Superfrost Plus Microscope Slides</td>
			<td>Fisherbrand</td>
			<td>1255015</td>
			<td>To collect the DRG section&#160;</td>
		</tr>
		<tr>
			<td>Marking pens</td>
			<td>Fisherbrand</td>
			<td>133794</td>
			<td>&#160;Mark the orientation of base OCT</td>
		</tr>
		<tr>
			<td>Scigen&#160;Tissue-Plus O.C.T. Compound</td>
			<td>Fisherbrand</td>
			<td>&#160;23730571</td>
			<td>Embedding medium for frozen tissue specimens to ensure optimal cutting temperature (O.C.T.).</td>
		</tr>
	</tbody>
</table>

a procedure for mouse dorsal root ganglion cryosectioning

在炎症和神经性疼痛、瘙痒以及其他周围神经系统疾病的研究中，高质量的小鼠背根神经节 （DRG） 低温恒温器切片对于适当的免疫化学染色和 RNAscope 研究至关重要。然而，由于 DRG 组织的样本量很小，因此在载玻片上始终如一地获得高质量、完整和扁平的低温恒温器切片仍然是一个挑战。到目前为止，还没有文章描述DRG冷冻切片的最佳方案。该协议提供了一种分步方法，以解决与DRG冷冻切片相关的常见困难。本文解释了如何从DRG组织样品中去除周围的液体，将DRG切片朝向相同的方向放置在载玻片上，并在不弯曲的情况下压平载玻片上的切片。尽管该协议是为冷冻切除DRG样品而开发的，但它可以应用于许多其他样品量小的组织冷冻切片。

目前的研究从一只小鼠 L4 DRG 中收集了大约 16 个连续的高质量 DRG 切片。获得的部分没有任何失真。 图 1 描述了冷冻切片的分步过程。从组织切片中去除多余的液体如图 2 所示。 OCT包埋组织的过程如图3所示。 图 4 显示了载玻片上 DRG 部分的正确位置。 图5 显示了小鼠DRG切...

Watch this Scientific Journal Video about A Procedure for Mouse Dorsal Root Ganglion Cryosectioning at JoVE.com

作者聚焦：小鼠背根神经节冷冻切片程序  

这里介绍的是持续获得高质量背根神经节低温恒温器切片的发展。

小鼠背根神经节冷冻切片的程序

High-quality mouse dorsal root ganglion (DRG) cryostat sections are crucial for proper immunochemistry staining and RNAscope studies in the research of ...

始终如一地获得高质量、完整和扁平的小鼠背根神经节低温恒温器切片仍然是一个挑战。该研究旨在回答如何获得理想的小鼠背根神经节低温恒温器切片。该协议提供了一种简单的分步技术，用于小鼠DRG的低温恒温器切片到高质量，完整和扁平的小鼠DRG低温恒温器切片。本文解释了如何从小鼠DRG样品组织中去除周围的液体，将DRG切片放置在面向相同方向的载玻片上，并在不弯曲的情况下压平DRG切片。一个主要的挑战是鼠标 DRG 的 OCT 部分往往会弯曲成卷状，因此很难放置在载玻片上。由于小鼠DRG的尺寸较小，从一个样品中获取足够的切片也具有挑战性。

a-procedure-for-mouse-dorsal-root-ganglion-cryosectioning

Research

JoVE Journal

Neuroscience

A Procedure for Mouse Dorsal Root Ganglion Cryosectioning

5.7K 次观看.  Capital Medical University. 始终如一地获得高质量、完整和扁平的小鼠背根神经节低温恒温器切片仍然是一个挑战。该研究旨在回答如何获得理想的小鼠背根神经节低温恒温器切片。该协议提供了一种简单的分步技术，用于小鼠DRG的低温恒温器切片到高质量，完整和扁平的小鼠DRG低温恒温器切片。本文解释了如何从小鼠DRG样品组织中去除周围的液体，将DRG切片放置在面向相同方向的载玻片上，并?...

视频: 作者聚焦：小鼠背根神经节冷冻切片程序  

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 ...

科研

神经科学

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 ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

一个程序植入微丝的有组织阵列的单机录音清醒，举止动物

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

测量脊髓突触前抑制小鼠背根电位记录<em&gt;在体内</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

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 ...

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 ...

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

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

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 ...