テクニカル サポート、博士魏斉彼 (東呉大学、中国・蘇州) と博士ヤン寧喬 (陝西師範大学、西安、中国) に感謝します。この作品は中国の国家自然科学基金 (グラント 31272311、81373295、81473420)、優先度学術プログラム開発の江蘇高等教育機関 (グラント号のプロジェクト資金によって支えられました。ysxk-2016)。

動物操作機関動物ケアおよび使用委員会 (IACUC) モデル動物研究センターの南京大学によって承認されました。
1. ソリューションの準備
<ol><li>HEPES Tyrode ソリューション (H-T) 137.0 mM NaCl、KCl 2.7 mM、1.8 mM CaCl2、1 mM MgCl2∙6H2O、5.6 mM D-グルコースおよび 10 mM HEPES、使用準備 pH 7.3 7.4。</li>
	<li>カルシウムなし HEPES Tyrode ソリューションを準備 (Ca2 +-無料の H T) 140.6 mM NaCl、2.7 mM KCl、1 mM MgCl2∙6H2O、5.6 mM D-グルコース 10 mM HEPES、pH 7.3 7.4 を使用します。</li>
	<li>124 mM KCl (高 K+) 15.7 mM の NaCl、KCl、124.0 mM 1.8 mM CaCl2, 1 mM MgCl2∙6H2O, 5.6 mM D-グルコースと 10 mM HEPES、pH を使用してを使用して HEPES Tyrode ソリューション準備 7.3 7.4。</li>
</ol>2. 実験準備
<ol><li>予熱時間 T と 37 ° C の水浴を使用した高 K+ソリューション。</li>
	<li>第 1 報システム、データ集録ハードウェアとコンピューターを入れます。</li>
	<li>慎重に各 H T 溶液 5 mL ですべて第 1 報室を埋めます。</li>
	<li>20 ml の 4 ° C H T と Ca2 +の 2 つのペトリ皿を埋める-H T ソリューションをそれぞれ、無料、氷の上を格納します。</li>
	<li>H T 溶液 20 mL でコーティング 10 cm シャーレを入力し、部屋の温度でそれを維持します。</li>
</ol>3. マウスの腸間膜動脈解離
<ol><li>頚部転位によって 8-12 週齢の c57bl/6 j 女性または男性マウスを安楽死させます。上向きの腹部とマウスを突き止めます。</li>
	<li>70% エタノールで腹部を湿らせます。鼠径部から腹側正中線に沿ってはさみで皮膚を切って、両側の脚に下方の最初の切開開始から切開を加えます。両面に皮膚を引っ張ってください。腹膜を開く同じような切開を行います。</li>
	<li>食道、大腸、体内から血管を餌と消化管を完全に隔離する他の結合組織を切るはさみを使用しています。</li>
	<li>鉗子、血液を洗浄する冷 H T ステップ 2.4 に用意され、数回 H T ソリューションで組織を優しくすすぎを含む皿に分離セグメントを移動します。</li>
	<li>2.5、準備したコーティングのシャーレにセグメントを分離を転送し、室温で上腸間膜動脈解離を行います。</li>
	<li>胃、腸、回腸、盲腸、時計回りの方向を滑らかにし、胃と盲腸の左側と右側の側では、それぞれピンします。</li>
	<li>腸間膜血管ベッドをストレッチし、公開解剖した腸間膜動脈にピンと腸を修正します。 注: これらの条件の下で、動脈は静脈の上にです。</li>
	<li>実体顕微鏡の伝送光源をオンにし、顕微鏡下で動脈を切り裂きます。全体の組織がソリューションに没頭していることを確認します。</li>
	<li>動脈周囲の脂肪組織を鉗子でクランプし、解剖はさみですべての結合組織を遮断し、動脈を分離します。避けるために、動脈を負傷します。</li>
</ol>4. 動脈取付
<ol><li>転送し、冷たい Ca2 +に上腸間膜動脈樹を浸す-余分な動脈を鉗子でクランプによって H T ソリューション (2.4 の手順で準備) を無料します。</li>
	<li>腸間膜のアーケードの腸の壁に近位動脈の 1.4 mm 部分を切断し、この動脈セグメントの両方の側面を慎重に開く 2 つの鉗子を使用します。</li>
	<li>ステンレス鋼ワイヤの長さ 2.5 cm の 2 つのセグメントを準備し、同じ皿にそれらを置きます。</li>
	<li>優しく、鉗子を用いた動脈の片方の端をクランプし、慎重に別の鉗子の助けを借りて 1 つずつ動脈の内腔に 2 本のワイヤを挿入します。ワイヤーがまっすぐに保持されますおよび血管内皮細胞には触れないでを確認します。</li>
	<li>2 つの鉗子を使用すると、スレッドの容器の外 2 つのスチール ワイヤを同時にクランプし、慎重に H T ソリューション (ステップ 2.3) で以前満ちていた第 1 報区域にシャーレから船を転送します。</li>
	<li>顎を離れてのネジ取り付け用のスペースを作る。2 つの鉗子を使用して 2 つの挿入された線のいずれかの両方の側面をクランプし、顎の隙間 (図 1 a) に容器を置きます。</li>
	<li>マイクロメータ (図 1 b) に接続されている顎のネジに固定線の両側を折り返します。</li>
	<li>左側のネジを時計回りにねじることによって修正します。右手の鉗子を使用してワイヤーをまっすぐにし、右側のネジを時計回りに (図 1) をねじることによって修正します。船は常に顎ギャップ内のことを確認してくださいが、損傷を避けるために顎を触れないでください。</li>
	<li>マイクロメータ (図 1) を使用して 2 つの顎を閉じる。2 つの顎が十分に近い彼らに触れないでお互い、固定線の上部に固定されていない線であるが、ことを確認します。</li>
	<li>右手の鉗子を使用して、慎重に探触子を強制的に接続されている顎の角でフロントレンズアセンブリのワイヤーを折り、右側にネジ (図 1E) 周りを時計回りに包んでください。ネジを固定します。線の左側にこの手順を繰り返し、左側にあるネジ (図 1 f) を修正します。</li>
	<li>慎重にマイクロメータ (図 1) を回転させることにより少し離れての顎を移動します。船のストレッチを避けます。鉗子を使用すると、探触子側でワイヤーの水平方向の平面にマイクロメータ側でワイヤーを移動します。2 つの顎の間のギャップはちょうど 2 つのワイヤを適応できるように、慎重にマイクロメータを回転します。</li>
	<li>他のチャンバーに動脈をマウントする手順 4.2-4.11.すべての部屋を装置に接続、チャンバー カバー、100% 酸素供給・温度プローブを接続、37 ° C に加熱を開始グラフ作成ソフトウェアを開き、録音を開始する、グラフ ビューウィンドウの開始ボタンを押します。</li>
	<li>約 20 分を平衡します。</li>
</ol>5. 正規化
注: 実験条件を標準化するために、血管の生理学的応答性信頼性の高いを取得する、正規化手順は必要な15です。アクティブな力と容器の内部の円周の関係, によると線第 1 報システムはマウントされた容器5,8の内部の円周 (IC) を評価するために標準の正規化プログラム 9。簡潔に、マイクロメータの IC (μ m) の計算、読み取って、トランスデューサーと X の値として値を入力するには、Y の値として力、すなわち、壁張力 (mN/mm)、休憩を出力します。プログラムは (X, Y) の曲線を戻り、100 mmHg (IC100) の経壁圧に対応する IC を計算します。船が正規化された内部の円周 (IC の1) に設定されているアクティブな応答性が最大。
<ol><li>デバイス上の力をすべてのチャネル 0 に設定し、別の 1-2 分の平衡します。</li>
	<li>正規化の設定から選択、「DMT メニュー、および次ようパラメーターを設定。 接眼レンズ校正 (mm/div): 0.36;圧 (kPa) をターゲット: 13.3;IC1/IC100: 0.9。オンライン平均時間 (秒): 2;遅延時間 (秒): 60。DMT 正規化の設定ウィンドウを閉じるに[ok]ボタンをクリックします。</li>
	<li>対応するチャネルの DMT の正規化] ウィンドウを開くには、 DMT メニューから目的のチャンネルを選択します。ウィンドウに定数値を入力すると、次のように: 組織エンドポイント a1: 0.1;組織エンドポイント a2: 4;線径 (μ m): 40。ウィンドウには、1.40 mm として計算される容器の長さが表示されます。</li>
	<li>適切な組織の部屋のマイクロメータをお読みください。マイクロメータの読みのボックスに値を入力し、ポイントの追加] ボタンをクリックします。この値 (X0) X の初期値です。60 秒遅延時間後力とこのメーターの値に対応する効果的な圧力 (ERTP) がウィンドウに表示されます。同時に、マイクロメータを読むボックスがアクティブになります。</li>
	<li>反時計回りの方向でマイクロメータを回すことによって正規化されている容器をストレッチします。マイクロメータを読むボックスにマイクロメータ値を入力し、ポイントの追加] ボタンをクリックします。60 の遅延時間を待ち再度 s。</li>
	<li>ステップ 5.5 を繰り返し、続行船をストレッチし、「マイクロメータ X1」、その IC1容器を伸ばすために必要な計算されたマイクロ メーター設定である値が表示されるまで、マイクロメータの値を追加します。</li>
	<li>1の値を X にマイクロ メーターを設定します。 注: 正規化された張力は通常 1-2 mN です。</li>
</ol>6. 動脈収縮記録
注: H T とこのセクションで使用される高 K+ソリューションを含む、すべてのソリューションは、手順 2.1 で調製しました。
<ol><li>正規化の後、15 〜 20 分のために部屋に容器を平衡します。 注: これらはこの手順では、ソリューションを変更する必要はありません。</li>
	<li>高 K+ソリューションの容器に 2 回挑戦します。<ol>
<li>船に挑戦、3-4 回 H T 溶液 5 mL で洗浄後、10 分のための収縮を誘発する高 K+溶液 5 mL と H T ソリューションを交換してください。 注: 一般的な収縮は、最大の力 3 mN および 2.5 mN12の周り一定の持続力。初挑戦 2.5 mN 以下の最大の力を生成するとともに、持続的な力を減少または 2 番目の課題は初めての用量よりもはるかに低い力を生成します、船は破棄され、さらに調査のため使用されません。</li>
	</ol>
</li>
	<li>収縮を誘発し高 K+溶液 5 mL の容器に挑戦します。5 分後に 1 μ M の neoliensinine の最終濃度で血管をリラックス室に neoliensinine 原液 (DMSO 10 mM)14 0.5 μ L を追加します。</li>
	<li>力が安定している場合 (これは通常、いくつかの分を) 濃度を増加する 2 μ m 追加 1 μ L に原液のたびに t を生成する 4、6、8、10 μ M の濃度を増加する商工会議所に別 0.5 μ neoliensinine 原液を追加彼の用量反応曲線。 注: 在庫と作業濃度は薬によって異なります。</li>
</ol>

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高血圧は、心血管などの重篤な合併症など腎臓疾患16広範な公衆衛生課題です。高血圧の病因を理解とより抗高血圧薬の探索は、この分野で緊急の課題になっています。血圧が生成され、循環の末梢血管抵抗によって維持されます。ポアズイユの法則によると比較的小さい動脈循環抵抗の大きい割合を生成し、血圧3,10の主要生産?...

ここで使用される小型船舶の第 1 報システムだった測定の内部直径 100 から 400 μ m 分離小型船舶 (約 2 mm 長い) に至るまでの小さな抵抗血管の等尺性収縮直径 40 μ m 2 本のワイヤーによって挿入されたし、順番にマイクロメータ側と探触子側の顎にマウントされています。この第 1 報手法だった 1972年1で最初に提案したし、Mulvany と彼の同僚2,3,の4,5,6が主に開発しました。今、安定装置、簡単なパフォーマンス標準正規化手順7,8,9と成熟した技術です。我々 はマウスの腸間膜動脈で測定にいくつかの変更でこのメソッドが利用されています。
血管平滑筋は、ほとんどすべての血管の壁を行します。その基本的な機能は、様々 な刺激に対する応答の収縮力を生成します。通常血管平滑筋収縮は血圧調節に欠かせない、栄養補完10。様々 な病気、高血圧、心不全、虚血.などの血圧測定結果の異常な規制いくつかの研究では、異常な血圧が血管平滑筋機能不全収縮7,11,12,13に関連付けられて常にことを示唆しています。第 1 報の方法では、血管収縮薬、阻害薬など様々 な刺激によるマウス血管の等尺性収縮の調査をことができます。収縮の成功の測定は、血圧維持と血管平滑筋疾患の新規治療アプローチを模索する病態のメカニズムを理解するのに役立ちます。
多くの中国のハーブは、血管疾患の臨床治療のため広く使用されています。ただし、その有効成分は通常不明のままに。したがって、有効成分の分離・同定は、新規治療薬の開発のため非常に重要です。第 1 報マルチ ワイヤ技術では、ハーブの有効成分をスクリーニングするための単純なアプローチを提供しています。小型船舶の第 1 報システムを使用してマウスの腸間膜動脈の収縮を調査するいくつかの研究を報告し、抗高血圧活動12,13,14天然化合物を識別しました。ここでは、詳細な説明第 1 報の方法のためのプロトコルし、ロータス (ハス春まき) 種子の胚から分離した neoliensinine の弛緩効果を評価14。

<table>
	<tbody>
		<tr>
			<td>Multi wire myograph system</td>
			<td>DMT</td>
			<td>610-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel wire</td>
			<td>DMT</td>
			<td>400447</td>
			<td></td>
		</tr>
		<tr>
			<td>Geuder dissection scissor</td>
			<td>DMT</td>
			<td>400431</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps</td>
			<td>DMT</td>
			<td>300413</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab/8SP</td>
			<td>ADInstruments</td>
			<td>ML785</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td>ADInstruments</td>
			<td>LabChart 5</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>SigmaAldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>SigmaAldrich</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>SigmaAldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2&#183;6H2O</td>
			<td>SigmaAldrich</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>SigmaAldrich</td>
			<td>G6152</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sangon Biotech</td>
			<td>A100511-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>SigmaAldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>SigmaAldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
	</tbody>
</table>

isometric contractility measurement of the mouse mesenteric artery using wire myography

ワイヤー第 1 報技術を使用して脱分極の応答、GPCR アゴニスト/阻害薬.での血管平滑筋の収縮性を評価するには血管平滑筋の生理学的機能、高血圧など血管系の疾患の病態解明と平滑筋弛緩薬の開発に関する多くの研究で広く使用されます。マウス疾患モデル、遺伝子組み換え系統の大きなプールで広く使用されているモデル動物であります。詳細にマウスの腸間膜動脈の等尺性収縮を測定する方法を紹介します。マウス腸間膜動脈の 1.4 mm のセグメントは分離され、内腔を通して 2 つの鋼線を渡すことによって第 1 報室にマウントされています。平衡と正規化の手順の後、血管収縮アッセイの前に 2 回高 K+ソリューションによってし。医薬品開発におけるこのメソッドのアプリケーションの例としてマウスの腸間膜の新規天然物質の neoliensinine、中国のハーブ、ハスの実 (ハス春まき。) の胚から分離した弛緩効果を測定動脈。第 1 報室に取り付けられた容器セグメント高 K+溶液で刺激を受けました。力の張力が安定した持続的な段階に達したら、neoliensinine の累積線量は商工会議所に追加されました。それは高血圧に対する潜在的な活性をクマが示唆その neoliensinine 平滑筋収縮に対する用量依存性弛緩効果があったとわかりました。さらに、血管できます取り付け後、少なくとも 4 時間を生き残るため、何回も、我々 はお勧めの高 K+ソリューションによる収縮性が維持された、創薬スクリーニングの時間のかかるプロセスの線第 1 報システムを使用すること。

第 1 報配線システムを使用してマウスの腸間膜動脈の等尺性収縮を測定し、, ロータス (ハス春まき) 種子の胚から精製した neoliensinine の弛緩効果を評価14. マウスの腸間膜動脈の分離、結合組織の洗浄、1.4 mm のセグメントにカットします。Ca2 +鋼のワイヤー 2 本で挿入された動脈セグメント-シャーレ、H T ソリューションを無料し、し?...

Watch this Scientific Journal Video about Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography at JoVE.com

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

線第 1 報技術を使用して、血管平滑筋機能や画面の新しい薬を調査します。今回はマウスの腸間膜動脈の等尺性収縮を測定して血管平滑筋の弛緩を新しいスクリーニングのための詳しいプロトコル。

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

isometric-contractility-measurement-mouse-mesenteric-artery-using

Research

JoVE Journal

Medicine

14.6K ビュー.  Nanjing University. 線第 1 報技術を使用して、血管平滑筋機能や画面の新しい薬を調査します。今回はマウスの腸間膜動脈の等尺性収縮を測定して血管平滑筋の弛緩を新しいスクリーニングのための詳しいプロトコル。

ビデオ: Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

中大脳動脈閉塞のマウスモデル

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

医学

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

マウスの永久中大脳動脈結紮の応用

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

自動ワイヤの筋運動記録法を用いた腸間膜動脈の収縮と弛緩の研究

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

Epidural Intracranial Pressure Measurement in Rats Using a Fiber-optic Pressure Transducer

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and cardiac arrest. This elevation may result in further neurological injury, in the form of transtentorial herniation1,2,3,4, midbrain compression, neurological deficit or increased cerebral infarct2,4. Current therapies are often inadequate to control elevated ICP in the clinical setting5,6,7 . Thus there is a need for accurate methods of ICP measurement in animal models to further our understanding of the basic mechanisms and to develop new treatments for elevated ICP.
In both the clinical and experimental setting ICP cannot be estimated without direct measurement. Several methods of ICP catheter insertion currently exist. Of these the intraventricular catheter has become the clinical 'gold standard' of ICP measurement in humans8. This method involves the partial removal of skull and the instrumentation of the catheter through brain tissue. Consequently, intraventricular catheters have an infection rate of 6-11%9. For this reason, subdural and epidural cannulations have become the preferred methods in animal models of ischemic injury. 
Various ICP measurement techniques have been adapted for animal models, and of these, fluid-filled telemetry catheters10 and solid state catheters are the most frequently used11,12,13,14,15. The fluid-filled systems are prone to developing air bubbles in the line, resulting in false ICP readings. Solid state probes avoid this problem (Figure 1). An additional problem is fitting catheters under the skull or into the ventricles without causing any brain injury that might alter the experimental outcomes. Therefore, we have developed a method that places an ICP catheter contiguous with the epidural space, but avoids the need to insert it between skull and brain. 
An optic fibre pressure catheter (420LP, SAMBA Sensors, Sweden) was used to measure ICP at the epidural location because the location of the pressure sensor (at the very tip of the catheter) was found to produce a high fidelity ICP signal in this model. There are other manufacturers of similar optic fibre technologies13 that may be used with our methodology. Alternative solid state catheters, which have the pressure sensor located at the side of the catheter tip, would not be appropriate for this model as the signal would be dampened by the presence of the monitoring screw. 
Here, we present a relatively simple and accurate method to measure ICP. This method can be used across a wide range of ICP related animal models.

A novel technique to record the pressures within the skull is described. The minimally invasive method uses a fibre-optic pressure sensing system to accurately measure intracranial pressure (ICP) in anaesthetized rats without causing significant brain trauma. The technique may be used in a wide range of experimental models.

光ファイバ圧力トランスデューサを使用したラットの硬膜外頭蓋内圧測定

Elevated intracranial pressure (ICP) is a significant problem in several forms of ischemic brain injury including stroke, traumatic brain injury and ...

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 ...

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

圧力筋運動記録法を用いた抵抗腸間膜動脈における筋原性応答および血管作用を評価します

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Isolating and Using Sections of Bovine Mesenteric Artery and Vein as a Bioassay to Test for Vasoactivity in the Small Intestine

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that system. Changes in blood flow to the small intestine can result in effects on the absorptive functions of the organ. Particular interest in toxins liberated from feedstuffs through fermentative and digestive processes has developed in ruminants as an area where productive efficiencies could be improved. The video associated with this article describes an in vitro bioassay developed to screen compounds for vasoactivity in isolated cross-sections of bovine mesenteric artery and vein using a multimyograph. Once the blood vessels are mounted and equilibrated in the myograph, the bioassay itself can be used: as a screening tool to evaluate the contractile response or vasoactivity of compounds of interest; determine the presence of receptor types by pharmacologically targeting receptors with specific agonists; determine the role of a receptor with the presence of one or more antagonists; or determine potential interactions of compounds of interest with antagonists. Through all of this, data are collected real-time, tissue collected from a single animal can be exposed to a large number of different experimental treatments (an in vitro advantage), and represents vasculature on either side of the capillary bed to provide an accurate picture of what could be happening in the afferent and efferent blood supply supporting the small intestine.

The small intestine is frequently exposed to toxins that can influence blood flow and negatively impact nutrient absorption. Using a multimyograph and mesenteric artery and vein isolates, compounds or toxins of interest can be screened for vasoactivity.

バイオアッセイとしてウシ腸間膜動脈と静脈の切片を分離して使用すると、小腸で血管作用をテストするために

Mammalian gastrointestinal systems are constantly exposed to compounds (desirable and undesirable) that can have an effect on blood flow to and from that ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

マウス肺における圧力 - 容積曲線の測定

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

血管周囲ドラッグデリバリーパッチの移植に大腿動脈ワイヤ傷害のマウスモデル

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

マウスにおける加速アテローム性動脈硬化症の誘導:ワイヤー傷害モデル

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...