この作品は、エルンスト ・ ウント ベルタ Grimmke 財団とドイツの Ophthalmologische 協会 (犬) からの助成金によって支えられました。

本研究の実験プロシージャによって、動物ケア委員会のラインラント = プファルツ州、ドイツが承認されました。動物の世話は、眼科における動物の使用のためのビジョンと眼科 (ARVO) ステートメントで研究・ ビジョン研究制度のガイドラインと協会に適合。動物は、動物実験のための EU 指令 2010年/63/EU によると扱われました。生後 3-4 ヶ月男性の c57bl/6 j マウス (のジャクソンの実験室、バー ハーバー、私、米国) は、実験に使われました。動物は、標準条件 (温度 23 ± 2 ° C、湿度範囲 55 ± 10%、12 時間の明暗サイクル) の下で収容された、標準的なマウスの食事と水広告自由にアクセスしていた。
1. マウス網膜の分離
<ol><li>準備テーブルに解剖器具を置き、すべての楽器がクイック準備を有効にするかどうかはチェックします。</li>
	<li>CO2吸入によるマウスを犠牲に。</li>
	<li>スチールはさみとマウスの首をはねる、頭蓋骨 sagitally をはさみの同じ組み合わせを使用して 2 つの半分に切る、皮膚と脳目はさみ (図 1) を使用して取り外します。</li>
	<li>目はさみを使って軌道の骨だけを残して頭蓋骨骨を断ちます。</li>
	<li>冷たい促進バッファーを含んでいる解剖皿に軌道を転送します。</li>
	<li>氷上解剖皿を維持または 10 分ごとのバッファーを変更します。</li>
	<li>目はさみを使用して軌道の骨を切り取って、タイプ 4 の精密ピンセットと学生 Vannas 春はさみ (図 2) を使用して眼窩組織とともに眼球を慎重に取り外します。</li>
	<li>タイプ 5 の精密ピンセットと眼動脈の主な枝を損傷しないように注意を取って、Vannas capsulotomy はさみを使用して外眼筋、結合組織、ハーダー腺を慎重に取り外します。</li>
	<li>眼動脈眼窩枝は、最終的に 10-0 ナイロン縫合糸 (図 3) を使用して眼動脈の主な枝の近位端を縛る。</li>
	<li>4 精密ピンセット 10 s を使用して入力の 70% のエタノールを含むペトリ皿に目グローブを転送します。目グローブ解剖皿に戻し、新鮮なクレブス ソリューションでバッファーを置き換えます。角膜が白っぽいエタノール曝露後表示されます。</li>
	<li>30 ゲージ針周辺の角膜を穿刺そして Vannas capsulotomy はさみを使用して角膜を解剖します。優しく虹彩組織を切断、Vannas capsulotomy はさみ脈絡膜と網膜の間の 1 つの鋭い点を配置し、脈絡膜と強膜を切断します。(図 4) 視神経の周りいくつかの強膜の組織を残します。</li>
</ol>2. 灌流チェンバで網膜の取付
注: 網膜細動脈実験用灌流チェンバは自家製です。透明な貯水池で、流出管 (図 5) で構成されています。シリコン チューブの一方の端は histoacryl 接着剤と三方活栓に接続されているもう一方の端の部屋の下に釘付けに。三方活栓に新鮮なクレブス バッファーを含む注射器を接続し、バッファーで管を埋めます。
<ol><li>ニードル ホルダーとガラス ピペットを取るし、灌流チャンバーの下部に管の端にそれをプッシュします。100 μ m 径の先端を取得する 4 型ハサミ、ピペットの先端を破る。</li>
	<li>マイクロ ピペットの先端に焦点を当てるし、経由で注射器をチューブに接続されているフラッシュします。毛細血管が残骸によって妨げられていません注意してください。閉塞の場合に、削除し、フラッシュ チューブ別マイクロ ピペットを挿入します。</li>
	<li>冷たいクレブス バッファーと灌流チャンバーし、ステレオ顕微鏡室を置きます。</li>
	<li>灌流チェンバに透明なプラスチックのプラットフォームを配置します。このプラットフォームは、視神経と眼動脈の幅 1.8 mm のインデントを持って、直径 1.6 mm の 2 つの鋼線が配置されます。次に、プラットフォームに 4.0 mm 外径内径 2.8 mm のステンレス製リングを配置します。</li>
	<li>スプーンを使用して細心の注意で、視神経と眼動脈の接続されている網膜を解剖室から灌流チェンバに転送します。この手順は、潜在的なを防ぐために重要な組織のストレッチとして、実験に支障をきたすことがあります。</li>
	<li>眼動脈の近位端の縫合部分を切断、マイクロ ピペットに 10-0 ナイロン縫合糸の前もって形成された 2 つのループを配置、ピペットと眼動脈を cannulate します。マイクロ ピペット (図 6) に動脈を結ぶ、罰金ポイント ピンセットを使用して透明なプラットフォーム上に網膜を配置します。</li>
	<li>優しく 2 つの細かい点ピンセットで前方レンズ被膜を開いて涙、レンズを抜くし、刀を使用して全体のレンズの被膜を断ちます。</li>
	<li>その後、網膜の視神経にパルス プラナから半分の距離に 4 つの放射状切開を作るし、下部にそれを修正する網膜上にステンレス製のリングを配置します。網膜は、(図 7) 実験の準備が整いました。</li>
</ol>3. 網膜細動脈の実験のための準備
<ol><li>光学顕微鏡の下で灌流チェンバを配置します。目的は 100 X 水浸対物レンズです。</li>
	<li>2 つビデのために- とアウト-flow をペリ ポンプに接続し、商工会議所でクレブス バッファー 95% O2と酸素し、37 ° C で 5% CO2炭酸循環100 と 120 mL/min の流量を使用します。</li>
	<li>貯留層シリコン ホースを介して三方活栓にピペットへ接続すると、シリコン チューブを接続します。それから、リザーバーは 50 mm Hg に対応する高さにクレブス バッファーがまだ三方活栓を開いていません。</li>
	<li>顕微鏡、網膜面に焦点を当てるの目標に目を通します。血管や赤血球を検索します。血管が完全に折りたたまれていること、時々 だけいくつかの赤血球は表示可能です。</li>
	<li>血管が見つかったら、それに焦点を当てるし、三方活栓を開きます。容器の直径がすぐに増やす必要があります、場合は、細動脈や小静脈の場合 centripetally、赤の血液細胞のいずれかを遠心に移動します。細動脈は直径同じ順序の静脈に比べて小さいです。</li>
	<li>よく見える壁と血管が見つかったら、次の 30 分 (図 8) の平衡期間の実験のためにも流用できます。平衡期間中に細動脈の弱い本質的な緊張の結果内腔の直径の減少の 10% 未満で開発します。</li>
</ol>4. 実験を実行します。
<ol><li>お風呂のソリューションに追加することによって、トロンボキサン擬態 9,11-dideoxy-9α 11α methanoepoxy プロスタグランジン F2α (U46619) と船の生存率をテストします。このエージェントは、≥ 10 の濃度で直径を休んでから約 50% によってマウス網膜細動脈を収縮-6 M。</li>
	<li>一度船を実行可能な確認は、ポンプを循環させることにより風呂からアゴニストを洗浄し、お風呂に新鮮なクレブス バッファーを供給します。</li>
</ol>

<ol>
	<li>Toda, N., Nakanishi-Toda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitric+oxide:+ocular+blood+flow,+glaucoma,+and+diabetic+retinopathy.">Nitric oxide: ocular blood flow, glaucoma, and diabetic retinopathy.</a> Prog Retin Eye Res. 26 (3), 205-238 (2007).</li><li>Schuster, A. K., Fischer, J. E., Vossmerbaeumer, C., Vossmerbaeumer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+coherence+tomography-based+retinal+vessel+analysis+for+the+evaluation+of+hypertensive+vasculopathy.">Optical coherence tomography-based retinal vessel analysis for the evaluation of hypertensive vasculopathy.</a> Acta Ophthalmol. 93 (2), e148-e153 (2015).</li><li>Cherecheanu, A. P., Garhofer, G., Schmidl, D., Werkmeister, R., Schmetterer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+perfusion+pressure+and+ocular+blood+flow+in+glaucoma.">Ocular perfusion pressure and ocular blood flow in glaucoma.</a> Curr Opin Pharmacol. 13 (1), 36-42 (2013).</li><li>Faraci, F. M., Sigmund, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+biology+in+genetically+altered+mice+:+smaller+vessels,+bigger+insight.">Vascular biology in genetically altered mice : smaller vessels, bigger insight.</a> Circ Res. 85 (12), 1214-1225 (1999).</li><li>Kornfield, T. E., Newman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+Retinal+Blood+Flow+Using+Fluorescently+Labeled+Red+Blood+Cells.">Measurement of Retinal Blood Flow Using Fluorescently Labeled Red Blood Cells.</a> eNeuro. 2 (2), (2015).</li><li>Guevara-Torres, A., Joseph, A., Schallek, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label+free+measurement+of+retinal+blood+cell+flux,+velocity,+hematocrit+and+capillary+width+in+the+living+mouse+eye.">Label free measurement of retinal blood cell flux, velocity, hematocrit and capillary width in the living mouse eye.</a> Biomed Opt Express. 7 (10), 4229-4249 (2016).</li><li>Schallek, J., Geng, Y., Nguyen, H., Williams, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+topography+of+retinal+pericytes+in+the+living+mouse+retina+using+in+vivo+adaptive+optics+imaging+and+ex+vivo+characterization.">Morphology and topography of retinal pericytes in the living mouse retina using in vivo adaptive optics imaging and ex vivo characterization.</a> Invest Ophthalmol Vis Sci. 54 (13), 8237-8250 (2013).</li><li>Gericke, A., et al. <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+the+muscarinic+acetylcholine+receptor+subtype+mediating+cholinergic+vasodilation+in+murine+retinal+arterioles.">Identification of the muscarinic acetylcholine receptor subtype mediating cholinergic vasodilation in murine retinal arterioles.</a> Invest Ophthalmol Vis Sci. 52 (10), 7479-7484 (2011).</li><li>Gericke, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+role+of+alpha1-adrenoceptor+subtypes+in+murine+ophthalmic+arteries.">Functional role of alpha1-adrenoceptor subtypes in murine ophthalmic arteries.</a> Invest Ophthalmol Vis Sci. 52 (7), 4795-4799 (2011).</li><li>Bohmer, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+alpha(1)B+-adrenoceptor+subtype+mediates+adrenergic+vasoconstriction+in+mouse+retinal+arterioles+with+damaged+endothelium.">The alpha(1)B -adrenoceptor subtype mediates adrenergic vasoconstriction in mouse retinal arterioles with damaged endothelium.</a> Br J Pharmacol. 171 (16), 3858-3867 (2014).</li></ol>

著者は競合する金融興味を持ってないです。

マウス網膜における血管反応の測定は、網膜血管のサイズが小さいため困難です。紹介したテクニックを持つ網膜細動脈は透過光顕微鏡による可視化します。これは、孤立した網膜は半透明なので可能です。テクニックの利点は、高光学解像度です。計算された空間分解能は 11 px/μ m です。ただし、白色光を使用する光学系の実際の解像度は 200 と 300 nm、アッベの回折限界で説明されている?...

網膜の血流障害は、糖尿病網膜症、高血圧性網膜症、緑内障1,2,3などの様々 な眼疾患の病態に関与しています。したがって、網膜の血管反応性の測定を目的とした研究がこれらの疾患の病態を理解することが重要、新しい治療法を開発するために近づきます。
マウスのゲノムに遺伝子操作の可能性があるためマウス心血管系4研究の広く使用されている動物モデルとなっています。しかし、網膜血管 (≤ 30 μ m) のサイズが小さいためマウス網膜における血管反応性の測定は難しい。たとえば、生体内計測表面技術の光学解像度で限られている、まさに搭載時 ≤ 30 μ m 径未満の少量の血液で直径または血の流れの変化を検出することがのみできます。蛍光染料または適応光学スキャン ・光検眼鏡5,6を使用して共焦点顕微鏡など、追加の高度なデバイスまた、ローカルの識別を目的とした体内測定の解釈は網膜血管のメカニズムは、麻酔薬、によって混同することができますシグナリング全身血圧と球後血管の影響で変更します。
したがって前のヴィヴォ高光学解像度とマウスの網膜の血管の反応を測定する方法を開発しました。ここに記載した技術光顕微鏡を介して送信される網膜細動脈の描出が可能します。ラットにも使用することができます、この方法は、眼血管研究の技術をターゲット遺伝子の利点へのアクセスを提供します。

<table>
	<tbody>
		<tr>
			<td>Steel Scissors</td>
			<td>Carl Roth GmbH</td>
			<td>3576.1</td>
			<td>1x 140 mm</td>
		</tr>
		<tr>
			<td>Eye Scissors</td>
			<td>Geuder</td>
			<td>G-19390</td>
			<td>1x straight, 10.5 cm</td>
		</tr>
		<tr>
			<td>Precision tweezers, straight with fine tips</td>
			<td>Carl Roth GmbH</td>
			<td>LH68.1</td>
			<td>2x type 4</td>
		</tr>
		<tr>
			<td>Precision tweezers, straight with extra fine tips</td>
			<td>Carl Roth GmbH</td>
			<td>LH53.1</td>
			<td>2x type 5</td>
		</tr>
		<tr>
			<td>Vannas capsulotomy scissors</td>
			<td>Geuder</td>
			<td>19760</td>
			<td>1x straight, 77 mm</td>
		</tr>
		<tr>
			<td>Student Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>91501-09</td>
			<td>1x curved,</td>
		</tr>
		<tr>
			<td>Barraquer Needle Holder</td>
			<td>Geuder</td>
			<td>G-17500</td>
			<td>1x curved, 120 mm</td>
		</tr>
		<tr>
			<td>Needle</td>
			<td>Becton, Dickinson and Company</td>
			<td>305128</td>
			<td>1x 30 G</td>
		</tr>
		<tr>
			<td>Glass Capillaries (for producing micropipettes)</td>
			<td>Drummond Scientific Company</td>
			<td>9-000-1211</td>
			<td>1x (1.2 x 0.8 mm; outer/inner diameter)</td>
		</tr>
		<tr>
			<td>Nylon Suture</td>
			<td>Alcon</td>
			<td>198001</td>
			<td>1x 10-0</td>
		</tr>
		<tr>
			<td>Nunclon cell culture dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>153066</td>
			<td>1x 35 mm diameter</td>
		</tr>
		<tr>
			<td>Nunclon cell culture dish</td>
			<td>Thermo Fisher Scientific</td>
			<td>172931</td>
			<td>1x 100 mm diameter</td>
		</tr>
		<tr>
			<td>Discofix C</td>
			<td>Braun</td>
			<td>16500C</td>
			<td>10 cm</td>
		</tr>
		<tr>
			<td>Histoacryl adhesive</td>
			<td>B. Braun Surgical, S.A.</td>
			<td>1050052</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pericyclic pump&#160; (CYCLO II)</td>
			<td>Carl Roth GmbH</td>
			<td>EP76.1</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Vertical Pipette Puller Model 700C</td>
			<td>David Kopf Instruments</td>
			<td></td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Microscope (Vanox-T AH-2)</td>
			<td>Olympus</td>
			<td></td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Water immersion objective LUMPlanFL, 1.0 NA</td>
			<td>Olympus</td>
			<td></td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Digital camera (TK-C1381)</td>
			<td>JVC</td>
			<td></td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Perfusion chamber</td>
			<td>self-made</td>
			<td></td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Drugs and Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Carl Roth GmbH</td>
			<td>K928.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate (CaCl2)</td>
			<td>Carl Roth GmbH</td>
			<td>5239.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Kalium chloride (KCl)</td>
			<td>Carl Roth GmbH</td>
			<td>6781.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Kalium dihydrogen phosphate (KH2PO4)</td>
			<td>Carl Roth GmbH</td>
			<td>3904.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulphate (MgSO4)</td>
			<td>Carl Roth GmbH</td>
			<td>261.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Carl Roth GmbH</td>
			<td>9265.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate (NaHCO3)</td>
			<td>Carl Roth GmbH</td>
			<td>0965.3</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-(D)-(+)- Glucose monohydrate</td>
			<td>Carl Roth GmbH</td>
			<td>6780.1</td>
			<td></td>
		</tr>
		<tr>
			<td>9,11-dideoxy-9&#945;,11&#945;-methanoepoxy prostaglandin F2&#945; (U-46619)</td>
			<td>Cayman Chemical</td>
			<td>16450</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetylcholine chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A6625-25G</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation steps for measurement of reactivity in mouse retinal arterioles ex vivo

血管不全と通常の網膜血流の変化、様々 な視力を脅かす眼疾患、糖尿病網膜症、高血圧性網膜症と緑内障などの発症の主な要因です。したがって、網膜微小血管の準備、基本的な病態生理学的メカニズムを記述する生理学的および薬理学的研究とデザイン疾患の治療に極めて重要なツールです。眼科研究マウス モデルの普及にもかかわらず網膜血管反応性に関する研究がこの種の不足しています。この矛盾の主な理由はであるこれらの網膜血管のサイズが小さいため挑戦的な分離のプロシージャ 〜 ≤ 内腔の直径 30 μ m。前のヴィヴォ近く生理条件下でマウス網膜血管反応の研究をできる分離および準備の技術を設けてこれらの網膜微小血管の機能研究のための直接分離の問題を回避するために.現在実験準備は特にマウス網膜細動脈を参照、ただしこの方法容易にラットから微小血管を利用します。

U-46619 は、c57bl/6 j 背景の野生型マウスから網膜細動脈の濃度依存性血管収縮反応を生産しました。10-6 M の濃度で内腔の直径の減少安静時直径から ≈50% であった。図 9 aは、1 つの網膜細動脈の代表的な濃度応答曲線を示します。細動脈 U46619 と preconstricted、累積アセチルコリン投与誘発濃度依存性内腔の直径上昇 ≈ 25 %10-5 M、そ?...

Watch this Scientific Journal Video about Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo at JoVE.com

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

多くのビジョンを脅かす眼疾患は、網膜血管の機能不全に関連付けられます。したがって、網膜血管応答の測定は、根本的な病態生理学的メカニズムを調査することが重要です。この記事では、マウスの網膜細動脈の分離と血管作動性物質の血管径に及ぼす影響を評価するために準備のための詳しいプロトコルについて説明します。

マウス網膜細動脈Ex Vivoでの反応性の測定の準備手順

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

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Research

JoVE Journal

Medicine

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

7.1K ビュー.  University Medical Center of the Johannes Gutenberg University Mainz. 多くのビジョンを脅かす眼疾患は、網膜血管の機能不全に関連付けられます。したがって、網膜血管応答の測定は、根本的な病態生理学的メカニズムを調査することが重要です。この記事では、マウスの網膜細動脈の分離と血管作動性物質の血管径に及ぼす影響を評価するために準備のための詳しいプロトコルについて説明します。

ビデオ: Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

腫瘍溶解性ウイルスと生きている組織のex vivoでの感染

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

医学

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly hydrated, forming a substantial endothelial surface layer (ESL) that contributes to the maintenance of endothelial function. As the endothelial glycocalyx is often aberrant in vitro and is lost during standard tissue fixation techniques, study of the ESL requires use of intravital microscopy. To best approximate the complex physiology of the alveolar microvasculature, pulmonary intravital imaging is ideally performed on a freely-moving lung. These preparations, however, typically suffer from extensive motion artifact. We demonstrate how closed-chest intravital microscopy of a freely-moving mouse lung can be used to measure glycocalyx integrity via ESL exclusion of fluorescently-labeled high molecular weight dextrans from the endothelial surface. This non-recovery surgical technique, which requires simultaneous brightfield and fluorescent imaging of the mouse lung, allows for longitudinal observation of the subpleural microvasculature without evidence of inducing confounding lung injury.

In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.

マウス肺内皮表面層の in vivo測定における

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an extended perfusion period is challenging. We have added the kidney to our previous ex vivo perfused liver experiment model to reproduce a more accurate physiological state for prolonged experiments without using live animals. Five intact livers and kidneys were retrieved post-mortem from sacrificed pigs on different days and perfused for a minimum of 6 hr. Hourly arterial blood gases were obtained to analyze pH, lactate, glucose and renal parameters. The primary endpoint was to investigate the effect of adding one kidney to the model on the acid base balance, glucose, and electrolyte levels. The result of this liver-kidney experiment was compared to the results of five previous liver only perfusion models. In summary, with the addition of one kidney to the ex vivo liver circuit, hyperglycemia and metabolic acidosis were improved. In addition this model reproduces the physiological and metabolic responses of the liver sufficiently accurately to obviate the need for the use of live animals. The ex vivo liver-kidney perfusion model can be used as an alternative method in organ specific studies. It provides a disconnection from numerous systemic influences and allows specific and accurate adjustments of arterial and venous pressures and flow.

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Study of the animal organ physiology can be achieved by performing an experimental ex vivo perfusion system. The addition of a porcine kidney, as a homeostatic organ to our previously developed ex vivo liver perfusion model can be a principal step to achieve a better physiological environment.

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

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. ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

アン<em&gt;エクスビボ</emヒト乳癌でホルモンの作用を研究するために&gt;モデル

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

共焦点検眼鏡イメージングとマウス緑内障における細胞の形態計測：神経変性中に網膜ミクログリアの活性化のインビボダイナミクス

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

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 ...

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function.
The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.

Here we describe and compare two positions for obtaining the apical four-chamber view in mice. These positions enable the quantification of the right ventricular function, provide comparable results, and can be used interchangeably.

マウスにおける右心室拡張期パラメータの心エコー測定

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

増殖糖尿病網膜症における線合併症の体外培養モデル

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

インビボおよびインビトロ実験用グシュカン(GSK)顆粒の調製

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

マウス網膜細動脈Ex Vivoでの反応性の測定の準備手順

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腫瘍溶解性ウイルスと生きている組織のex vivoでの感染

マウス肺内皮表面層の in vivo測定における

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

細胞追跡のためのin vivoでの¹⁹ F MRI における

アン

共焦点検眼鏡イメージングとマウス緑内障における細胞の形態計測：神経変性中に網膜ミクログリアの活性化のインビボダイナミクス

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

マウスにおける右心室拡張期パラメータの心エコー測定

増殖糖尿病網膜症における線合併症の体外培養モデル

インビボおよびインビトロ実験用グシュカン(GSK)顆粒の調製