この研究は、ジョンズホプキンス大学のがんセンターも国立科学財団助成番号0651981とアレクサンダーとマーガレットスチュワートトラストによって資金を供給された。著者は、IRBと患者の研究だけでなく、ヘルプや博士セウォンカンと患者の試験中に彼の部門の支援に博士ローダアラニの貢献を感謝したい。

1。セットアップ<ol><li>赤外線カメラと赤外線画像の取得と保管だけでなく、コンピュータに接続されているデータ集録カード用のPCを搭載した温度管理された試験室は、図1Aに示されています。 </li><li>室温と皮膚の表面温度は、患者調査や測定データの間にデータ収集カードに接続された熱電対によって監視されているコンピュータ上に格納されています。 </li></ol> 2。画像収集<ol><li>病変が冷却効果がなく、熱画像で検出することができないので、正方形の接着剤のマーカーは、関心とその周辺（図1b）の色素性病変をローカライズするために使用されます。 </li><li>我々は、デジタルカメラ（キヤノンPowerShot G11）（図1b）と色素性病変と接着剤窓の明るい光の像を取得する。 </li><li>デジタルカメラ（DermLiteフォトシステム）に接続さdermatoscopeは、偏光のイメージをキャプチャするために使用されます。 </li><li>我々は、図1Aに示すようにマーリンmidwave（3-5μm）の赤外線カメラでcを定常状態赤外線画像を取得</li><li>私たちは、1分間の間、病変だけでなく、50mmの直径周辺領域を含む、患者の皮膚の領域に冷たい空気の流れを適用する。 </li><li> 1分後、我々は3-4分以内に室温（熱回復期）（図1c - d）で皮膚が再暖かくできるようにするには、この冷却ストレスを取り除く。 </li><li>熱回復期、色素性病変の赤外線画像は2秒おき（図1c - d）がキャプチャされます。 </li><li>試験期間中に撮影したすべてのIRの画像は（白色光と偏光の画像に加えて）保存し、LabVIEWソフトウェアを使用して格納されます。 </li></ol> 3。画像処理<ol><li> IR画像は、皮膚表面に正確に過渡的な温度分布を得るために、専用のMatlabのコードを用いて分析している。この目的のために、我々はいくつかの校正手順とマルチモーダル画像解析システムを導入する。 </li><li>我々は、接着剤、マーカーの角をローカライズするために明るい光の像に画期的な検出アルゴリズムを適用することから始まります。次に、我々は、基準IR画像の対応点を同定した。 </li><li>患者の不本意な身体/手足の動きを補償するために、我々は、回復期に赤外線画像シーケンスを整列させるための二次運動モデルのランドマークとして、これらの点を使用。 </li><li>我々はランダムウォーカー、ユーザが空間的に病変の輪郭を描くマスク画像を作成するために、シード点を配置することでセグメンテーションを導くことができるインタラクティブな画像分割アルゴリズムを使用してください。 </li><li>かつて我々は病変の形状を決定する、我々は、登録された赤外画像のそれぞれに対応する領域を識別します。 </li><li>我々はそれぞれ、病変内のランダムな点を選択して、離れて病変と健康な組織を代表する病変から。 </li><li>私たちは、健康な皮膚と病変の応答の過渡熱応答を比較する。 </li><li>デジタル、ダーモスコピー、色分けされた冷却励起後に周囲の条件と2秒で記録された病変と周辺地域のIR画像、および病変と健常組織の過渡熱応答：私たちは、すべてのデータを示す表を用意。 </li></ol> 4。代表的な結果： <img src="/files/ftp_upload/2679/2679fig1.jpg" alt="図1" /> 図1。周囲温度での領域の色素性病変のクラスタとイメージング、Cに適用されるテンプレートのフレームと大きな体表面積のa）臨床試験室での赤外線イメージングシステムのHRIS、b）は写真）を参照赤外線画像、D）冷却およびeの後に同じ領域）黒色腫の病変と周囲の拡大セクション<img src="/files/ftp_upload/2679/2679fig2.jpg" alt="図2" /> 図2。私たちの熱画像システムと試験室。 <img src="/files/ftp_upload/2679/2679fig3.jpg" alt="図3" /> 図3。渦管から冷たい空気の流れを吹くことによって病変と周囲の皮膚組織を冷却。

<ol>
	<li>Elder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+progression,+early+diagnosis+and+prognosis+of+melanoma.">Tumor progression, early diagnosis and prognosis of melanoma.</a> Acta Oncol. 38, 535-547 (1999).</li><li>Wartman, D., Weinstock, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+we+overemphasizing+sun+avoidance+in+protection+from+melanoma.">Are we overemphasizing sun avoidance in protection from melanoma.</a> Cancer Epidemiol Biomarkers Prev. 17, 469-470 (2008).</li><li>Pirtini Cetingul, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Using high resolution infrared imaging to detect melanoma and dysplastic nevi [dissertation]. , (2010).</li><li>Jones, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reappraisal+of+the+use+of+infrared+thermal+image+analysis+in+medicine.">A reappraisal of the use of infrared thermal image analysis in medicine.</a> IEEE Trans. Med. Imaging. 17, 1019-1027 (1998).</li><li>Anbar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+thermal+imaging+today-shifting+from+phenomenological+thermography+to+pathophysiologically+based+thermal+imaging.">Clinical thermal imaging today-shifting from phenomenological thermography to pathophysiologically based thermal imaging.</a> IEEE Eng. Med. Biol. Mag. 17, 25-33 (1998).</li><li>Anbar, M., Gratt, B. M., Hong, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermology+and+facial+telethermography.+Part+I:+history+and+technical+review.">Thermology and facial telethermography. Part I: history and technical review.</a> Dentomaxillofacial Radiology. 27, 61-67 (1998).</li><li>Jones, B. F., Plassmann, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+infrared+thermal+imaging+of+human+skin.">Digital infrared thermal imaging of human skin.</a> IEEE Eng. Med. Bio. 21, 41-48 (2002).</li><li>Qi, H., Diakides, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Infrared imaging in Medicine. , (2007).</li><li>Pirtini Cetingul, M., Herman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+skin+lesions+from+the+transient+thermal+response+using+infrared+imaging+technique.">Identification of skin lesions from the transient thermal response using infrared imaging technique.</a> IEEE 5th Int. Symp. on Biomedical Imaging: From Nano to Macro 1-4. , 1219-1222 (2008).</li><li>Cetingul, P. i. r. t. i. n. i., M, ., Herman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+thermal+signature+of+a+melanoma+lesion.">Quantification of the thermal signature of a melanoma lesion.</a> Int. Journal of Thermal Science. 50, 421-431 (2011).</li><li>Pirtini Cetingul, M., Herman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+heat+transfer+model+of+skin+tissue+for+the+detection+of+lesions:+sensitivity+analysis.">A heat transfer model of skin tissue for the detection of lesions: sensitivity analysis.</a> Physics in Medicine and Biology. 55, 5933-5951 (2010).</li><li>Pirtini Cetingul, M., Herman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+skin+lesions+using+transient+thermal+imaging.">Quantitative evaluation of skin lesions using transient thermal imaging.</a> Proc. Int. Heat Transfer Conf. , (2010).</li></ol>

利害の衝突は宣言されません。

結果は、冷却ストレスを印加することによって我々は病変と周囲の正常組織の間に温度差を強化することをお勧めします。また、ために熱画像中の患者の小さな動きから、我々は正しく、基準状態と熱回収時の温度分布の温度差を測定するために画像をオーバーレイするトラッキングモーションを適用する必要がありました。モーショントラッキングがなければ、悪性病変と健常組織との間の温度差を検出し、測定することはできなかったでしょう。これらの結果と正確なモーショントラッキングの必要性は、動的な熱画像の利点を証明する明確に単独で定常状態の情報に基づいて、IRイメージング法を用いて黒色腫を診断しようとすると、研究者が過去に直面する困難を説明する。 それはときに目の肥えた小病変赤外線カメラ（IR焦点面アレイの画素数）の空間分解能が重要であることに留意すべきである。空間分解能と初期の赤外線カメラの温度感度の両方は、過去の早期の悪性黒色腫の検出に困難を占めている、限られていた。我々のアプローチと事前のサーマルイメージングの試みの主な違い - 適度に成功した - 私たちは正確にアクティブ冷却に依存している動的なイメージ作成プロセスに加えてこのシステムの温度差を測定するためのキャリブレーションと画像処理ステップのシーケンスです。

<table border="1"><tbody><tr><td> 機器の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>マーリンMWIRカメラ</td><td> FLIR </td><td> - </td></tr><tr><td>キヤノンPowerShot G11 </td><td>キヤノン</td><td> - </td></tr><tr><td> DermLiteフォトシステム</td><td> DermLite </td><td> - </td></tr><tr><td>渦管</td><td> EXAIR </td><td> - </td></tr><tr><td>空気タンク</td><td> Airgasの</td><td> - </td></tr></tbody></table>

quantitative visualization and detection of skin cancer using dynamic thermal imaging

2010年には約68720メラノーマは死亡1で、その結果8650前後で、米国内だけで診断される。日付に、黒色腫のための唯一有効な治療法は外科的切除のまま、したがって、拡張された生存の鍵は、早期発見2,3です。すぐに特別なケアへのアクセスで、毎年診断された患者と制限の多数を考えると、診断を支援するために生体内診断機器で目的の開発が不可欠である。皮膚がん、特に非侵襲的な診断ツールを、検出するための新しい技術は、数々の研究室で検討されています。このようなデジタル写真、ダーモスコピー、マルチスペクトルイメージングシステム（MelaFind）、レーザーベースのシステム（走査型共焦点レーザー顕微鏡、レーザードップラー血流イメージング、光コヒーレンストモグラフィ）、超音波、磁気共鳴画像法などの手術の方法、技術、と一緒にすることはテストされている。各テクニックは、有効性および精度と使いやすさとコストの考慮事項の間で妥協をもたらすその多くは独自の長所と短所を、提供しています。これらの技術との比較についての詳細は文献4で利用可能です。 赤外線（IR）イメージングは​​局所の皮膚温度を測定することにより、特定の病気の兆候を診断する有用な方法であることが示された。正常な機能から疾患または偏差が再び皮膚5,6の温度に影響を与えるボディの温度の変化を伴っていることを示す証拠の大きな体がある。人体の温度と皮膚についての正確なデータは頻繁に病気によって引き起こされる通常の条件から、特に偏差、で、発熱と体温調節のための責任のプロセスに関する情報を豊富に提供することができます。しかし、IRイメージングは広く、温度の測定精度と空間分解能が不十分であったと高度な画像処理ツールが使用できなくなったときに、7,8数十年前に技術の早期の使用による医学では認識されていません。この状況は1990年代後半 - 2000年代に劇的に変化。 IR計測機器の進歩により、デジタル画像処理アルゴリズムとフィールドのブレークスルー許可されて科学者だけでなく、空間だけでなく、皮膚9の時間的な熱挙動を解析することができるダイナミックなIRイメージング、、の実装。 私たちの研究では、我々は、スクリーニングに重点を置いたと黒色腫10-13の早期発見と、腫瘍の検出のための生体光計測技術では、非侵襲的、効果的なコストとして、理論的、実験的研究と組み合わせることで、IRイメージングの実現可能性を探る。本研究では、我々は生検のための臨床指標と色素性病変を持つ患者がイメージングのために選択されている患者の研究で得られたデータを示しています。私たちは、健康と悪性組織との間の熱応答の違いを比較し、生検の結果と我々のデータを比較した。我々は黒色腫の病変の増加代謝活性は、動的な赤外線撮像によって検出することができると結論づけた。

Watch this Scientific Journal Video about Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging at JoVE.com

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

我々はその増加代謝活性と悪性の色素性病変は、熱の定量化可能な量を生成する実証し、冷却励磁に皮膚の過渡熱応答の測定は、早い段階で黒色腫の定量的な同定と他の皮膚癌を（対非増殖性母斑）が可能病気の段階。

定量的な可視化と動的な熱画像を用いた皮膚癌の発見

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

quantitative-visualization-detection-skin-cancer-using-dynamic

Research

JoVE Journal

Medicine

16.6K ビュー.  The Johns Hopkins University. 我々はその増加代謝活性と悪性の色素性病変は、熱の定量化可能な量を生成する実証し、冷却励磁に皮膚の過渡熱応答の測定は、早い段階で黒色腫の定量的な同定と他の皮膚癌を（対非増殖性母斑）が可能病気の段階。

ビデオ: Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

全血刺激のためのアッセイを用いて炎症性サイトカインIL -1βの正確かつ簡単に測定

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

医学

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

鳥類胚のモデルを使用して、がんの転移の定量的解析

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

がん治療のモニタリングのためのチェレンコフ発光イメージング（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that participate in regenerating tissue, tumor formation, or vasculogenesis, we devised a multi-colored cellular transplant model of tumor development in which cell populations originate from different fluorescently colored reporter gene mice and are transplanted, engrafted or injected in and around a developing tumor. These colored cells are then recruited and incorporated into the tumor stroma. In order to quantitatively assess bone marrow derived tumor stromal cells, we transplanted GFP expressing transgenic whole bone marrow into lethally irradiated RFP expressing mice as approved by IACUC. 0ovarian tumors that were orthotopically injected into the transplanted mice were excised 6-8 weeks post engraftment and analyzed for bone marrow marker of origin (GFP) as well as antibody markers to detect tumor associated stroma using multispectral imaging techniques. We then adapted a methodology we call MIMicc- Multispectral Interrogation of Multiplexed cellular compositions, using multispectral unmixing of fluoroprobes to quantitatively assess which labeled cell came from which starting populations (based on original reporter gene labels), and as our ability to unmix 4, 5, 6 or more spectra per slide increases, we&#39;ve added additional immunohistochemistry associated with cell lineages or differentiation to increase precision. Utilizing software to detect co-localized multiplexed-fluorescent signals, tumor stromal populations can be traced, enumerated and characterized based on marker staining.1

Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.

細胞の起源、タイプ、および相互作用の可視化のための蛍光組織移植に続いて定量的マルチスペクトル解析

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

定量リアルタイムPCRを用いて脳脊髄液マイクロRNAプロファイリング

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Measurement of Dynamic Scapular Kinematics Using an Acromion Marker Cluster to Minimize Skin Movement Artifact

The measurement of dynamic scapular kinematics is complex due to the sliding nature of the scapula beneath the skin surface. The aim of the study was to clearly describe the acromion marker cluster (AMC) method of determining scapular kinematics when using a passive marker motion capture system, with consideration for the sources of error which could affect the validity and reliability of measurements. The AMC method involves placing a cluster of markers over the posterior acromion, and through calibration of anatomical landmarks with respect to the marker cluster it is possible to obtain valid measurements of scapular kinematics. The reliability of the method was examined between two days in a group of 15 healthy individuals (aged 19-38 years, eight males) as they performed arm elevation, to 120&#176;, and lowering in the frontal, scapular and sagittal planes. Results showed that between-day reliability was good for upward scapular rotation (Coefficient of Multiple Correlation; CMC = 0.92) and posterior tilt (CMC = 0.70) but fair for internal rotation (CMC = 0.53) during the arm elevation phase. The waveform error was lower for upward rotation (2.7&#176; to 4.4&#176;) and posterior tilt (1.3&#176; to 2.8&#176;), compared to internal rotation (5.4&#176; to 7.3&#176;). The reliability during the lowering phase was comparable to results observed during the elevation phase. If the protocol outlined in this study is adhered to, the AMC provides a reliable measurement of upward rotation and posterior tilt during the elevation and lowering phases of arm movement.

This report presents details of how to adopt the acromion marker cluster method of obtaining scapular kinematics when using a passive marker motion capture device. As has been described in the literature, this method provides a robust, non-invasive, three-dimensional, dynamic and valid measurement of scapular kinematics, minimizing skin movement artifact.

皮膚の動きのアーティファクトを最小化するために肩峰マーカークラスタを使用して、動的な肩甲骨運動学の測定

The measurement of dynamic scapular kinematics is complex due to the sliding nature of the scapula beneath the skin surface. The aim of the study was to ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

同所膵臓癌マウスモデルのダイナミックコントラスト強化磁気共鳴画像

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

マウスモデルを用いた急性電離放射線による皮膚毒性の定量的評価のための光学分光拡散

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

光シート顕微鏡による劇症心筋炎のマウスモデルにおける白血球浸潤の定量的可視化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing continuous remodeling with the competing activity of osteoblasts, which produce the new bone matrix, and osteoclasts, whose function is to eliminate mineralized bone. [18F]-NaF is a positron emission tomography (PET) radiotracer that enables visualization of bone metabolism. [18F]-NaF is chemically absorbed into hydroxyapatite in the bone matrix by osteoblasts and can thus noninvasively detect osteoblastic activity, which is occult to conventional imaging techniques. Kinetic modeling of dynamic [18F]-NaF-PET data provides detailed quantitative measures of bone metabolism. Conventional semi-quantitative PET data, which utilizes standardized uptake values (SUVs) as a measure of radiotracer activity, is referred to as a static technique due to its snapshot of tracer uptake in time.&#160; Kinetic modeling, however, utilizes dynamic image data where tracer levels are continuously acquired providing tracer uptake temporal resolution. From the kinetic modeling of dynamic data, quantitative values like blood flow and metabolic rate (i.e., potentially informative metrics of tracer dynamics) can be extracted, all with respect to the measured activity in the image data. When combined with dual modality PET-MRI, region-specific kinetic data can be correlated with anatomically registered high-resolution structural and pathologic information afforded by MRI. The goal of this methodological manuscript is to outline detailed techniques for performing and analyzing dynamic [18F]-NaF-PET-MRI data. The lumbar facet joint is a common site of degenerative arthritis disease and a common cause for axial low back pain.&#160; Recent studies suggest [18F]-NaF-PET may serve as a useful biomarker of painful facetogenic disease.&#160; The human lumbar facet joint will, therefore, be used as a prototypical region of interest for dynamic [18F]-NaF-PET-MRI analysis in this manuscript.

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. We present detailed methodologies for performing and analyzing dynamic [18F]-NaF-PET-MRI data in a patient with facetogenic low back pain using the lumbar facet joints as a prototypical region of interest.

顔面性腰痛患者における動的骨回転率の評価に関する定量的[18F]-Naf-PET-MRI分析

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...