著者らは、レイ・リー氏、HCリョン氏、ウォレス氏に対し、光源と粉末のインスフレーターを作る際の親切な支援に感謝したいと考えている。動物イメージングの支援のための教員コア施設。研究助成金協議会(17300319)によって支援されました。

本研究で行われた実験は、香港大学の生きた動物の教育研究用使用委員会(CULATR)によって承認されています。ルシファーゼメッセンジャーRNA(mRNA)の0.5%を含む噴霧凍結乾燥(SFD)によって調製された乾燥粉末製剤は、5%合成ペプチドPEG12KL4および94.5%のマンニトール(w/w)を肺16におけるmRNA発現を実証するために本研究で使用される。SFD粉末の質量中央値空気力学直径(MMAD)は2.4μmです。噴霧乾燥(SD)マンニトール粉末は、粉末分散液16に用いられる空気量の影響を調べるため使用される。SD粉末のMMADは1.5μmです。
1. 乾燥粉末インスフレーターの製造と乾燥粉末のローディング
<ol><li>(オプション)乾燥粉末(バイアル内)の静電荷と200 μLノンフィルター丸いゲルローディングピペットチップを中和します。メーカーの指示に従って、帯電防止銃または脱イオン機能を備えたバランスを使用してください。</li>
	<li>約4cm x 4cmのサイズの計量用紙を用意します。紙を斜めに半分に折り、折り返します。</li>
	<li>計量紙に1〜2mgの乾燥粉末を計量する。</li>
	<li>ゲルローディングピペットチップに、チップの広い開口部を通して粉末を充填します。粉末が先端の狭い端付近に緩い凝集体を形成するまで粉末を軽くたたきます(図1A)。粉末の分散を妨げる可能性があるため、粉末をしっかりと詰め込むのは避けてください。</li>
	<li>パウダーロードされたチップを3方向のストップコックを通して1 mLのシリンジに接続します(図1B)。シリンジのサイズは、粉末を分散させるために使用される空気の量に応じて変更することができます。取り付け時にチップとシリンジを垂直に持ち、粉がこぼれないようにします。投与がすぐに行われない場合は、パラフィルムを使用して先端の開口部を密封し、投与するまで適当な状態で一時的に保管する。</li>
</ol>2. 挿管装置の製造
<ol><li>光源(図2)<ol>
<li>発光ダイオード(LED)トーチと直径0.8~1mmのフレキシブル光ファイバを使用して、カスタムメイドの光源を用意します。</li>
		<li>LEDトーチのクリアレンズに、光ファイバが通り過ぎるのがやっとになるように、ハンドドリルまたはドリルビットで中央のオリフィスを作ります。</li>
		<li>オリフィスを通して光ファイバを挿入します。LEDトーチのスイッチを入れ、光ファイバのもう一方の端で最大の明るさを得るために、位置と挿入深度を調整します。</li>
		<li>透明エポキシ接着剤で光ファイバを位置に貼り付ける。</li>
	</ol>
</li>
	<li>導くカニューレ(図3)<ol>
<li>1 mLプラスチック製のパスツールピペット(図3A)を取り、両端にピペットを持ちなさい。</li>
		<li>アルコールランプ(またはブンゼンバーナーなどの実験室の他の熱源)を使用して、ピペットの真ん中を火炎の上に5〜10cmに置いて加熱する(図3B)。ピペットを回転させて、均一に加熱します。</li>
		<li>プラスチックが柔らかく変形可能になったら、ピペットを炎から離し、ピペットを軽く伸ばします。</li>
		<li>中央に伸ばしたピペットを、はさみでパートAとパートB(図3C-E)に切ります。細かいチップピペットとしてパーツAを使用し、ガイドカニューレとしてパートBを使用します。導くカニューレで挿管が成功する可能性を高めるために、パートBの終わりにベベル(動物を傷つけやすいリスクを高める可能性のある鋭すぎない)を作ります(図3F)。200 μLのゲルローディングピペットチップ(粉末ローディング用)を導くカニューレに挿入すると、カニューレを1~2mm突き出す必要があります。 注:挿管のための適切な次元(内径および外径)を有する導くカニューレ(パートB)は、17ゲージ針の中に収まる一方で、その中に21ゲージの針を収めることができます。適切な寸法を達成するためにピペットを伸ばすに複数の試みが必要な場合があります。</li>
		<li>(オプション):導くカニューレの広い端にある小さな開口部を切り取り、光ファイバを保持しやすくするために、より柔軟にします(図3F)。この開口部はまた液体エアロゾルの管理のためのマイクロスプレイヤーの付属品を可能にする。</li>
	</ol>
</li>
</ol>3. 挿管
<ol><li>腹腔内注射によりケタミン(100mg/kg)およびキシラジン(10mg/kg)でマウス(BALB/c、7-9週)を麻酔します。</li>
	<li>プレキシガラス製のプラットフォームを準備し、クランプ付きスタンドに取り付けます(図4A)。麻酔マウスをプラットフォーム上(約60°の傾き)に置きます。プラットフォームの高さと傾斜角度は、スタンドのクランプの位置によって調整できます。</li>
	<li>ナイロンフロスに切歯を掛けてマウスを吊り下げます(図4B)。テープまたはゴムバンドでマウスの位置を固定します。</li>
	<li>誘導カニューレの開口部を持つファイバーレベルの先端を使用して挿管前に誘導カニューレに光ファイバを挿入します。LEDトーチをオンにして点灯します。</li>
	<li>マウスの舌を一対の鉗子で軽く突き出して気管を露出させる。</li>
	<li>もう一方の手を使用して、内部の光ファイバーと導くカニューレを保持します。口腔を通してそれらを挿入します。光ファイバからの照明により、気管の開口部を声帯間のオリフィスとして可視化することができます。</li>
	<li>ガイドカニューレのベベルを開口部の中線に向かって位置合わせします(図5A)。気管開口部でカニューレの最高級先端を狙って、光ファイバーで導くカニューレを気管にそっと挿管します。</li>
	<li>挿管時に、光ファイバを素早く取り外し、導くカニューレを気管の中に残します(図5B)。正常な呼吸を観察する必要があります。</li>
	<li>導くカニューレの開口部に細かい先端ピペット(パートA)を持ち、マウスの肺に空気の小さなパフ(約0.2 mL)を注入します。マウスの胸部にわずかなインフレが適切な挿管を示す。粉末投与前に細かいチップピペットを取り除きます。</li>
</ol>4. 粉末管理
<ol><li>ステップ 1.5 で説明したように、シリンジに接続されている粉末のチップを保持します。シリンジとチップの間の気流が切断されていることを確認します。</li>
	<li>0.6 mLの空気を引き出すために、シリンジプランジャーを後方に引っ張ります。 注:粉末を分散させるために使用される空気の量は、粉末の特性と粉末の積み込みの量に依存します。これは、結果のセクションで詳しく説明します。</li>
	<li>三方向ストップコックのバルブを回して、シリンジと粉末を積んだ先端の間の気流を接続します。</li>
	<li>粉末を装填したチップを、マウスの気管に既に入れられていた導きカニューレに挿入します(図5C)。導くカニューレを保持し、肺にエアロゾルとして粉末を分散させる1つの連続的なアクションで注射器プランジャーを強引に押します。 メモ:デバイスの前方運動は、動物が傷つくことを避けるために最小限に抑える必要があります。</li>
	<li>先端を取り出し、先端の中の粉が空になっているかどうかを確認します。ない場合は、ステップ 4.1 から 4.4 を繰り返します。 メモ:過度のタッピングのために粉末がしっかりと詰め込みすぎると、適切に分散していない可能性があります。</li>
	<li>投与が完了したら、導くカニューレを気管から取り出します。</li>
	<li>マウスは、気道の封鎖を避けるために、舌を半分突き出した後、縦方向に縦方向に配置して回復させます。</li>
</ol>

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著者らは開示する利益相反を持っていません。

本論文では乾燥粉末の浸潤と気管内挿管用のカスタムメイドの装置を紹介する。粉末ローディングステップでは、乾燥粉末を200 μLのゲルローディングピペットチップに積み込みます。先端の狭い端で粉の緩いパッキングを可能にするために、先端を軽くタップすることが重要です。ただし、粉末が固く詰めすぎると、先端に詰まり、適切に分散することはできません。特に低密度で相対湿度...

肺投与経路は、局所的および全身的な行動の両方の治療薬を提供する上で様々な利点を提供する。肺疾患の治療のために、高い局所薬物濃度は肺分娩によって達成され、それにより必要な用量を減少させ、全身的な副作用の発生率を低下させる。さらに、肺における比較的低い酵素活動は、早期薬物代謝を減少させることができる。肺はまた、大きく、よく浸透した表面積、極めて薄い上皮細胞層および肺毛細血管における高血中量による全身作用のための薬物吸収のために有効である1。
吸入乾燥粉末製剤は、喘息、慢性閉塞性肺疾患、糖尿病および肺ワクチン接種2、3、4などの様々な疾患の予防および治療のために広く検討されている。固体状態の薬物は、一般的に液体の形態よりも安定しており、乾燥粉末吸入器はネブライザー5、6よりも携帯性と使いやすさである。吸入乾燥粉末製剤の開発において、安全性、薬物動態プロファイルおよび治療効果は、肺投与7に続く前臨床動物モデルにおいて評価される必要がある。乾燥粉末を積極的に吸い込むことができる人間とは異なり、乾燥粉末を小動物に肺分娩することは困難である。動物の肺に乾燥粉末を送達する効率的なプロトコルを確立する必要があります。
マウスは経済的で繁殖性が高いため、研究動物モデルとして広く使用されています。彼らはまた、扱いやすく、多くの疾患モデルが確立されています。マウスの肺に乾燥粉末を投与する2つの主要なアプローチがあります: 吸入と気管内投与.吸入のために、マウスは乾燥粉末がエアロゾル化され、動物がセデレーション8、9なしでエアロゾルで呼吸する全身または鼻のみのチャンバーに置かれる。高価な機器が必要であり、薬物送達効率が低い。全身室は技術的にはそれほど難しいかもしれませんが、鼻のみの暴露室は身体表面への薬物の暴露を最小限に抑えることができます。いずれにせよ、肺に送達される用量を正確に制御し、決定することは依然として困難である。乾燥粉末は主に、粘液性クリアランスが顕著である鼻咽頭領域に堆積する10.また、チャンバー内のマウスは、食糧および水供給11を制約し、奪われるため、投与プロセス中に大きなストレスを受けている。気管内投与の場合、一般的に気管内に直接物質を導入することをいう。これを達成するための2つの異なる技術があります:気管切開術と口内挿管。前者は、侵襲的で粉末投与にめったに使用されない気管の切開を行う外科的処置を必要とする。ここでは、2 番目のテクニックのみを説明します。吸入方法と比較して、気管内投与は、薬物損失を最小限に抑える12,13の高い送達効率のためにマウスでの肺送達のためにより一般的に使用される方法である。これは、マウスに数ミリグラム以内の粉末を正確に少量を提供する簡単で迅速な方法です。マウスはヒトに対して解剖学的および生理的に区別され、挿管過程では麻酔が必要であるが、気管内投与は上気道をバイパスし、肺吸収、生体利用能および治療効果14、15などの乾燥粉末製剤の生物学的活性を評価するより効果的な方法を提供する。
乾燥粉末を気管内に投与するには、マウスを挿管する必要があり、これは困難である可能性があります。本論文では、カスタムメイドのドライパウダーインスフレーターと挿管装置の製造について説明する。マウスの肺における乾燥粉末の挿管および浸潤の手順が実証されている。

<table>
	<tbody>
		<tr>
			<td>BALB/c mouse</td>
			<td></td>
			<td></td>
			<td>Female; 7-9 weeks old; Body weight 20-25 g</td>
		</tr>
		<tr>
			<td>CleanCap Firefly Luciferase mRNA</td>
			<td>TriLink Biotechnology</td>
			<td>L-7602</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Powder Insufflator</td>
			<td>PennCentury</td>
			<td>Model DP-4M</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine 10%</td>
			<td>Alfasan International B.V.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Light emitting diode (LED) torch</td>
			<td>Unilite Internation</td>
			<td>PS-K1</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol (Pearlitol 160C)</td>
			<td>Roquette</td>
			<td>450001</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-filter round gel loading pipette tip (200 &#181;L)</td>
			<td>Labcon</td>
			<td>1034-800-000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon floss</td>
			<td>Reach</td>
			<td>30017050</td>
			<td></td>
		</tr>
		<tr>
			<td>One milliliter syringe without needle</td>
			<td>Terumo</td>
			<td>SS-01T</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical fibre</td>
			<td>Fibre Data</td>
			<td>OMPF1000</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG12KL4 peptide</td>
			<td>EZ Biolab</td>
			<td></td>
			<td>(PEG12)-KLLLLKLLLLKLLLLKLLLLK-NH2</td>
		</tr>
		<tr>
			<td>Plastic Pasteur fine tip pipette</td>
			<td>Alpha Labotatories</td>
			<td>LW4061</td>
			<td></td>
		</tr>
		<tr>
			<td>Three-way stopcock</td>
			<td>Braun</td>
			<td>D201</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine 2%</td>
			<td>Alfasan International B.V.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Zerostat 3 anti-static gun</td>
			<td>MILTY</td>
			<td>5036694022153</td>
			<td></td>
		</tr>
	</tbody>
</table>

intratracheal administration of dry powder formulation in mice

吸入可能な乾燥粉末製剤の開発では、前臨床動物モデルにおけるそれらの生物学的活性を評価することが不可欠である。本論文では、マウスにおける乾燥粉末製剤の非侵襲的な方法を紹介する。3方向のストップコックを介して1 mLシリンジに接続された200 μLのゲルローディングピペットチップから成る乾燥粉末ローディング装置が提示されます。少量の乾燥粉末(1〜2mg)をピペットチップに装填し、0.6mLの空気をシリンジに分散させます。ピペットチップは使い捨てで安価であるため、異なる乾燥粉末製剤を事前に異なるチップにロードすることができます。様々な製剤は、デバイスの洗浄および用量補充なしで同じ動物実験で評価することができ、それによって時間を節約し、残留粉末からの交差汚染のリスクを排除する。粉末分散の程度は、ピペットチップに残っている粉末の量によって検査することができる。カスタムメイドの光源と導くカニューレを使用したマウスの挿管のプロトコルが含まれています。適切な挿管は、マウスの深部肺領域への乾燥粉末製剤の気管内送達に影響を与える重要な要因の1つである。

乾燥粉末インスフレーターを使用して動物の肺に粉末エアロゾルを送達する場合、使用される空気の量は、安全性と粉末分散効率に影響を与えるため、非常に重要です。この方法を最適化するために、異なる量の空気(0.3mL、0.6mLおよび1.0mL)を用いて乾燥粉末(1mgの噴霧乾燥マンイトール)を分散させ、マウスの体重を投与後48時間監視した(図6)。0.3 mLおよび0.6 mLの空気の使?...

Watch this Scientific Journal Video about Intratracheal Administration of Dry Powder Formulation in Mice at JoVE.com

Intratracheal Administration of Dry Powder Formulation in Mice

吸入用の乾燥粉末製剤は、呼吸器疾患の治療に大きな可能性を秘めています。ヒト研究に入る前に、前臨床試験における乾燥粉末製剤の有効性を評価する必要がある。気管内経路を介したマウスにおける乾燥粉末の投与の簡便かつ非侵襲的な方法が提示される。

マウスにおける乾燥粉末製剤の気管内投与

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

intratracheal-administration-of-dry-powder-formulation-in-mice

Research

JoVE Journal

Medicine

9.8K ビュー.  The University of Hong Kong. 吸入用の乾燥粉末製剤は、呼吸器疾患の治療に大きな可能性を秘めています。ヒト研究に入る前に、前臨床試験における乾燥粉末製剤の有効性を評価する必要がある。気管内経路を介したマウスにおける乾燥粉末の投与の簡便かつ非侵襲的な方法が提示される。

ビデオ: Intratracheal Administration of Dry Powder Formulation in Mice

Improving IV Insulin Administration in a Community Hospital

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4 The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5 It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6 Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p&lt;0.001). From this pivotal trial and two others,7,8 Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia. 
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG &lt; 40 mg/dL), clinical hypoglycemia (BG &lt; 70 mg/dL), and hyperglycemia (BG &gt; 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG &lt; 70 mg/dL) with the use of the computer glucose management system was well under 1%.

When compared to the previous paper protocol, implementation of a computerized glucose management system results in a substantial increase in blood glucose concentration measurements within the target range. Using a computerized glucose management system to monitor blood glucose levels, decreases in severe hypoglycemia (&lt;40 mg/dL), clinical hypoglycemia (&lt;70 mg/dL), and hyperglycemia (&gt;180 mg/dL) also can be observed.

コミュニティ病院でIVのインスリン投与を向上させる

Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose ...

医学

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

胎児のマウスモデルにおける生物活性剤の気管内投与のための新しい手術アプローチ

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

アウェイクマウスに対する中枢神経系治療薬の経鼻投与

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Intubation-mediated Intratracheal (IMIT) Instillation: A Noninvasive, Lung-specific Delivery System

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences between the murine and human respiratory systems, especially in their upper respiratory tracts (URT). In some models, these differences in the murine nasal cavity can have a significant impact on disease progression and presentation in the lower respiratory tract (LRT) when using intranasal instillation techniques, potentially limiting the usefulness of the mouse model to study these diseases. For these reasons, it would be advantageous to develop a technique to instill bacteria directly into the mouse lungs in order to study LRT disease in the absence of involvement of the URT. We have termed this lung specific delivery technique intubation-mediated intratracheal (IMIT) instillation. This noninvasive technique minimizes the potential for instillation into the bloodstream, which can occur during more invasive traditional surgical intratracheal infection approaches, and limits the possibility of incidental digestive tract delivery. IMIT is a two-step process in which mice are first intubated, with an intermediate step to ensure correct catheter placement into the trachea, followed by insertion of a blunt needle into the catheter to mediate direct delivery of bacteria into the lung. This approach facilitates a &#62;98% efficacy of delivery into the lungs with excellent distribution of reagent throughout the lung. Thus, IMIT represents a novel approach to study LRT disease and therapeutic delivery directly into the lung, improving upon the ability to use mice as surrogates to study human respiratory disease. Furthermore, the accuracy and reproducibility of this delivery system also makes it amenable to Good Laboratory Practice Standards (GLPS), as well as delivery of a wide range of reagents which require high efficiency delivery to the lung.

Intubation-mediated Intratracheal IMIT Instillation: A Noninvasive, Lung-specific Delivery System

Intubation-mediated intratracheal (IMIT) instillation of reagents is an excellent, noninvasive method for studying respiratory disease, as well as a method for instilling therapeutic reagents directly into the lung. It is a rapid and highly reproducible method which is suitable for preclinical testing.

挿管媒介気管内（IMIT）点眼：非侵襲的、肺特異的送達システム

Respiratory disease studies typically involve the use of murine models as surrogate systems. However, there are significant physiologic differences ...

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

ラットにおける同所性肝細胞癌の局所領域治療のための腫瘍溶解性ウイルスのTransarterial管理

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

乾燥粉末および医薬品の霧状エアロゾルを吸入すると、鼻のみの露光装置を用いてマウスに配信します

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

A Pleural Effusion Model in Rats by Intratracheal Instillation of Polyacrylate/Nanosilica

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study these pulmonary diseases. Previous pleural effusion models paid more attention to biological factors rather than nanoparticles in the environment. Here, we introduce a model to make pleural effusion in rats by intratracheal instillation of polyacrylate/nanosilica, and a method of nanoparticle isolation in the pleural effusion. By intratracheal instillation of polyacrylate/nanosilica with concentrations of 3.125, 6.25 and 12.5 mg/kg&#8729;mL, the pleural effusion in rats presented on day 3, peaked at days 7-10 in 6.25 and 12.5 mg/kg&#8729;mL groups, then slowly decreased and disappeared on day 14. When the concentration of polyacrylate/nanosilica increased, the pleural effusion is produced more and faster. This pleural fluid was detected by ultrasound examination or CT chest scanning and confirmed by dissection of rats. Silica nanoparticles were observed in the rats' pleural effusion by transmission electron microscope. These results showed that the exposure to polyacrylate/nanosilica leads to the induction of pleural effusion, which was consistent with our previous report in humans. Additionally, this model is beneficial for the further study of nanotoxicology and the pleural effusion diseases.

Here, we present a protocol to construct a pleural effusion model in rats by intratracheal instillation of polyacrylate/nanosilica.

ポリアクリル酸/ナノシリカの気管内投与によるラットの胸水貯留モデル

Pleural effusion is a prevalent clinical finding of many pulmonary diseases. Having a useful animal pleural effusion model is very important to study ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

直接気管内リポ多糖刺激によるマウスの急性肺損傷の誘導

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

ウサギにおける完全なダクリオアデネクトー切除術を用いた重症ドライアイモデルの確立

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...