Edith Suss-Toby is thanked for her assistance in the time-lapse microscopy and image analysis. Nofar Rada is thanked for the artistic work.

6週齢の雌C57BL / CJマウス（ハーラン、エルサレム、イスラエル）の四は、実験動物管理仕様の評価と認定のための協会によると、実験に使用しました。全ての実験手順は、施設内動物管理使用委員会と農業の規制の部門に合わせて行われました。 1.収穫脊髄<ol><li> CO 2チャンバーを用いて、マウスを安楽死させます。それが剪断力に起因する神経節根を損傷する可能性がありますように頸椎脱臼を避けてください。ここで上からは、無菌状態下にあるすべての手順を実行します。 </li><li> 70％エタノールで動物を下に浸します。このステップは、殺菌のためと内臓を通じてドラッグから髪を防止するために重要です。 </li><li>エタノールから乾燥するためにマウスを残します。神経毒性に起因する内臓とエタノールの任意の接触を防ぎます。 </li><li>マウスを乾燥させた後、腹臥位でピンを使用して、それを配置（下向き）。各後肢および前肢のピンを配置します。 </li><li>鉗子を使用して、腰椎の後方の首からcraniocaudally伸びる正中切開を行います。次に、ピンセットを用いて、左右の皮膚フラップを上げ、皮下組織を露出させます。 </li><li>頭頸接合部に到達するまで、マウスの頭蓋骨を触診します。重い鉗子を使用して、頭頸接合部（ 第 7頸椎）で子宮頸部筋肉や背骨を切断動物に対して垂直なハサミを使用して。尾側脊椎を解剖し、腰椎レベル（後肢レベル）に到達するまで、重い鉗子で背骨を切り出します。 </li><li>重い鉗子を使用して、腰椎レベル（ 第 5腰椎）での完全な尾切断を実行します。 </li><li> （フェイスアップ）、マウスをロールオーバー。 DRGは、子宮頸部の下側の椎骨レベルから抽出されていないため、切開部をカバーする必要はありません。 </li><li>撤回、腹部に首から正中線で切り倒し皮膚は横方向にしてから腹膜を開くためにカット。 </li><li> 一括腹膜内部の内臓（肝臓、脾臓、膵臓、胃、および腸）を取り外します。次に、後腹膜臓器（ すなわち 、腎臓、膵臓）を削除。頭蓋は、胸壁を開きます。 </li><li>鉗子や外科用メスを使用して、左右対称に離れて脊柱からリブの5ミリメートルを残して、リブをカット。この時点で、脊柱は、身体の残りの部分から分離されています。 </li><li>二回の冷（4°C）新鮮なリン酸緩衝生理食塩水（PBS）で血液から脊柱を洗ってください。 </li></ol> 2.分離後根神経節（DRG） <ol><li> 4Xの倍率で実体顕微鏡を使用してください。 </li><li>非接着性の非吸収プラットフォーム（Telfa /ナイロン）での背骨の位置を決めます。同じ頭尾の向きで脊椎表向きに置きます。 </li><li>任意の脊椎筋肉や結合組織を除去します。彼らのような神経のためのランドマークとしてリブを使用してください背骨を残します。リブはまた、手術器具から神経を保護します。 DRGは、椎骨の頚椎、胸椎、腰椎のサイトに置かれています。 </li><li>次に、はさみを使用すると、正中線で椎体をカットし、椎体の穏やかな横方向の後退により、脊髄およびDRGの根を公開します。 </li><li>肋間空間では、DRGに横リブに沿って内側に末梢神経に従ってください。 </li><li> DRGを識別します。これは、神経の上に横たわる「目玉焼き」の黄身のように見えます。 </li><li>神経節に神経遠位2-3ミリ残しDRGに肋間神経末端をカットし、後退のために使用されます。この「テール」は、収穫後に削除されます。 </li><li>ピンセットを用いて神経をつかみます。 DRG本体自体をつまんすると、神経細胞の損傷を引き起こし、避けるべきです。 </li><li>その前方および後方根を経由して、脊髄への取り付けに沿って近位にDRGに近づきます。 </li><li> EFを引っ張ることによって、DRGに穏やかな後退を適用しますferent神経（肋間神経切り株）は、横方向、およびDRGに近い前方および後方根をカット。 </li><li> 10％ウシ胎児血清（FCS）を補充した新鮮な氷冷DMEM、1％ペニシリン - ストレプトマイシン、1％非必須アミノ酸、1％ピルビン酸ナトリウムを充填した35 mmのペトリ皿にDRGを保ちます。 </li></ol>ペトリ皿に3注入<ol><li> ECMの凝固を防ぐために、予め冷却したピペットチップを使用して、氷の上で次の手順を実行します。 </li><li> 2〜4倍の倍率で実体顕微鏡下では、紙のグリッド上の35ミリメートルのガラスボトムシャーレを置きます。 重要：液体状態にECMを維持するために氷の上で作業してください。グリッドの中心に成長因子枯渇ECMの1ミリメートルの直径のスポット（約20μl）を作成します。 </li><li>直接可視化の下では、皿の底に近いECMスポットの中心にDRGを配置します。 注意：このプロトコルで使用される癌細胞であるマウスの膵臓癌細胞（KPC）establis他の場所で19説明したようにKPCマウスから新たに単離された腫瘍標本からHED。 </li><li>コンフルエント培養物から収穫40,000癌細胞は、PBSで一度洗って、そして氷上で40μlのECMで再懸濁します。 </li><li> DRGから各方向に500μmで実体顕微鏡の尺度を使用して、グリッドの直接可視化の下で。この時点で、ゆっくりと10,000細胞/10μlのECMをシード。行列の行列や剥離を自分の広がりを避け、皿底に近い細胞を注入するために2-10μlの予冷チップを使用してください。 （ 図1）。 </li><li>固化する10〜15分間層流フード内で皿を残します。 ECMの乾燥を避けます。 </li><li>ゆっくりとプレートの側壁に対して、前述したように調製し、DMEMを追加します。 ECM（約2ml）をカバーするのに十分なメディアを追加します。 </li><li>バック37℃のインキュベーターに皿を置きます。 </li><li>次の日に新鮮な培地で培地を交換してください。 </li><li> 2日ごとに培地を交換してください。&lt;/李&gt; </ol> 4.データ収集、タイムラプスビデオ顕微鏡<ol><li>移植後7日目に、タイムラプス顕微鏡用の皿を取ります。特定の細胞が原因で高速に移行する以前の取得を必要とするかもしれないことに注意してください。 </li><li>ライブイメージングの間、37±0.1℃、5％CO 2の平均温度で閉じた環境で皿を置きます。 </li><li>顕微鏡上に配置された電荷結合素子カメラを用いて細胞を記録します。 </li><li> 3〜6個の異なる細胞から細胞移動を追跡するために最大72時間、デジタル画像ごとに10分を取ります。 </li><li> 24時間後に培地を交換してください。 </li><li>顕微鏡ソフトウェアを使用してデータ収集及び単純な分析を行います。 </li></ol> 5.データ解析<ol><li>より高度なアプリケーション（2Dまたは3Dで、すなわち 、細胞トラッキング）について（そのようなIMARIS 4Dなど）特殊なソフトウェアを使用しています。注：このソフトウェアは、セルのxy座標を決定任意のは、運動の原点からの距離、および速度とともに、時間を与えられました。 </li><li> 10分間隔で、その後の位置の間の移動距離を測定することによって、平均速度を計算します。神経突起からの細胞フロントの距離であり、DRGに向かって細胞の一方向の動きを説明するフォワード移行指数（FMI）を計算します。 </li></ol>

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This research was supported by the ICRF Barbara S. Goodman endowed research career development award (2011-601-BGPC) and a grant from the US-Israel Binational Science Foundation. No other financial disclosures from the authors are to be made.

この記事では、神経ニッチ、DRGモデルにおける癌微小環境を再現するin vitroモデルを提示します。ビデオは、マウスにおけるDRG、その抽出、およびECMで最終的に、その培養として、解剖学的ランドマークを認識することから始めて、すべての手順を示しています。共培養癌細胞と一緒にDRGにも提示されています。 in vitroでの神経周囲ニッチ微小環境を研究するためのこのモデル?...

直接浸潤、リンパ節転移、および造血スプレッド：固形腫瘍は、主に次の3通りの方法で広めます。しかし、頻繁に神経に沿って、普及を無視して、がんの広がりの第4の手段があります。癌性神経浸潤（CNI）は、特に頭頸部の癌において、前立腺1 2、3及び膵臓癌の広がりの周知の経路である。4-8 CNIは、先頭、膵臓腺癌を有する個体の80％以上で起こります後腹膜腫瘍を腹腔神経節神経を介して広がります。これらの神経向性癌細胞は、中枢神経系（CNS）に向かって神経に沿って一方向に移行するためのユニークな能力を有する。9この知見は、神経周囲の微小環境は、腫瘍細胞によって利用され得ることを示唆している悪性の増殖を支持する因子を提供します。 CNIの研究のためのいくつかのin vitroモデルの一つは、後根神経節（DRG）/癌細胞モデルです。このモッズELが頻繁に神経間質細胞と癌細胞との間のパラクリン相互作用を研究するために使用される。10-18このモデルでは、マウスまたはヒトの癌細胞株は、新たに解離し、培養DRG調製物に隣接する細胞外マトリックス（ECM）中で増殖させます。 このビデオの記事では、膵管腺癌におけるインビトロCNIのアプリケーションを示しています。

<table border="0" cellpadding="0" cellspacing="0" width="710">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:417px;">Equipments:</td>
			<td style="width:121px;"></td>
			<td style="width:108px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Operating microscope</td>
			<td>Leica</td>
			<td>M205</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tiime Lapse System</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F4142&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical blade</td>
			<td>Sigma-Aldrich</td>
			<td>Z309036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Sigma-Aldrich</td>
			<td>S3271</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35mm petri dishes, glass bottom</td>
			<td>de groot</td>
			<td>60-627860</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company&#160;</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">70% ethanol</td>
			<td>sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cold PBS</td>
			<td>Biological industries</td>
			<td>02-023-1A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Biological industries</td>
			<td>01-055-1A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FCS</td>
			<td>Rhenium</td>
			<td align="right">10108165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin and streptomycin</td>
			<td>Biological industries</td>
			<td>01-031-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Pyruvate</td>
			<td>Biological industries</td>
			<td>03-042-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Biological industries</td>
			<td>03-020-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Growth factor depleted matrigel</td>
			<td>Trevigen</td>
			<td>3433-005-01</td>
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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.

<html>ビデオ顕微鏡画像を使用して、DRGは、癌細胞がDRGに向かってそれらのコロニーから離れて移行中に注入した後、神経突起を5~7日間発芽分かります。移植後7日目では、癌細胞は神経突起（ 図2）と接触します。 プロトコルで使用される膵臓癌細胞の前方移動指数は、他の細胞株（QLL2、B16F）（ <strong...

Watch this Scientific Journal Video about In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model at JoVE.com

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.

後根神経節モデル：癌性神経浸潤のin vitroモデルでは

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 ...

in-vitro-modeling-cancerous-neural-invasion-dorsal-root-ganglion

Research

JoVE Journal

Neuroscience

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

9.0K ビュー.  Rambam Medical Healthcare Campus. This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

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

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

拡散うつ病を使用してエピソードと慢性片頭痛の神経免疫シグナルのモデリングインビトロ</em

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

神経科学

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.

根切断術後の後根の軸索のライブイメージング

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.

培養大人の背根神経節ニューロンの軸索再生の遺伝学的研究

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

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.

後根潜在的記録によりマウスにおける脊髄シナプス前抑制を測定する<em&gt;インビボ</em

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

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

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

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

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

根神経節ニューロンと差別化された脂肪由来幹細胞を背：<em&gt;インビトロ</em末梢神経再生を研究するために&gt;共培養モデル

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

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.

後根神経節ニューロンのアライメントや髄鞘形成を強化するためのアプローチ

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

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

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

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

げっ歯類での後根神経節の脊髄および単離の油圧押出

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

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

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

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

後根神経節インジェクションおよび後根感覚軸索再生のためのモデルとして傷害をつぶします

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.

腰部後根神経節のアクセスと豚注入の椎

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.

単一の坐骨神経とフローサイトメトリーを使用して単一のマウスの後根神経節における免疫系細胞の解析

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

後根神経節モデル：癌性神経浸潤のin vitroモデルでは

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拡散うつ病を使用してエピソードと慢性片頭痛の神経免疫シグナルのモデリングインビトロ