我々の研究は、NIH、国立眼研究所の学内研究プログラムによってサポートされています。

使用されるすべての機器や試薬は無菌である。全ての動物実験は、動物のケアによって承認され、NEI / NIH（動物実験のプロトコルのNEI - 570）で委員会（ACUC）を使用して、NIHのガイドラインと規則に従って行ったれました。 1。 1日目に網膜神経節細胞のベースライン逆行性標識法（RGCs） この手順の目的は、三日間視神経挫滅の前にマウスの上丘に神経トレーサー色素を注入することにより逆行性に網膜神経節細胞を標識することです。染料は逆行網膜神経節細胞に取り込まれ、生きているRGCs（図1）のマーカーを提供する。このアプローチは、ほとんど変化1-5実行可能なRGCsの再現可能なラベルが得られます。 <ol><li>深くマウスを麻酔。頭頂部の毛をきれいに。小さな定位固定装置にマウスを置きます。保護oinmentは、両眼に適用されます。頭部の皮膚を消毒三回ヨードと70％アルコールのスクラブを使用して、各。 </li><li>頭蓋骨を露出する皮膚に切開を加えて、3％の過酸化水素を使用して、それが乾燥して清潔に保つ。 </li><li>ブレグマを識別し、マークします。ブレグマと各半球7の正中線に横0.5ミリメートルの後ろに2.9ミリメートルで、上丘、上記の穴を開けます。掘削中に、穴が二次熱損傷を防止するために掘削されたサイトへ生理食塩水適用されます。 </li><li>定位測定装置とハミルトンシリンジを用いて、脳の骨表面から1.6mmの深さでそれぞれの半球の上丘に非常にゆっくりとFluoroGold神経トレーサー色素（3％の1μlの生理食塩水に蛍光色素）を注入する。 </li><li>切開部位を縫合。鎮痛のためのブプレノルフィンの皮下注射をする。 </li><li>暖かく、乾燥した地域にマウスを移動し、直立姿勢を維持し、そしてそのホームケージにそれを返すことができるようになるまでそれを監視する。標識手順の後の最初の3日間は、全身性鎮痛薬（例えば、ブプレノルフィン）および局所抗生物質軟膏1日2回与えられているとマウスが密接に監視されています。 </li></ol> 2。 4日目の視神経挫滅この手順では、我々は、網膜神経節細胞の緩やかな死につながる軸索（図2）、の主要な損傷を引き起こすことが視神経に挫滅が適用されます。 <ol><li>トレーサー色素アプリケーションの3日後には、マウスが深く麻酔です。解剖顕微鏡を使って、片方の眼の結膜は、春のはさみのペアを使用して、4時の位置についてで切開しています。 </li><li>ゆっくりと軌道筋肉を偏向し、それらを脇に置く。目の世界からの出口で白い視神経を公開。あらゆる血管を傷つけないように細心の注意を払って。 </li><li>クロスアクションのピンセットの助けを借りて、約3秒間眼球から約2 mmに視神経に挫滅を適用します。出血を引き起こすために眼動脈を傷つけないように細心の注意が払われています。 </li><li>圧挫終了後、切開を縫合されています。 </li><li>マウスは、麻酔から回復する前に、鎮痛のためにブプレノルフィンの皮下注射をする。 </li><li>それは直立姿勢を維持し、そのホームケージにそれを返すことができるようになるまで、暖かく乾燥した領域にマウスを移動し、それを監視する。圧挫の手順の後の最初の3日間は、全身性鎮痛薬（例えば、ブプレノルフィン）および局所抗生物質軟膏1日2回与えられているとマウスが密接に監視されています。 </li></ol> 3。網膜を収穫し、11日目でRGC生存率を分析するこの手順の目的は、retinaetoは、網膜神経節細胞の生存率を分析し収穫することです。 <ol><li> 11日目（ONC後7日目）で、マウスをCO 2注入し、頚椎脱臼により安楽死されています。 </li><li>目が軌道に圧力をかけてピンセットの助けを借りて摘出されています。 </li><li>目が2時間4％パラホルムアルデヒド溶液で固定し、PBSで3回洗浄する。網膜を解剖。網膜の解剖の手順は、すでに6。Gustmann、S.らによって2010年にJoveのことで以前に公開されているので、ここでは説明されません。 </li><li>生存網膜神経節細胞のイメージは網膜の定義された領域で蛍光顕微鏡を用いて撮影され、網膜神経節細胞の密度をカウントすることができます。視神経挫滅の有無に蛍光標識された網膜神経節細胞の代表的な結果を図3に示されています。 </li></ol> 4。代表的な結果： 七日視神経挫滅後の網膜神経節細胞の生存を、分析するために、網膜は、収穫固定​​し、平らにし、マウントされた。網膜像は、蛍光顕微鏡を用いて撮影した。無傷通常のRとの比較etina（ノーマル、左）、実行可能なRGCsの数は（緑、逆行上丘に注入FluoroGold神経トレーサー色素で標識された）視神経挫滅（右、ONC）（図3）と網膜で有意に低いです。 。 <img src="/files/ftp_upload/2685/2685fig1.jpg" alt="図1" /> 図1。網膜神経節細胞のベースライン逆行性標識法 視神経挫滅後の異なる時点で実行可能な網膜神経節細胞をカウントするためには、網膜神経節細胞（視神経前に脳三日間の上丘に神経トレーサー色素（緑色）を注入して逆行性に標識される赤）圧挫。網膜神経節細胞の軸索は上丘に存在するので、トレーサーの染料は逆行RGCsで取り上げられ、生活RGCs用マーカーを提供する。 <img src="/files/ftp_upload/2685/2685fig2.jpg" alt="図2" /> 図2。視神経挫滅損傷 網膜神経節細胞の生存率の状況を分析するために、圧挫は、軸索への一次損傷を引き起こすために視神経（赤）に適用されます。これは、網膜神経節細胞の緩やかな死につながる。トレーサー色素（緑）注射の3日後には、マウスは麻酔です。片眼の結膜を切開される。軌道筋肉をそらし、それを脇に置く。目の世界からの出口で視神経を公開。クロスアクションのピンセットを使用して約3秒間眼球から約2 mmに視神経に挫滅を適用します。 <img src="/files/ftp_upload/2685/2685fig3.jpg" alt="図3" /> 図3。視神経挫滅の有無に蛍光標識された網膜神経節細胞 七日視神経挫滅後の網膜神経節細胞の生存を、分析するために、網膜は、収穫固定​​し、平らにし、マウントされた。網膜像は、蛍光顕微鏡を用いて撮影した。無傷正常網膜（左、ノーマル）、実行可能なRGCsの数（緑、逆行上丘に注入FluoroGold神経トレーサー色素で標識された）と比較して視神経挫滅（右、ONCと網膜で有意に低かった）（図3）。スケールバー：50μm以下

<ol>
	<li>Yoles, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GM1+reduces+injury-induced+metabolic+deficits+and+degeneration+in+the+rat+optic+nerve.">GM1 reduces injury-induced metabolic deficits and degeneration in the rat optic nerve.</a> Investigative ophthalmology &#38; visual science. 33, 3586-3591 (1992).</li><li>Fisher, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccination+for+neuroprotection+in+the+mouse+optic+nerve:+implications+for+optic+neuropathies.">Vaccination for neuroprotection in the mouse optic nerve: implications for optic neuropathies.</a> J Neurosci. 21, 136-142 (2001).</li><li>Levkovitch-Verbin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RGC+death+in+mice+after+optic+nerve+crush+injury:+oxidative+stress+and+neuroprotection.">RGC death in mice after optic nerve crush injury: oxidative stress and neuroprotection.</a> Investigative ophthalmology &#38; visual science. 41, 4169-4174 (2000).</li><li>Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-B+inhibits+apoptosis+via+VEGFR-1-mediated+suppression+of+the+expression+of+BH3-only+protein+genes+in+mice+and+rats.">VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats.</a> The Journal of clinical investigation. 118, 913-923 (2008).</li><li>Tang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+effect+of+PDGF-CC+rescues+neurons+from+apoptosis+in+both+brain+and+retina+by+regulating+GSK3beta+phosphorylation.">Survival effect of PDGF-CC rescues neurons from apoptosis in both brain and retina by regulating GSK3beta phosphorylation.</a> The Journal of experimental medicine. 207, 867-880 (2010).</li><li>Gustmann, S., Dunker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo-like+organotypic+murine+retinal+wholemount+culture.">In vivo-like organotypic murine retinal wholemount culture.</a> J Vis Exp. , (2010).</li><li>Franklin, K., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li></ol>

利害の衝突は宣言されません。

視神経挫滅モデルマウスは、RGC死と生存の過程を研究するのに便利です。このモデルはまた、しばしばRGCアポトーシスと生存の異なる試薬と遺伝子の影響を調査するために使用されます。このモデルの利点の1つは、最小限のバリエーションを持つ再現性の高い学位を持っているということです。 しかし、特別な注意はこのモデルではいくつかのステップが必要です。彼?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Stereotaxic apparatus</td>
<td>&nbsp;</td>
<td>ASI Instruments</td>
<td>SAS-4100</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>PZMIV-BS </td>
<td>&nbsp;</td>
<tr>
<td>Scalpel handle</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>10003-12 </td>
<td>&nbsp;</td>
<tr>
<td>Scalpel Blades #15</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>10015-00</td>
<td>&nbsp;</td>
<tr>
<td>Microdrill</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>18000-17</td>
<td>&nbsp;</td>
<tr>
<td>Suture needle</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>12050-03</td>
<td>&nbsp;</td>
<tr>
<td>Spring scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>16144-13</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools </td>
<td>11252-50</td>
<td>&nbsp;</td>
<tr>
<td>Hamilton syringe</td>
<td>&nbsp;</td>
<td>Hamilton Company </td>
<td>88400</td>
<td>&nbsp;</td>
<tr>
<td>Gauze sponges</td>
<td>&nbsp;</td>
<td>Office Depot</td>
<td>674889</td>
<td>&nbsp;</td>
<tr>
<td>Fluorochrome</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>39286</td>
<td>&nbsp;</td>
<tr>
<td>Alcohol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>459844</td>
<td>70%</td>
</tr>
<tr>
<td>H2O2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3410</td>
<td>3%</td>
</tr>		</tbody>
		</table>
		</div>

an optic nerve crush injury murine model to study retinal ganglion cell survival

視神経への損傷は不可逆的な視力喪失につながる網膜神経節細胞（RGCs）、の緩やかな死が続く、軸索変性につながることができます。人間のような疾患の例としては、外傷性視神経障害、緑内障の視神経の変性などがあります。それは、視力低下や失明につながる視神経の頭の中で典型的な変化、進歩的な視神経の変性、および網膜神経節細胞の消失、制御されていない場合、によって特徴付けられる。 視神経クラッシュ（ONC）傷害のマウスモデルでは、外傷性視神経障害のための重要な実験的な疾患モデル、このモデルでは緑内障など、視神経が徐々に網膜神経節細胞のアポトーシスに導くに圧挫です。この疾患モデル、治療手段の開発に不可欠な神経細胞死と生存、の一般的なプロセスとメカニズムを研究するために使用することができます。さらに、薬理学的および分子的アプローチは、異なるタイプの視神経障害の治療に潜在的な治療薬を特定し、テストするには、このモデルで使用することができます。 ここでは、1日目における網膜神経節細胞のベースライン逆行性標識法（RGCs）、4日目（II）視神経挫滅、（III）網膜を収穫し、一日でRGC生存率を分析（I）のステップのデモンストレーションでステップを提供する11、および（IV）代表の結果。

Watch this Scientific Journal Video about An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival at JoVE.com

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

このプロトコルは、逆行性に網膜神経節細胞、およびどのように続いて網膜神経節細胞の生存とアポトーシスを分析するために視神経挫滅するためにラベルを付ける方法を示しています。それは、緑内障を含む視神経障害の様々なタイプ、のための実験的疾患モデルです。

網膜神経節細胞の生存を研究するために視神経挫滅モデルマウス

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

an-optic-nerve-crush-injury-murine-model-to-study-retinal-ganglion

Research

JoVE Journal

Neuroscience

30.2K ビュー.  NIH. このプロトコルは、逆行性に網膜神経節細胞、およびどのように続いて網膜神経節細胞の生存とアポトーシスを分析するために視神経挫滅するためにラベルを付ける方法を示しています。それは、緑内障を含む視神経障害の様々なタイプ、のための実験的疾患モデルです。

ビデオ: An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Optic Nerve Transection: A Model of Adult Neuron Apoptosis in the Central Nervous System

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be accessed within the orbit of the eye and completely transected (axotomized), cutting the axons of the entire RGC population. Optic nerve 
transection is a reproducible model of apoptotic neuronal cell death in the adult CNS 1-4. This model is particularly attractive because the vitreous
 chamber of the eye acts as a capsule for drug delivery to the retina, permitting experimental manipulations via intraocular injections. The diffusion of chemicals 
through the vitreous fluid ensures that they act upon the entire RGC population. Moreover, RGCs can be selectively transfected by applying short interfering RNAs 
(siRNAs), plasmids, or viral vectors to the cut end of the optic nerve 5-7 or injecting vectors into their target, the superior colliculus 8. 
This allows researchers to study apoptotic mechanisms in the desired neuronal population without confounding effects on other bystander neurons or surrounding glia. 
An additional benefit is the ease and accuracy with which cell survival can be quantified after injury. The retina is a flat, layered tissue and RGCs are localized in 
the innermost layer, the ganglion cell layer. The survival of RGCs can be tracked over time by applying a fluorescent tracer (3% Fluorogold) to the cut end of the 
optic nerve at the time of axotomy, or by injecting the tracer into the superior colliculus (RGC target) one week prior to axotomy. The tracer is retrogradely transported, labeling 
the entire RGC population. Because the ganglion cell layer is a monolayer (one cell thick), RGC densities can be quantified in flat-mounted tissue, without the need 
for stereology. Optic nerve transection leads to the apoptotic death of 90% of injured RGCs within 14 days postaxotomy 9-11. RGC apoptosis has a 
characteristic time-course whereby cell death is delayed 3-4 days postaxotomy, after which the cells rapidly degenerate. This provides a time window for 
experimental manipulations directed against pathways involved in apoptosis.

Optic Nerve transection is a widely used model of adult CNS injury. Ninety percent of retinal ganglion cells (RGCs) whose axons are completely transected (axotomy) die within 14 days after axotomy. This model is easily amenable to experimental manipulations and highly reproducible.

視神経切断：中枢神経系における成人ニューロンのアポトーシスのモデル

Retinal ganglion cells (RGCs) are CNS neurons that output visual information from the retina to the brain, via the optic nerve. 
The optic nerve can be ...

神経科学

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

遺伝的に標識された網膜神経節細胞からの光は応答を記録するために隔離された網膜の準備

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

実験視神経の脱髄のラットモデルにおける視覚誘発電位の記録

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

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

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

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

網膜神経節細胞の定義されたサブセットの単一細胞RNA-Seq

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

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

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

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

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 ...

Partial Optic Nerve Transection in Rats: A Model Established with a New Operative Approach to Assess Secondary Degeneration of Retinal Ganglion Cells

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection is considered a useful and reproducible model. Compared with other optic nerve injury models used commonly for assessing secondary degeneration, e.g. complete optic nerve transection and optic nerve crush models, the partial optic nerve transection model is superior as it distinguishes primary from secondary degeneration in situ. Therefore, it serves as an excellent tool for evaluating secondary degeneration. This study describes a novel operative approach of partial optic nerve transection by directly accessing the area of the retrobulbar optic nerve through the orbital lateral wall of the eyeball. Moreover, we present a newly designed, low cost surgical instrument to assist with transection. As demonstrated by the representative results in distinguishing the boundary of primary and secondary injury areas, the new approach and instrument ensures high efficiency and stability of the model by providing adequate space for surgical operation. This in turn makes it easy to separate the meningeal sheath and ophthalmic vessels from the optic nerve before transection. An additional benefit is that this space-saving operative approach improves the investigators&#39; ability to administer drugs, carriers, or selective RGC tracers to the stump of the partially transected optic nerve, allowing the exploration of mechanisms behind secondary injury in RGCs, in a new way.

Secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. This study describes an innovative operative approach for partial optic nerve transection. The use of this space-saving operative approach extends the model's application range, and allows exploration of secondary injury mechanisms in RGCs in a new way.

ラットの視神経の部分断裂: 網膜神経節細胞の二次変性を評価する新しい手術アプローチとの確立モデル

Previous studies have shown that the secondary degeneration of retinal ganglion cells (RGCs) occurs commonly in glaucoma. Partial optic nerve transection ...

Ethanol-Induced Cervical Sympathetic Ganglion Block Applications for Promoting Canine Inferior Alveolar Nerve Regeneration Using an Artificial Nerve

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen films. Favorable clinical outcomes have been achieved when using these tubes for the treatment of damaged inferior alveolar nerve (IAN). A critical factor for the successful nerve regeneration using PGA-C tubes is blood supply to the surrounding tissue. Cervical sympathetic ganglion block (CSGB) creates a sympathetic blockade in the head and neck region thus increasing blood flow in the area. To ensure an adequate effect, the blockade must be administered with local anesthetics one to two times a day for several consecutive weeks; this poses a challenge when creating animal models for investigating this technique. To address this limitation, we developed an ethanol-induced CSGB in a canine model of long-term increase in blood flow in the orofacial region. We examined whether IAN regeneration via PGA-C tube implantation can be enhanced by this model. Fourteen Beagles were each implanted with a PGA-C tube across a 10-mm gap in the left IAN. The IAN is located within the mandibular canal surrounded by bone, therefore we chose piezoelectric surgery, consisting of ultrasonic waves, for bone processing, in order to minimize the risk of nerve and vessel injury. A good surgical outcome was obtained with this approach. A week after surgery, seven of these dogs were subjected to left CSGB by injection of ethanol. Ethanol-induced CSGB resulted in improved nerve regeneration, suggesting that the increased blood flow effectively promotes nerve regeneration in IAN defects. This canine model can contribute to further research on the long-term effects of CSGB.

We evaluated the effect of cervical sympathetic ganglion block on nerve repair using artificial nerve conduits. Male beagle dogs were each implanted with an artificial nerve across a 10-mm gap in the left inferior alveolar nerve; left cervical sympathetic ganglion was blocked by injecting 99.5% ethanol via lateral thoracotomy.

人工神経を用いた犬の下歯槽神経再生を促進するためエタノールによる頸部交感神経節ブロック ・ アプリケーション

Polyglycolic acid collagen (PGA-C) tubes are bio-absorbable nerve tubes filled with collagen of multi-chamber structure, which consist of thin collagen ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神経クラッシュ損傷のラットモデルにおける機能的評価のための3D運動学的解析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Multifactorial Assessment of Motor Behavior in Rats after Unilateral Sciatic Nerve Crush Injury

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In addition, in the research field of movement disorders, sciatic crush injury has been employed to trigger a dystonia-like phenotype in genetically predisposed DYT-TOR1A rodent models of dystonia. To achieve consistent, reproducible and comparable results after a sciatic nerve crush injury, a standardized method for inducing the nerve crush is essential, in addition to a standardized phenotypical characterization. Attention must be paid not only to the specific assortment of behavioral tests, but also to the technical requirements, the correct execution and consecutive data analysis. This protocol describes in detail how to perform a sciatic nerve crush injury and provides a behavioral test battery for the assessment of motor deficits in rats that includes the open field test, the CatWalk XT gait analysis, the beam walking task, and the ladder rung walking task.

We provide a protocol for the assessment of motor behavior via a behavioral test battery in rats after sciatic nerve crush injury.

一方的坐骨神経クラッシュ傷害後のラットにおける運動行動の多因子的評価

The induction of a peripheral nerve injury is a widely used method in neuroscience for the assessment of repair and pain mechanisms among others. In ...

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury are essential for translating novel therapeutic strategies from rodent models to clinical application due to their closer similarities to humans in size and anatomy. Here we describe some in vivo methods to evaluate the function and structure of the retinal ganglion cells (RGCs) and optic nerve (ON) in large animals, including visual evoked potential (VEP), pattern electroretinogram (PERG) and optical coherence tomography (OCT). Both goat and non-human primate were employed in this study. By presenting these in vivo methods step by step, we hope to increase experimental reproducibility among different labs and facilitate the usage of large animal models of optic neuropathies.

In Vivo Methods to Assess Retinal Ganglion Cell and Optic Nerve Function and Structure in Large Animals

Here we demostrate several in vivo tests (flash visual evoked potential, pattern electroretinogram and optic coherence tomography) in goat and rhesus macaque to understand the structure and function of the optic nerve and its neurons.

イン・ヴィヴォ 大型動物の眼球神経節細胞と視神経機能と構造を評価する方法

The optic nerve collects axons signals from the retinal ganglion cells and transmits visual signal to the brain. Large animal models of optic nerve injury ...