Thermostimulator TSA IIを使用して熱の痛みのテスト 1。はじめ<ol><li>この手順では、TSA II、サーモード（メドックアドバンスドメディカルシステムズ、ダーラム、ノースカロライナ州）で皮膚に熱刺激を提供する市販の熱感覚アナライザを使用しています。中央ユニットはサーモードする信号を生成して送信します。この信号は通常、中央装置に接続されているラップトップに保存されているプロプライエタリなソフトウェアによって制御されます。また、中央装置に接続されていることサーモードの加熱を停止するために調査対象を可能にする手持ち式コントロールです。 </li><li>七シーケンシャルと同一の熱刺激は、30代の間刺激の間隔で、配信されます。基礎サーモード温度は快適な35℃に設定されていますそれが冷却する速度が10℃/ sで設定されている間サーモードがヒートアップする速度は、1℃/ sで設定されています最大サーモードの温度は52℃で設定されています</li></ol> 2。準備とトレーニング課題<ol><li>頭部、腕と脚のための十分なサポートを提供するリクライニングチェアで快適に熱の痛みの試験の座席彼らのために科目を準備する。議長はまた、検査の身体部位へ簡単にアクセスを許可する必要があります。 </li><li>試験施設は、快適な室温になる、とのない気晴らしにはかなりと中立的な環境を提供する必要があります。 </li><li>実験を開始する前に、すべての参加者は熱の痛みのテストのすべての面でよく訓練されたはずです。訓練は、実験前にいつでも場所取ることができます。 </li><li>被験者と研究者間の相互作用は、データを収集するために必要なものに限定されるべきである。彼らはデータの一貫性を危険にさらす可能性があるため、いろいろジョークを言うと世間話は推奨されません。被験者は相互作用が意図的に実験中にやや単調で、中立的になることが通知されます。 </li><li>命令は、明確なシンプルで、かつ一貫してください。その痛みは主観的であり、いかなる権利または間違った答えがないことを強調する。それは一貫性を保つために最も重要です。 &quot;約苦痛になる&quot;という間に点として、熱痛覚閾値を定義し、&quot;ただ痛いとなった。&quot; </li><li>それは、被験者がテストセッション中に期待する内容を正確に知っていることが重要です。そのような不安のような交絡因子が生成される可能性があるため、任意の&quot;サプライズ&quot;は、あまり一貫性のあるデータが表示される場合があります。例えば、7つの熱の疼痛刺激、熱痛覚閾値を決定するために使用されている場合、被験者はこれを知っている必要があります。 </li><li>いつでも、被験者はハンドヘルドコントロールのボタンを押すことで任意暑くを得ることからサーモードを停止することができます。コントロールにいると被験者の不安を軽減し、データの信頼性が一段と向上します。。 </li><li>トレーニングセッションは、研究の被験者のパフォーマンスを判断する機会を提供しています。それは彼らの個々の疼痛閾値がない以上0.8以上で° C、そして理想的にはこれ以上0.6以下によって異なりますので、·最終つのテストサイクルの間にCを二から四テストサイクル中に被験者を訓練するようにするとよいでしょう。一貫して行うために失敗した被験者は、通常、試験から除外されます。 </li></ol> 3。痛覚閾値の決定<ol><li>痛覚閾値のテストサイクルを開始するには、被験者の皮膚上サーモードの位置をマークします。この実験ではマイクロダイアリシスのために使用したのと同じ炎症を起こしている皮膚の病変は、痛みのテストに対応してリリースされた生化学物質について学ぶために痛みのためにテストされます。 </li><li>その後、著しい皮膚の領域にサーモードを配置することにより、各サイクルを開始する。できる限り近づけるようにマーキングサーモードの位置と一致している。皮膚とサーモード間に温度が35に平衡を許可° C - 安全に痛みを引き起こすことはないでしょう温度。これは、約5秒かかりますと、コンピュータの画面上で35℃の測定値によって示されます。 </li><li>開始する前に、被写体までのハンドヘルド制御を与える。 </li><li>テスト中に、被験者の皮膚に完全に接触サーモードの全表面を保つために一定と穏やかな圧力を使用してください。 </li><li>今すぐ事前にプログラムのテストアルゴリズムを起動します。サーモードは1から加熱が開始されます° C / sのとすぐに主題の経験の痛みとして、彼らは手持ち型デバイスを押してくださいよ。 </li><li>ハンドヘルドデバイス上のボタンを押してから、サーモード温度引き起こす痛みは自動的に登録されますと35℃のそのベースラインの温度にC / sのダウン℃で10の割合で減少を開始します</li><li>温度プローブは自動的に35℃に保たれる℃で30秒C主題が再びハンドヘルドデバイスのボタンを押すまで、それは1から再び上昇° C / sで、始まる前に。サーモード温度が再び上昇しようとしている直前に、この現象が発生しようとしていること主題を思い起こさせる。これは、被験者が集中し続けます。 </li><li> seven温度の記録が登録されるまで、このシーケンスが繰り返されます。これらのテンペの平均を計算するratureの測定値は、これは、熱痛覚閾値である。 </li></ol>断続圧力プローブによる機械的な痛みのテスト 1。はじめ<ol><li>彼らは伝統的に使用されると市販のフォンフレイ毛よりも一貫性のある結果を提供するため、我々は、カスタムメイドのキャリブレーション断続圧力プローブを使用してください。プローブは、同一の円筒形のステンレス鋼線製（240ミクロ​​ンの直径）より広い中を自由に移動、様々な重みの銅棒（1、2、4、8、16、32、49、64、と81グラム）に搭載されている手アルミニウム管を開催。 </li><li>熱の痛みのテストのために概説同じ一般的な実験条件は、機械的な痛みのテストに適用されます。被験者は十分な準備と訓練でなければなりません。それは、プローブが鈍であり、穿刺肌をしないという科目を安心させるために特に重要です。これは、被験者が一貫性のあるデータをリラックスして提供するために役立ちます。 </li></ol> 2。機械的痛覚閾値の決定<ol><li>機械的痛覚閾値を決定するには、まず実験的に炎症病変の中心で、皮膚に対して垂直に軽量プローブの鋼線の先端を適用します。 </li><li>手持ちのアルミチューブは、それに触れることなく皮膚に向かって低下する。この方法で、アルミ管の内側の銅ロッドの全体重量は鋼線の先端にかかるようにしてください。 </li><li>調査対象は、プローブが&quot;はい&quot;または言って痛みを引き起こすかどうかを示します&quot;ない。&quot; </li><li>被験者の答えが&quot;ノー&quot;、その後、同じプローブまたは次のより重いプローブは次の使用される場合。このアルゴリズムは、昇順刺激シーケンスと呼ばれています。ランダムに次のプローブが重いか、同じ重量のかどうかを判断します。これは、アルゴリズムを知らされていない主題を維持します。 </li><li> &quot;はい。&quot;このシリーズの最後の2つのプローブの平均重量が最初に偏向点として記録されているサブジェクトが応答するまで昇順シーケンス内のプローブを適用します。 </li><li>被験者が応答した後、&quot;はい、&quot;降順刺激シーケンスを使用して起動し、以前のように軽いプローブまたは同じプローブのいずれかを使用します。ランダムに次のプローブを明るくしたり、同じ重量のかどうかを判断します。これは、アルゴリズムを知らされていない主題を維持します。 </li><li>被験者の答えが&quot;いいえ。&quot;このシーケンスの最後の2つのプローブの平均体重は、第二偏向点を構成するまで、降順シーケンスでプローブを適用する。 </li><li>被験者の答えは&quot;いいえ、&quot;昇順刺激の順序に戻ったとき。 seven偏向点が記録されるまで、昇順とシーケンスを降順が交互に。これらの7つのポイントの平均は、機械的な痛みの限界です。 </li></ol>

<ol>
	<li>Angst, M. S., Clark, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comment+on+Koltzenburg+et+al.:+Differential+sensitivity+of+three+experimental+pain+models+in+detecting+the+analgesic+effects+of+transdermal+fentanyl+and+buprenorphine.">Comment on Koltzenburg et al.: Differential sensitivity of three experimental pain models in detecting the analgesic effects of transdermal fentanyl and buprenorphine.</a> Pain. 126, 165-174 (2006).</li><li>Angst, M. S., Clark, J. D., Carvalho, B., Tingle, M., Schmelz, M., Yeomans, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+profile+in+human+skin+in+response+to+experimental+inflammation,+noxious+stimulation,+and+administration+of+a+COX-inhibitor:+A+microdialysis+study.">Cytokine profile in human skin in response to experimental inflammation, noxious stimulation, and administration of a COX-inhibitor: A microdialysis study.</a> Pain. , (2008).</li><li>Angst, M. S., Koppert, W., Pahl, I., Clark, J. D., Schmelz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+infusion+of+the+mu-opioid+agonist+remifentanil+in+humans+causes+hyperalgesia+during+withdrawal.">Short-term infusion of the mu-opioid agonist remifentanil in humans causes hyperalgesia during withdrawal.</a> Pain. 106, 49-57 (2003).</li><li>Lotsch, J., Angst, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mu-opioid+agonist+remifentanil+attenuates+hyperalgesia+evoked+by+blunt+and+punctuated+stimuli+with+different+potency:+a+pharmacological+evaluation+of+the+freeze+lesion+in+humans.">The mu-opioid agonist remifentanil attenuates hyperalgesia evoked by blunt and punctuated stimuli with different potency: a pharmacological evaluation of the freeze lesion in humans.</a> Pain. 102, 151-161 (2003).</li></ol>

このビデオでは、ヒトボランティアの炎症を起こしている皮膚の熱的および機械的疼痛閾値を評価するためのアルゴリズムを示しています。痛みの知覚は主観的です。十分な疼痛のデータの収集は、適切な試験施設（例えば、静かで心地よい）、内蔵のデータの品質管理対策（検査 - 再検査信頼性）、標準化された研究のプロトコールを厳守、及び交絡因子の回避による研究参加者の徹底的なトレーニングを必要とする（例：ストレス、不安）。そのような鎮痛薬の投与とそのような侵害受容性メディエーターのローカルリリースのような機械論的データの収集などの介入と対になって痛みの感受性に関する信頼性の高い主観的データは、痛みのメカニズムの貴重な洞察を提供し、それを防ぐか、または減衰させることを意味します。

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>TSA-II Neuro Sensory Analyzer</td>
<td>Tool</td>
<td>Medoc Advanced Medical Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Puntuated Pressure Probes</td>
<td>Tool</td>
<td>Custom-made</td>
<td>&nbsp;</td>
<td>Alternatively, commercially available von Frey Hairs can be used.</td>
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determining heat and mechanical pain threshold in inflamed skin of human subjects

可視化実験のジャーナルで前回の記事では、ヒトの組織特異的侵害受容と炎症生化学物質の収集のために皮膚の微小透析技術を実証している。この記事では、多くの場合、マイクロダイアリシスの手続きと並行して使用されている痛みのテストのパラダイムが表示されます。マイクロダイアリシスと痛みのテストを組み合わせることで痛みを発生し、伝播する責務キー生化学物質を識別できるように、行動学的および生化学的データの間の重要なリンクを提供します。 人間の研究参加者の炎症を起こしている皮膚に想起させる痛みの2つのモデルが示されています。最初のモデルは、熱の刺激の助けを借りて、痛みを呼び起こす。ここで説明したように痛みを誘発する熱は、主に小、非髄末梢侵害受容神経線維（C線維）によって媒介される。第二のモデルは、断続圧力の刺激を介して痛みを呼び起こす。断続圧力誘発痛みは主に小、有髄末梢侵害受容性神経線維（A -デルタ線維）によって媒介される。二つのモデルは、機構的別個のものと独立して痛みのシグナル伝達に関与する2つの主要な末梢神経線維の集団によって侵害受容処理を調べます。 熱の痛みは、TSA II、市販の熱感覚アナライザ（メドックアドバンスドメディカルシステムズ、ダーラム、ノースカロライナ州）の助けを借りて誘発される。刺激の構成と配信は、特定のソフトウェアの助けを借りて処理されます。 Thermodesは、サイズや形状で異なりますが、原理的には様々な速度で、時間の異なる期間のための加熱または冷却することができる金属板で構成されています。熱誘発疼痛を評価するアルゴリズムが多様です。ここに示した実験では、研究参加者は、それらが皮膚に接触するとサーモードは痛みを惹起しない温度で開始する所定の速度で加熱した状態で痛みを経験して開始するどの時点で示すように求められます。被験者が苦痛を経験して開始するサーモードの温度は、熱痛覚閾値を構成している。 機械的な痛みが断続プローブで誘発される。そのようなプローブは、いくつかのメーカー（フォンフレイ毛）から市販されている。しかし、フォンフレイ毛の精度を批判し、多くの研究者は、カスタムメイドの断続圧力プローブを使用している。実験に示すように異なる重みをここに8つのカスタムメイドの断続プローブは知覚の偏向点、すなわち、感じる痛みまたはその逆に痛みを感じていないからの変化を識別するために、連続した順序でアップダウンと呼ばれるアルゴリズムの手順を適用されます。知覚のたわみの原因となる平均的な重量は機械的な痛みのしきい値を構成している。

Watch this Scientific Journal Video about Determining heat and mechanical pain threshold in inflamed skin of human subjects at JoVE.com

Determining heat and mechanical pain threshold in inflamed skin of human subjects

人間の被験者の実験的炎症を起こしている皮膚に熱や機械的な痛みのしきい値を評価するアルゴリズムが示されています。 two痛みのテストのパラダイムは、独立して痛み、すなわち、非髄C線維と小さな有髄-デルタ線維を伝達する二つの主要な末梢神経線維の集団によって侵害受容処理を調べます。

熱とヒトの炎症を起こしている皮膚における機械的痛覚閾値の決定

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific ...

determining-heat-mechanical-pain-threshold-inflamed-skin-human

Research

JoVE Journal

Biology

20.7K ビュー.  Stanford University School of Medicine. 人間の被験者の実験的炎症を起こしている皮膚に熱や機械的な痛みのしきい値を評価するアルゴリズムが示されています。 two痛みのテストのパラダイムは、独立して痛み、すなわち、非髄C線維と小さな有髄-デルタ線維を伝達する二つの主要な末梢神経線維の集団によって侵害受容処理を調べます。

ビデオ: Determining heat and mechanical pain threshold in inflamed skin of human subjects

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

診断ツールを開発するマイクロエレクト​​ロメカニカルシステム（MEMS）の使用

生物学

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

マウス皮膚移植

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

ラット心臓における培養組織の移植

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis. In some women the severe episodes of cyclic pain change and the resultant pain becomes continuous and this condition becomes known as Chronic Pelvic Pain. This state can be present even after the appropriate medical or surgical therapy has been instituted. It can be associated with pain and tenderness in the muscles of the abdomen wall and intra-pelvic muscles leading to severe dyspareunia. Additional symptoms of irritable bowel and interstitial cystitis are common. A common sign of the development of this state is the emergence of cutaneous allodynia which emerges from the so-called viscero-somatic reflex. A simple bedside test for the presence of cutaneous allodynia is presented that does not require excessive time or special equipment. This test builds on previous work associated with changes in sensation related to gall bladder function and the viscera-somatic reflex(1;2).
The test is undertaken with the subject s permission after an explanation of how the test will be performed. Allodynia refers to a condition in which a stimulus that is not normally painful is interpreted by the subject as painful. In this instance the light touch associated with a cotton-tipped applicator would not be expected to be painful. A positive test is however noted by the woman as suddenly painful or suddenly sharp. The patterns of this sensation are usually in a discrete pattern of a dermatome of the nerves that innervate the pelvis.
The underlying pathology is now interpreted as evidence of neuroplasticity as a consequence of severe and repeating pain with changes in the functions of the dorsal horns of the spinal cord that results in altered function of visceral tissues and resultant somatic symptoms(3). 
The importance of recognizing the condition lies in an awareness that this process may present coincidentally with the initiating condition or after it has been treated. It also permits the clinician to evaluate the situation from the perspective that alternative explanations for the pain may be present that may not require additional surgery.

Demonstration of Cutaneous Allodynia in Association with Chronic Pelvic Pain

A demonstration of the bedside test for cutaneous allodynia and its clinical implications.

のデモンストレーション皮膚アロディニア慢性骨盤痛との関連で

Pelvic pain is a common condition that is associated with dysmenorrhea and endometriosis.  In some women the severe episodes of cyclic pain change and the ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

ヒト皮膚から大人上皮幹細胞の単離と培養

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 &#181;m&#160;x 200 &#181;m&#160;x 200 &#181;m using LED illumination, to 5 mm&#160;x 5 mm&#160;x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.

Digital Inline Holographic Microscopy DIHM of Weakly-scattering Subjects

The three-dimensional locations of weakly-scattering objects can be uniquely identified using digital inline holographic microscopy (DIHM), which involves a minor modification to a standard microscope. Our software uses a simple imaging heuristic coupled with Rayleigh-Sommerfeld back-propagation to yield the three-dimensional position and geometry of a microscopic phase object.

弱散乱被験者のデジタルインラインホログラフィ顕微鏡（DIHM）

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

シングルミトコンドリアスーパーオキシド無傷ハートや点滅の共焦点イメージング<em&gt;インビボ</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

ex vivoで培養マウス胚の皮膚およびメラノブラストの移行のライブイメージング

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

ギャップ結合細胞間コミュニケーション：毒物および毒素の有害効果を評価するための機能バイオマーカー、および天然物の健康上の利点

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

A Buoyancy-based Method of Determining Fat Levels in Drosophila

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat in Drosophila, but most are expensive and/or laborious and have clear limitations. Here, we present a method to quickly and cheaply determine organismal fat levels in L3 Drosophila larvae. The technique relies on the differences in density between fat and lean tissues and allows for rapid detection of fat and lean phenotypes. We have verified the accuracy of this method by comparison to body fat percentage as determined by neutral lipid extraction and gas chromatography coupled with mass spectrometry (GCMS). We furthermore outline detailed protocols for the collection and synchronization of larvae as well as relevant experimental recipes. The technique presented below overcomes the major shortcomings in the most widely used lipid quantitation methods and provides a powerful way to quickly and sensitively screen L3 larvae for fat regulation phenotypes while maintaining the integrity of the larvae. This assay has wide applications for the study of metabolism and fat regulation using Drosophila.

A Buoyancy-based Method of Determining Fat Levels in Drosophila

Here we present a method to measure organismal fat levels in the third instar (L3) larval stage of Drosophila melanogaster. This method exploits the comparatively low density of fat tissue to differentiate between larvae with altered fat stores. Buoyancy-based analysis is a valuable tool for rapid, reproducible, and economical screening.

で脂肪レベルを決定するの浮力に基づく方法<em&gt;ショウジョウバエ</em

Drosophila melanogaster is a key experimental system in the study of fat regulation. Numerous techniques currently exist to measure levels of stored fat ...