German Research Foundation (FOR2240 "(Lymph) Angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye" to CC and LMH; HE 6743/2-1 and HE 7643/3-1 to LMH; CU47/6-1 to CC), German Cancer Aid (to LMH and CC), GEROK program University of Cologne (to SLS and LMH), and EU COST BM1302 "Joining Forces to Corneal Regeneration" (to CC).

ヒト組織の使用を見直し、施設内倫理委員会または同等の承認を得なければなりません。ここで説明する作業は、地元の倫理委員会によって承認され、科学的審査の承認を持っていました。この作品は、ヘルシンキ宣言に従って実施しました。人間の強膜標本は、眼科学科のアイバンク、ケルン大学、ドイツで（最大死後時間24時間）、地球ドナーの目から得ました。 1.実験の準備<ol><li> 96％エタノールを調製し、別のチューブ中で緩衝化生理食塩水（PBS）をリン酸。 2％ウシ血清アルブミン（BSA）を含むPBSの推奨希釈率で一次抗体を調製し、氷上で全ての溶液を保持します。サンプルあたり100μlのを準備します。 </li><li>すべての楽器をきれいにし、鋭いメスを使用しています。同じメスで切断を繰り返した後、メスを変更します。 </li><li> 1-2コリブリ鉗子、ストレートマイクロ解剖ハサミ、＃10メスを取得または眼科用メスマイクロ羽、および4 26 G針。 </li><li>ポリスチレン板の周りにアルミホイルを巻きまたは組織を固定するためにコルク板を使用しています。双眼実体顕微鏡の下に働きます。必要に応じて層流の下に滅菌作業。 </li></ol> 2.強膜を準備<ol><li>静かに電球を保持し、強角膜部分に穿孔穿孔を行い、その後、トレパンを回転させます。均等にラウンドカットを実行するために表面に慎重かつ均等にトレパンを回転させます。 15.5ミリメートルサイズのトレパンを使用してください。湾曲したハサミで残りの添付ファイルをカットします。強角膜穿孔を削除します。これは、前方に開いた電球になります。 <ol><li>上向きに開口部を持つ綿棒に強膜を置きます。強膜からの両方の層（網膜およびブドウ膜）をやってのけるコリブリ鉗子を使用して、網膜およびブドウ膜を削除します。内側の強膜に色素性ブドウ膜を放置しないでください。乳頭から網膜およびブドウ膜を除去するために、湾曲したハサミを使用してください。レムを削除しますaining結膜、外眼筋、および表面的な強膜からのテノン嚢。 </li></ol></li><li>ストレート型はさみとコリブリ鉗子を使用して、必要なサイズの強膜サンプルを削除してください。約2cmに別の場所から2サイズ強膜サンプルを取ります。そのため、小さなサイズで、優しく強膜を保持し、ストレート型のはさみを使用して、正方形として必要なサイズを切り出します。強角膜穿孔は強膜サンプルの前縁を定義します。 <ol><li>繰り返しつかまれた組織は、共焦点顕微鏡分析のために適していない領域として鉗子でつかんでは避けてください。実験の種類に応じて、サンプルの必要な数を用意（ 例えば、前方対後方、 図1と比較）及び使用される抗体の量は。 </li></ol></li><li>後で使用するために、1.5ミリリットルチューブにサンプルを置きます。 15分間1.5ミリリットル96％エタノール中のサンプルを修正し、その後、5分間、それらを各3回を洗いますシェーカー上で1.5ミリリットルのPBSインチ凍結組織で作業する場合は、スナップ凍結する前にこの手順を実行します。 </li><li>新鮮な組織を使用してください。これが不可能な場合には、液体窒素中で強膜を凍結し、後で使用するために-20℃でそれを維持スナップ。凍結組織で作業する場合は、使用前に解凍。解凍と凍結の繰り返し繰り返しは避けてください。 </li><li> 2時間室温で1.5ミリリットルの5％BSAを含むPBS中で各サンプルを保管してください。このステップでは、積層するのに役立つサンプルの膨潤を誘発し、また、非特異的結合を防ぐことができます。 </li><li>サンプルが膨潤した後、積層に強膜の正方形を準備します。 例えば 、双眼実体顕微鏡顕微鏡の下に働きます。 26 G針を使用して、アルミ箔やコルク板に包まれたポリスチレン膜に後部強膜のサンプルを貼り付けます。 </li><li>彼らは、顕微鏡下に視野を妨げることはありませんので、針の端を曲げます。 </li><li>全層強膜SAMPを積層レ。 <ol><li>まず、コリブリ鉗子を使用して、前方の強膜のエッジを保持します。その後、慎重に＃10メス、眼科用メスマイクロ羽、またはへらの刃を用いて表面的な強膜の薄い層をカット。水平にメスを持ち、下地層からできるだけ薄く表層を切りました。 </li><li>強膜広場から薄い強膜層を分析。この方法は、線維柱帯切除術の際にフラップを準備する標準的な外科技術に匹敵し、30-80μmの厚い層（ 図2を比較）をもたらすべきです。 </li></ol></li><li> 例えば 、50μlのPBSをピペットを用いて組織にPBSを少量添加することにより、強膜の乾燥を防ぎます。 </li><li>防水色で例えばと層（表面的で深遠な）（はexternとインターンの方に）96ウェルプレートに100μlのPBSにカット層を入れて、両方の向きにラベルを付けます。 </li><li>組織が完全に積層されて繰り返して、2.7から2.9を繰り返します。&lt;/李&gt; </ol> 3.免疫組織化学を実行します<ol><li>推奨希釈に必要な一次抗体を100μlを追加します。ここでは、 例えば、使用CD 31（モノクローナルマウス抗ヒト）およびLYVE1抗体（ウサギ抗ヒト）、1の希釈の両方において（2％BSAを含む）PBS 100と一晩4℃でインキュベートします。 96ウェルプレート中の培地を変更するには、ウェルの壁にピペットを保持し、流体を除去。 </li><li>翌日、シェーカー上で200から300μlのPBSで5分間のサンプルを3回ずつ洗浄します。対応する二次抗体を100μlを追加します。ここでヤギ抗マウスフルオレセインイソチオシアネート（FITC）およびヤギ抗ウサギシアニン染料3（Cy3の）を使用し、1-2時間室温でサンプルをインキュベートします。 2％ヤギ血清を含むPBS中で300：二次抗体1を希釈。 </li><li>シェーカー上で200から300μlのPBS中で5分間、再び3回ずつサンプルをすすいでください。 </li><li>核染色を行い、 例えば </em&gt;の。 4 &#39;、6-ジアミジノ-2-フェニルインドール（DAPI）（希釈1：2,000、100μlの各ウェル中で、室温で10分間インキュベートする）、次いで、シェーカー上で200〜300μlのPBSで再度2回洗浄します。 </li><li> 、顕微鏡スライド上にサンプルを転送し、蛍光封入剤の1〜2滴でそれらを埋め込む、カバースリップを追加し、透明ニスでエッジを覆うことにより、それをシールし、4℃で保存し、後で使用するために、または直接共焦点でスライドを調べます顕微鏡。 </li><li>サンプルを分析するために、共焦点顕微鏡（または同等）を使用します。所望の倍率、 例えば 10-40X倍率を使用してください。 </li><li>サンプルの厚さを確認するには、Z-スタックを用いた共焦点顕微鏡で測定します。厚さは赤道と後方（導入を比較）異なるので可能な強膜の部分の量は、強膜の場所によって異なります。 </li></ol>

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The authors declare no financial disclosures. The contents of this article are solely the responsibility of the authors.

人間の強膜を積層することは、この組織の共焦点顕微鏡検査を行う方法です。このプロセスにおける重要なステップは、組織を固定するための代わりホルマリンエタノールの使用です。代わりに、固定のためにホルマリンのエタノールを使用する場合に我々の経験では、より良好な結果が得られます。ブラントメスは、プロシージャを悪化させるし、避けるべきです。同様に、強膜の乾燥ま?...

強膜は、密性結合組織で構成されている、目を覆う硬質の外側の層です。それは、眼内の構造を保護し、眼内圧を維持するのに役立ちます。このように、強膜は、明確なビジョンのために不可欠です。これは、リンパ管1,2を欠いており、それによって、それとリンパのない眼の内部3-7との間に、外リンパのない境界線を形成しています。また、それによって腱と解剖学的類似性を共有し、外眼筋のための結合部位を提供します。 強膜は、主にI型コラーゲンの密な束で構成され、コラーゲンタイプIII、IV、V、VI、VIII 8,9及びエラスチン10,11のより小さい数を有するため、この組織は、免疫組織化学のために使用することは容易ではありません。 （1）表在血管新生した上強膜、結膜およびテノン嚢の下に発見され、側面と目に向けて：解剖学的に、強膜は、3つの主要な層に分離することができます電子バック軌道に直面して、目の。 （2）強膜間質、強膜の主要部分を、 （3）直接ブドウ膜の上に位置する薄い着色層であるラミナフスカ、。強膜に関する当社の解剖学的知識は、20 世紀の前半から主茎。当時、研究者は、主にインドのインク注入12と血管キャスティング13-15を使用して、血管系の解剖学を研究しました。その後、それは血管造影の研究16-19で調査しました。 その時以来、古い技術が改善されていると新しいものは、私たちは前の解剖学的知識を補完することができましたが開発されています。我々はリンパ血管内皮特異的なヒアルロン酸受容体1（LYVE1）20またはポドプラニン21のような信頼性のリンパマーカーを持っていたので、例えば、それは約10年となっています。共焦点顕微鏡は、異なるTIの解剖学的特徴を研究するための新たな可能性を提供しています目のssues。複数の汚れが細胞のマーカーを区別するための又は血管及び他の解剖学的構造に関連する細胞の局在のために使用されることが可能になります。サンプルはより大きなサイズのものであり、ときに特定の細胞型の検索では、私たちはサンプルをスキャンすることを可能にするとき、それは概要を説明します。 Zスタック技術を、共焦点顕微鏡は、100〜200マイクロメートルまでのサンプルのために使用することができます。強膜は、筋肉の挿入および後部磁極11で1ミリメートルの背後に0.3ミリメートルの厚さが異なります。その厚さと不透明さの両方のために、強膜は、伝統的な方法を用いた共焦点顕微鏡観察には適していません。 これを解決するには、強膜組織は、共焦点顕微鏡との分析を可能にするために積層しました。この技術は、人間の強膜の両方の生理学的および病理学的状況のより良い理解を得るために有用です。

<table>
	<tbody>
		<tr>
			<td>96% ethanol</td>
			<td>Merck Chemicals, Darmstadt, Germany</td>
			<td>P075.4</td>
			<td></td>
		</tr>
		<tr>
			<td>binocular stereo microscope&#160;</td>
			<td>Motic, Hongkong, China</td>
			<td>n.a</td>
			<td></td>
		</tr>
		<tr>
			<td>26G needles&#160;</td>
			<td>Terumo, Leuven, Belgium</td>
			<td>303800</td>
			<td></td>
		</tr>
		<tr>
			<td>15.5mm trepan</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>n.a</td>
			<td></td>
		</tr>
		<tr>
			<td>no.10 scalpel&#160;</td>
			<td>Feather, pfm medical, Osaka, Japan</td>
			<td>2E+08</td>
			<td></td>
		</tr>
		<tr>
			<td>ophthalmic scalpel micro feather&#160;</td>
			<td>Feather, pfm medical, Osaka, Japan</td>
			<td colspan="2">no. 7657BR</td>
		</tr>
		<tr>
			<td>CD 31 antibody (monoclonal mouse anti human)</td>
			<td>Dako, USA</td>
			<td>IR610</td>
			<td></td>
		</tr>
		<tr>
			<td>LYVE1 antibody&#160; (polyclonal rabbit anti human)</td>
			<td>Zytomed, Germany</td>
			<td colspan="2">RBK014-05</td>
		</tr>
		<tr>
			<td>goat anti mouse FITC antibody</td>
			<td>Sigma Aldrich, Steinheim, Germany</td>
			<td>F0257</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rabbit Cy3 antibody</td>
			<td>Dianova, Germany</td>
			<td colspan="2">111-165-003</td>
		</tr>
		<tr>
			<td>Goat Serum normal</td>
			<td>Dako, Glostrup, Denmark</td>
			<td colspan="2">X090710-8</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Carl Roth GmbH, Karlsruhe, Germany</td>
			<td>6335.1</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides&#160;</td>
			<td>Engelbrecht, Ederm&#252;nde, Germany</td>
			<td colspan="2">WC7695002</td>
		</tr>
		<tr>
			<td>Coverslips 24x24mm</td>
			<td>Th. Gayer, Lohmar, Germany</td>
			<td>7695026</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO fluorescent mounting medium&#160;</td>
			<td>DAKO, USA</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM Meta 510 confocal microscopy&#160;</td>
			<td>Carl Zeiss AG, Jena, Germany</td>
			<td>n.a</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a laminating technique to perform confocal microscopy of the human sclera

強膜はカバーし、目を保護する密な結合組織です。これは、主に高密度のコラーゲン束（タイプI、III、IV、V、VI、及びVII）からなります。 、その自己蛍光、不透明さ、及び厚さは、共焦点顕微鏡法のために適切であることが見出されていません。免疫組織化学のためにパラフィンに包埋ホルマリン固定強膜を使用して、ここに提示されている1つの代替アプローチは、抗原回復のための組織を予熱する場合は特に、技術的な課題があります。強膜は、細胞及び血管の両方において比較的悪いため、大きな組織サンプルを使用することは、望む細胞を防ぎ、血管および他の解剖学的部位に関連し、その局在化を理解するのに役立つように調べました。共焦点顕微鏡下でより大きな組織サンプルの分析を可能にするために、積層技術は、強膜の薄層を作成するために行きました。 CD31血管およびリンパ管の内皮hyaluの結果の分析を以下のローナン受容体1（LYVE1）科学的な審査の承認が得られたために陽性細胞は、この方法の利点と限界が議論されています。

ここで行う代表的な実験では、この特定の積層技術の使用に由来する明白な利点があります。最初の実験は、3つの代表的な写真（ 図3）内の強膜上の血管叢の多様なネットワークを示す図です。血管はCD31陽性です。 第二の実験は、特定のLYVE1 +上強膜の細胞とCD31陽性の血管との関係では、免疫細胞を示してい...

Watch this Scientific Journal Video about Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera at JoVE.com

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used.

人間の強膜の共焦点顕微鏡法を実行するために積層技術を用いて、


The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and ...

using-laminating-technique-to-perform-confocal-microscopy-human

Research

JoVE Journal

Medicine

7.5K ビュー.  University of Cologne. Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used. 

ビデオ: Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. Conventional methods assess neurological deficits and electrophysiology and quantitative sensory testing quantifies functional alterations to detect neuropathy. However, the earliest damage appears to be to the small fibres and yet these tests primarily assess large fibre dysfunction and have a limited ability to demonstrate regeneration and repair. The only techniques which allow a direct examination of unmyelinated nerve fibre damage and repair are sural nerve biopsy with electron microscopy and skin-punch biopsy. However, both are invasive procedures and require lengthy laboratory procedures and considerable expertise. Corneal Confocal microscopy is a non-invasive clinical technique which provides in-vivo imaging of corneal nerve fibres. We have demonstrated early nerve damage, which precedes loss of intraepidermal nerve fibres in skin biopsies together with stratification of neuropathic severity and repair following pancreas transplantation in diabetic patients. We have also demonstrated nerve damage in idiopathic small fibre neuropathy and Fabry's disease.

Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity.

角膜共焦点顕微鏡：末梢神経障害で小型ファイバ病理を定量化するための新しい非侵襲的方法

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

医学

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

人間の神経筋システムを研究するために経頭蓋磁気刺激の活用

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Probe-based Confocal Laser Endomicroscopy of the Urinary Tract: The Technique

Probe-based confocal laser endomicroscopy (CLE) is an emerging optical imaging technology that enables real-time in vivo microscopy of mucosal surfaces during standard endoscopy. With applications currently in the respiratory1 and gastrointestinal tracts,2-6 CLE has also been explored in the urinary tract for bladder cancer diagnosis.7-10 Cellular morphology and tissue microarchitecture can be resolved with micron scale resolution in real time, in addition to dynamic imaging of the normal and pathological vasculature.7
The probe-based CLE system (Cellvizio, Mauna Kea Technologies, France) consists of a reusable fiberoptic imaging probe coupled to a 488 nm laser scanning unit. The imaging probe is inserted in the working channels of standard flexible and rigid endoscopes. An endoscope-based CLE system (Optiscan, Australia), in which the confocal endomicroscopy functionality is integrated onto the endoscope, is also used in the gastrointestinal tract. Given the larger scope diameter, however, application in the urinary tract is currently limited to ex vivo use.11 Confocal image acquisition is done through direct contact of the imaging probe with the target tissue and recorded as video sequences. As in the gastrointestinal tract, endomicroscopy of the urinary tract requires an exogenenous contrast agent&mdash;most commonly fluorescein, which can be administered intravenously or intravesically. Intravesical administration is a well-established method to introduce pharmacological agents locally with minimal systemic toxicity that is unique to the urinary tract. Fluorescein rapidly stains the extracellular matrix and has an established safety profile.12 Imaging probes of various diameters enable compatibility with different caliber endoscopes. To date, 1.4 and 2.6 mm probes have been evaluated with flexible and rigid cystoscopy.10 Recent availability of a &lt; 1 mm imaging probe13 opens up the possibility of CLE in the upper urinary tract during ureteroscopy. Fluorescence cystoscopy (i.e. photodynamic diagnosis) and narrow band imaging are additional endoscope-based optical imaging modalities14 that can be combined with CLE to achieve multimodal imaging of the urinary tract. In the future, CLE may be coupled with molecular contrast agents such as fluorescently labeled peptides15 and antibodies for endoscopic imaging of disease processes with molecular specificity.

Probe-based confocal laser endomicroscopy enables real-time microscopy of the human urinary tract during cystoscopy, providing dynamic, intravital imaging of pathological states such as bladder cancer with cellular resolution. Endomicroscopy may augment the diagnostic accuracy of standard white light endoscopy and provide intraoperative image guidance to improve surgical resection.

尿路のプローブベースの共焦点レーザーEndomicroscopy：テクニック

Probe-based  confocal laser endomicroscopy (CLE) is an emerging optical imaging technology  that enables real-time in vivo microscopy of mucosal surfaces ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脳Microslicesの興奮毒性刺激<em&gt;インビトロ</em&gt;脳卒中のモデル

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

変更されたベルイストローム法を用いたヒト骨格筋生検手順

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Construction of Vapor Chambers Used to Expose Mice to Alcohol During the Equivalent of all Three Trimesters of Human Development

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal Alcohol Spectrum Disorders (FASDs). At the most severe end of the spectrum is Fetal Alcohol Syndrome (FAS), characterized by growth retardation, craniofacial dysmorphology, and neurobehavioral deficits. Studies with animal models, including rodents, have elucidated many molecular and cellular mechanisms involved in the pathophysiology of FASDs. Ethanol administration to pregnant rodents has been used to model human exposure during the first and second trimesters of pregnancy. Third trimester ethanol consumption in humans has been modeled using neonatal rodents. However, few rodent studies have characterized the effect of ethanol exposure during the equivalent to all three trimesters of human pregnancy, a pattern of exposure that is common in pregnant women. Here, we show how to build vapor chambers from readily obtainable materials that can each accommodate up to six standard mouse cages. We describe a vapor chamber paradigm that can be used to model exposure to ethanol, with minimal handling, during all three trimesters. Our studies demonstrate that pregnant dams developed significant metabolic tolerance to ethanol. However, neonatal mice did not develop metabolic tolerance and the number of fetuses, fetus weight, placenta weight, number of pups/litter, number of dead pups/litter, and pup weight were not significantly affected by ethanol exposure. An important advantage of this paradigm is its applicability to studies with genetically-modified mice. Additionally, this paradigm minimizes handling of animals, a major confound in fetal alcohol research.

We demonstrate the construction of alcohol vapor chambers using readily available materials that simultaneously house 6 mouse cages. We further describe their use in a mouse model of fetal alcohol exposure equivalent to all 3&#160;trimesters of human pregnancy. This paradigm exposes animals during gestation and postnatal days 1-12.

人間開発の3つのすべての学期の等価時にはアルコールにマウスを公開するために使用蒸気チェンバースの建設

Exposure to alcohol during development can result in a constellation of morphological and behavioral abnormalities that are collectively known as Fetal ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

共焦点光学イメージングによるローズベンガル光血栓<em&gt;インビボ</em&gt;：単一の容器ストロークのモデル

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.

The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

遠隔測定装置を使用した高脊髄損傷で動物に自律神経反射異常の総合的研究を行うためにアルゴリズムの開発

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the ...

Topical Airway Anesthesia for Awake-endoscopic Intubation Using the Spray-as-you-go Technique with High Oxygen Flow

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can jeopardize their spontaneous breathing, topical anesthesia of the airway is a popular technique. The spray-as-you-go technique represents one of the simplest opportunities to anesthetize the airway mucosa. The application of local anesthetic through the working channel of the flexible endoscope is a widespread practice for anesthetists as well as pulmonologists. There is neither need for additional devices nor special training as a pre-requisite to perform this technique. However, a known clinical problem is the coughing and gagging reflex that may occur when the liquid anesthetic strikes the airway mucosa and other sensitive structures like the vocal cords. This can be avoided by the use of oxygen applied through the working channel with the aim of fogging the local anesthetic into finer particles. Furthermore, the oxygen flow provides a higher oxygen supply and contributes to a better view, dispersing mucus secretions and blood away from the lens. Using an atomizer with a high oxygen flow of 10 L/min we maximized these benefits, caused less coughing and had more satisfied and therefore cooperative patients. Possible, but very rare complications of using oxygen flow including gastric insufflation, organ rupture or barotrauma did not arise. We attribute the complication-free use of high oxygen flow to the design of the set, which permits flow and pressure release.

The topical anesthetic lidocaine was atomized using a high oxygen flow through the working channel of a flexible intubating endoscope to achieve topical airway anesthesia for awake endotracheal intubation. We prefer this modified spray-as-you-go technique for endoscopic intubation to classical bolus application because of higher patient satisfaction and better compliance.

高酸素流量でスプレーとして-ユー・ゴー技術を使用して目を覚まし、内視鏡挿管のための局所麻酔気道

A patient&#39;s willingness to cooperate is an absolute precondition for successful awake intubation of the trachea. Whilst drug-sedation of patients can ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

架橋 (TXL) 近視治療の組織の評価ツールとしてウサギの強膜の第 2 高調波発生信号

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

人間の強膜の共焦点顕微鏡法を実行するために積層技術を用いて、

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