著者の謝辞があります。

低ビジョン アカデミーの倫理委員会で承認された研究のプロトコルとすべての科目はヘルシンキ宣言に従って書面による同意署名します。この研究は、ラフバラからシェフィールド大学倫理的な承認を受けた。
注: 脈絡自家移植 Limoli 網膜修復法 (LRRT) による乾燥 - 加齢黄斑変性症患者の包含と除外基準は、表 1に示すです。
1. 乾燥 - 加齢黄斑変性症患者の診断
<ol><li>共焦点走査型レーザー検眼鏡、SD-10 月、私と診断を確認します。</li>
	<li>遠くと近くの各グループの BCVA を評価します。メジャー メーカー ポイント単位 (Pts) で近いビジョン (クローズ アップ) のために。BCVA 時間 0 (T0) 90 (T90) を測定し、180 日間初期の治療糖尿病網膜症の研究と比較して (T180) ログマー 4 メートルで (EDTRS) グラフ。</li>
	<li>レコード電気幼魚、薄明視と明所視細胞の活動、または国際臨床視覚電気生理学 (ISCEV)11会によって 2009 年に設定された基準によると、フラッシュの ERG。</li>
</ol>2. anesthetization
注: LRRT の中に麻酔のゴールド スタンダードは局所麻酔、麻酔と鎮静のサブほぞの含浸による補強です。特定のケースで全身麻酔をお勧めします。
<ol><li>角膜を取得し、外用局所麻酔薬を適用することで結膜の麻酔注入 4% リドカイン 1% ロピバカインと手術前に、の滴の 15 〜 20 分。</li>
	<li>サブ結膜に直接浸潤麻酔を注入とテノン嚢下のスペース。</li>
	<li>局所浸潤の腹部の脂肪組織を抽出すると、前とサブ結膜と角膜輪部から 12 mm サブほぞのスペースの両方を使用します。Carbocaine または 1,200 IU エピネフリン混合 marcain の局所麻酔薬を採用してください。</li>
	<li>繰り返される小さなボリを通じて麻薬性鎮痛薬としてフェンタニルを使用して正しく行うことができる麻酔薬を用いた術中の鎮静を提供します。投与量は、一般的にボーラスあたりミダゾラムの 1 mg とフェンタニルの 0.025 mg です。</li>
</ol>3. Limoli 網膜修復技術準備
注: この手法は、subscleral スペース1,6,7,12軌道自家脂肪を移植する Pelaez の介入のバリアントを表します。外科的移植細胞が周囲の組織、脈絡膜と網膜18,19,20,21,22 で神経栄養因子や原因プロパティの多くの GFs を作り出すことができます。 ,23,24,25。LRRT、深い sclerectomy による移植自家細胞と脈絡膜の間の距離を削減し、脈絡膜流れ9、パラクリン自家細胞分泌を促進するために茎と脈絡膜の接触面積を拡大 10,14。
<ol><li>脈絡膜と強膜、Limoli 網膜修復手法 (LRRT)15,16,17と呼ばれるプロシージャの間の細胞移植手術の前にそれぞれの目の適切な消毒を行います。</li>
	<li>コールマンらと sovrachoroidal スペース15,16,17の SVF で、腹部の脂肪からのローレンスの法 (図 1) を用いて、ADSCs を移植します。</li>
	<li>血小板と潜入脂肪椎弓根は、次の手順により得られた PRP ゲルから派生します。</li>
	<li>遠心分離機の血6,12と多血小板血漿 (PRP) を収集します。血小板顆粒への刺激は、脂肪椎弓根6,12GF リリースします。</li>
</ol>4. 技術仕様および戦略
注: 脂肪質のティッシュの収集し、ローレンスとコールマン技術17(材料表) によると、患者の腹部の皮下層から精製します。
<ol><li>手動で収穫 3 mm 鈍カニューレを使用して、各患者の腹部の皮下層からの脂肪組織の 10 mL は、ローレンスとコールマン技術17 (図 2 a ・ 2 b) によるとロック注射器に接続します。</li>
	<li>20 ° C (図 2C) で 1,500 × g で 5 分間遠心分離して血や脂肪、油、液体から脂肪質のティッシュの純粋な SVF を区切ります。SVF は、ADSCs17の非常に豊富です。</li>
	<li>22 G 針と PRP の準備のための別々 のチューブは、ひと末梢血の 8 mL を収集します。</li>
	<li>20 ° c (図 2D) 1,500 × g で 5 分間収集した血液を遠心分離します。LRRT、その後で変更結果 ADSC の増殖は、脈絡膜血流の増加を支持する、自家脂肪移植の生存率と脂肪7、によってだけ分泌されるこれらの要因の作用のより包括的な変調で 11,17。</li>
	<li>眼窩脂肪から得られる移植を収容し、SVF から PRP、ローレンスに従って得られる ADSCs の混合物ではこのポケットの残留量が飽和に脈絡ポケット (詳細については手順 4 で、特定 4.4 と 4.5) を構築し、コールマン技術17。</li>
</ol>5 LRRT (Limoli 網膜修復技術) によって視細胞移植: 外科手術と技術的な詳細
<ol><li>6-0 シルク縫合糸、サルの角膜輪部近くで強膜を固定します。</li>
	<li>5.5「ウェストコット Tenetomy 曲線はさみを使用して、劣った一時的な角膜輪部から 11 ミリメートルで結膜下と subtenonian スペースを開きます。</li>
	<li>Limoli バジーレ結膜リトラクターを強膜手術野をするこの空間に挿入します。</li>
	<li>角膜輪部からの 8 mm の強膜側にフラップをカット済み、5 mm 三日月ナイフの角度面取りを使用しています。フラップのヒンジは、ラジアルと外科医の左側に常にです。</li>
	<li>角膜輪部から 8 mm で、サルの象限には、クレッセント ナイフ、斜め傾斜面を使用して、放射状のヒンジ側に約 5 mm の深い強ドアを開きます。脈絡膜のスレートの色を表示するのには十分な深さで sclerectomy を実行します。</li>
	<li>後続の視細胞移植で血液の循環を促進するために、フラップの遠位部で少し蓋を削除することによってギャップを作成します。</li>
	<li>眼科ピンセットで下斜筋上のギャップから眼窩脂肪を抽出します。抽出された脂肪は十分にその注入後生き残るためにそれを許可する血管であることを確認します。</li>
	<li>脈絡膜のベッドとドアの近位端に脈絡膜 6/0 polyglactin 繊維縫合糸に自家脂肪弁をそっと置きます。</li>
	<li>脂肪の茎またはその栄養血管の圧縮を避けるために強膜フラップを縫合します。</li>
	<li>(血液材料の遠心分離、コンポーネント、および血小板顆粒26の分離によって得られた) PRP ゲル 1 mL で脂肪の椎弓根の間質に潜入 30 G 斜め (30 °) カニューレを使用します。</li>
	<li>縫合糸を結膜の側面を準備します。結膜のリトラクターを削除します。</li>
	<li>6/0 polyglactin ファイバーを用いた結膜を縫合します。</li>
	<li>閉じる前に、フラップ、脈絡膜と脈絡膜の自家移植との間の subscleral スペースに自家脂肪移植を併用した小さな柔軟なプラスチック製のチューブを挿入するスペースを残します。</li>
	<li>飽和自家脂肪移植、脈絡膜、強膜フラップの残留容量 0.5 cc SVF (ADSCs のリッチ)、以前 3.2 で、小さな柔軟なプラスチック製のチューブで強膜のポケットに挿入準備します。</li>
	<li>残余空間を飽和後の縫合を閉じます。</li>
	<li>手術後、抗生物質アジスロマイシン 500 mg との 3 日間を管理します。また、組み合わせて抗生物質やステロイド、クロラムフェニ コールなどベタメタゾン、約 15-20 日目ドロップ療法を提供します。 注: ADSCs、SVF PRP から脂肪細胞から成っている、自家今得られている26。脈絡膜の流れに自己細胞の傍分泌の分泌を刺激するために深い sclerectomy 移植自家細胞と脈絡膜の間の距離を減らします。同じ目的のためには、茎と脈絡膜の間の接触のエリアを展開します。</li>
</ol>

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ARVO 2015 年に 5 月 3-7-デンバー、コロラド州 - 米国を提示しました。

本研究の主な目的は、脂肪細胞、ADSCs、SVF で PRP の視細胞移植 VA と AMD の影響を受けた目の乾燥で網膜の感度を時間をかけて改善するかどうかを評価するためだった。別の主な目的は、GF を用いた治療法がいくつかの病気の患者にとって役に立つかもしれないいくつかの臨床研究が示唆しているので、最近の文献に基づいて、これらの細胞の可能な治療効果を実証することでした。
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複数の慢性疾患を治療する患部に幹/前駆細胞の全身またはローカル注入から成る細胞療法は最後の十年の1で細心の注意を描画されます。1990 年代、網膜萎縮2における潜在的治療役割で成長因子 (GFs) を調べた。実際には、多くのヒトの細胞はアポトーシス、すなわち、プログラムされた死細胞3のブロックを遅らせたりすることができます特定の蛋白質である GFs を生成できます。
それは漸進的かつ不可逆的な細胞死が視細胞層と、その結果、中央視覚機能4の損失への損傷を含む萎縮性網膜疾患を乾燥 - 加齢黄斑変性症 (AMD) には知られています。AMD は、先進国および日付に有効な治療法がないすべての黄斑変性の 80% を占める 55 歳以上の人の失明の主要な原因です。
いくつかの研究では、自家の GFs を取得できる様々 なソースがあることを示しています。これらは異なる種類の細胞、脂肪由来幹細胞 (ADSCs) の脂肪5 間質血管の分数 (SVF) に含まれる、血小板血小板血漿 (PRP) から派生した眼窩脂肪由来間質細胞の脂肪を含む、構成します。 ,6,7。現在の GF セットにより網膜 neuroenhancement フィラトフ、Meduri、Pelaez、調査と Limoli は自家脂肪移植 (AFT) が効果的な8,9,10であることを示しています。
また、事前調査は網膜電図 (ERG) データの重要な改善を示したと、AMD の影響を受けた目の乾燥11にポスト脈絡自家移植が記録されます。脈絡で外科的移植組織は変調のアポトーシス6,7,12を遅らせること、網膜細胞のパラクリン分泌です。核の外側の層の厚さを考慮したモルモットの網膜の病理組織学的検討は GFs が網膜に栄養効果を持っていることを示しています。したがって、GFs の直接または間接的な使用は、分子の誘導剤と阻害剤6,7,12のバランスのとれた関係を治療上の利点をもたらすことができる可能性があります。
このメソッドの目的は、脂肪細胞、SVF で PRP ADSCs の視細胞移植が最も向上するかどうかを評価するために修正視力 (BCVA) と microperimetry (MY) 反応 AMD の影響を受けた目の乾燥です。本研究は文献6,7,12,13によると、GF 生産に基づいて再建の治療効果を発揮することを目的と。

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Blunt cannula, 3 mm.&#160;</td>
			<td style="width:179px;">Mentor, Santa Barbara, CA.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luer-LokTM syringe.&#160;</td>
			<td style="width:179px;">BD Biosciences, Franklin Lakes, NJ.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Regen-BCT tube.&#160;</td>
			<td style="width:179px;">RegenKit; RegenLab, Le Mont-sur-Lausanne, CH.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Centrifuge</td>
			<td style="width:179px;">&#160;RegenPRP Centri. RegenLab, Le Mont-sur-Lausanne, CH.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD Venflon Pro Safety 22G x 1.00 inch (0.9 mm x 25 mm).&#160;</td>
			<td style="width:179px;">BD Biosciences, Franklin Lakes, NJ.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SPSS Statistics Version 19.0</td>
			<td style="width:179px;">IBM Corp., Armonk, NY, USA.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Confocal scanning laser ophthalmoscope&#160;</td>
			<td style="width:179px;">Nidek Inc, Fremont, CA</td>
			<td style="width:119px;">Nidek F10&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cirrus 5000 Spectral Domain-Optical Coherence Tomography</td>
			<td style="width:179px;">Carl Zeiss Meditec AG, Jena, Germany</td>
			<td style="width:119px;">&#160;SD-OCT&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Maia 100809 Microperimetry&#160;</td>
			<td style="width:179px;">CenterVue S.p.A., Padua, Italy</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ocular electrophysiology electromedical system,</td>
			<td style="width:179px;">C.S.O., S.r.l., Scandicci, Italy</td>
			<td style="width:119px;"></td>
			<td>&#160;Retimax for ERG&#160;</td>
		</tr>
	</tbody>
</table>

regenerative therapy by suprachoroidal cell autograft in dry age-related macular degeneration: preliminary in vivo report

自己細胞の視細胞移植は最高矯正視力 (BCVA) を向上させることが、microperimetry (MY) によって影響を受ける目のレスポンスを時間をかけて加齢による黄斑変性症 (AMD) を乾燥かどうかを調べることを本研究の目的は、生産と周囲の組織の成長因子 (GFs) が分泌。患者は各研究グループにランダムに割り当てられました。すべての患者は、乾燥 AMD BCVA (ログマー) の解像度の最小角度の 1 対数以上と診断されました。脈絡自家移植 Limoli 網膜修復法 (LRRT) では 11 患者から 11 目を含んだグループ A で実施されました。テクニックは、脂肪細胞、間質の血管分数と脈絡で多血小板血漿血小板から得られる脂肪幹細胞の注入によって行われました。逆に、14 人の患者の 14 目を含む B 群は対照群として使用されました。それぞれの患者の診断は、共焦点走査型レーザー検眼鏡、スペクトル ドメイン光コヒーレンストモグラフィー (SD 10 月) によって確認されました。グループ A の BCVA によって改善 0.581 に 0.504 0.376 ログマーと 90 日間で 180 日 (+32.20%) 術後。さらに、私のテスト 180 日 12.59 db 11.44 db 増加します。定数 GF 分泌脈絡膜の流れを確保するためは、脈絡膜の背後に接ぎ木した異なった細胞のタイプができました。したがって、結果を示す移植グループの視力 (VA) を増やすことができます 6 ヵ月後コントロール群よりも。

ここに示す手順を使用して、BCVA (ログマー) の解像度の最小角度の 1 対数以上と、AMD の影響を受けた患者の中でも乾燥の 2 つのグループの研究で在籍していた。グループ A に対し受信脈絡自家移植 Limoli 網膜修復法 (LRRT) による 11 患者の 11 目を含むグループ B、14 患者の 14 目を含む、対照群として使われました。
スチュ?...

Watch this Scientific Journal Video about Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report at JoVE.com

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

乾燥 - 加齢黄斑変性症の視細胞の細胞移植による再生療法:体内の予備的なレポート

本研究の目的は、間質血管の一部に含まれている脂肪由来幹細胞と血小板由来血小板豊富な血しょう Limoli 網膜修復技術の視細胞移植が視力を改善できるかどうかを評価するためには、乾燥 - 加齢黄斑変性症の影響を受ける目の網膜感度応答。

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

regenerative-therapy-suprachoroidal-cell-autograft-dry-age-related

Research

JoVE Journal

Medicine

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

10.0K ビュー.  Low Vision Research Centre of Milan. 本研究の目的は、間質血管の一部に含まれている脂肪由来幹細胞と血小板由来血小板豊富な血しょう Limoli 網膜修復技術の視細胞移植が視力を改善できるかどうかを評価するためには、乾燥 - 加齢黄斑変性症の影響を受ける目の網膜感度応答。

ビデオ: Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

ビボ犬筋肉の機能アッセイに

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

医学

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

がんの生産と活性酸素種の検出（ROS）

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

神経筋電気刺激による筋力トレーニングのマウスモデル

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

細胞追跡のためのin vivoでの19 F MRI における

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

経時インドシアニングリーン血管造影による加齢黄斑変性症のマウスモデルにおける脈絡膜血管系の異常を検出する

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

シーケンシャル<em&gt;インビボ</em&gt;骨折修復中に、骨形成幹細胞/前駆細胞のイメージング

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

マウスにおける変形性関節症の痛みのMonoiodoacetateモデル

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

加速度計を使用して日常生活に上肢のパフォーマンスを定量化する方法

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Mechanical Micronization of Lipoaspirates for Regenerative Therapy

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of cell products using collagenase. Here, we present a protocol to generate an injectable mixture of SVF cells and native extracellular matrix from adipose tissue by a purely mechanical process. Lipoaspirates are put into a centrifuge and spun at 1,200 x g for 3 min. The middle layer is collected and separated into two layers (high-density fat at the bottom and low-density fat on the top). The upper layer is directly emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance under the oil layer is collected and defined as the extracellular matrix (ECM)/SVF-gel. The oil on the top layer is collected. Approximately 5 mL of oil is added to 15 mL of high-density fat and emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance is also ECM/SVF-gel. After the transplantation of the ECM/SVF-gel into nude mice, the graft is harvested and assessed by histologic examination. The result shows that this product has the potential to regenerate into normal adipose tissue. This procedure is a simple, effective mechanical dissociation procedure to condense the SVF cells embedded in their natural supportive ECM for regenerative purposes.

Here, we present a protocol to obtain stromal vascular fraction from adipose tissue through a series of mechanical processes, which include emulsification and multiple centrifugations.

成人再生療法のための機械的微細化

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of ...