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

9.7K 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

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 ...

医学

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 ...

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 ...

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

期産期新生児ラットにおける幹細胞の気管内植在

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...

A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered drug. This has direct translational impact because surfactant and drugs are administered locally into the lungs. Though the literature has many publications describing minimally invasive transoral intubation of adult mice and rats in therapeutic experiments, this approach in neonatal rat pups is lacking. The small size of orotracheal region/pharynx in the pups makes visualization of laryngeal lumen (vocal cords) difficult, contributing to the variable success rate of intratracheal drug delivery. We hereby demonstrate effective oral intubation of neonatal rat pup - a technique that is non-traumatic and minimally-invasive, so that it can be used for serial administration of drugs. We used an operating otoscope with an illumination system and a magnifying lens to visualize the tracheal opening of the rat neonates. The drug is then instilled using a 1 mL syringe connected to a pipette tip. The accuracy of the delivery method was demonstrated using Evans blue dye administration. This method is easy to get trained in and could serve as an effective way to instill drugs into trachea. This method could also be used for administration of inoculum or agents to simulate disease conditions in animals and, also, for cell-based treatment strategies for various lung diseases.

This technique of instilling drugs directly into the trachea of neonatal rodents is important in studying the impact of locally administered drugs or biologicals on neonatal lung diseases. Additionally, this method can also be used for inducing lung injury in animal models.

肺疾患を治療するための新生児げっ歯類における薬物の気管内点滴のための低侵襲方法

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered ...

Customizable Disposable Dosators to Deliver Dry Powder Aerosol Drugs to Mice

Disposable Dosators Intended for Dry Powder Delivery to Mice

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus metering and dosing, and a propellant-free dispersal mechanism. To develop pharmaceutical dry powder aerosol products, robust in vivo testing is essential. Typically, initial studies involve using a murine model for preliminary evaluation before conducting formal studies in larger animal species. However, a significant limitation in this approach is the lack of suitable device technology to accurately and reproducibly deliver dry powders to small animals, hindering such models&#39; utility. To address these challenges, disposable syringe dosators were developed specifically for intrapulmonary delivery of dry powders in doses appropriate for mice. These dosators load and deliver a predetermined amount of powder obtained from a uniform bulk density powder bed. This discrete control is achieved by inserting a blunt needle to a fixed depth (tamping) into the powder bed, removing a fixed quantity each time. Notably, this dosing pattern has proven effective for a range of spray-dried powders. In experiments involving four different model spray-dried powders, the dosators demonstrated the ability to achieve doses within the range of 30 to 1100 &#181;g. The achieved dose was influenced by factors such as the number of tamps, the size of the dosator needle, and the specific formulation used. One of the key benefits of these dosators is their ease of manufacturing, making them accessible and cost-effective for delivering dry powders to mice during initial proof-of-concept studies. The disposable nature of the dosators facilitates use in animal procedure rooms, where cleaning and refilling reusable systems and weighing materials is inconvenient. Thus, developing disposable syringe dosators has addressed a significant hurdle in murine dry powder delivery for proof-of-concept studies, enabling researchers to conduct more accurate and reproducible preliminary studies in small animal models for pulmonary drug delivery.

Author Spotlight: Developing a Disposable Dosator for Preclinical Testing of Dry Powder Inhalers in Small Animal Models 

Pharmaceutical dry powder development necessitates reliable in vivo testing, often using a murine model. Device technology for accurately and reproducibly delivering dry powder aerosols to mice is restricted. This study presents disposable dosators for pulmonary drug delivery at mouse-relevant doses, aiding initial proof-of-concept research.

マウスへの乾燥粉末送達を目的としたディスポーザブルドセーター

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus ...