This work was supported by AI101263 from the National Institutes of Health (T.V.B), and by a grant from The Hartwell Foundation (M.L.M). M.L.M. is a member of the Duke Comprehensive Cancer Center.

注：ラットの使用を含むすべての実験手順は、デューク大学の動物実験委員会によって承認されたプロトコルに従って行われた。 気管挿管カニューレの作製<ol><li>ペンチを切断と2インチ14のG angiocathether針の針端をカット。 </li><li>針の内腔がラジオペンチで閉じピンチ。 </li><li>ペンチ、オープンルーメン部のエッジまで挟持部を切断し、次いで、30で残りの端部の両側縁をカット - 針縁に対して45°の角度。 </li><li>ストレートスリップジョイントプライヤーのオープンエリアにカニューレの4ミリメートルとわずかに上向きの曲線が最後に形成されるまで上向きに引っ張る - ボトム3を配置します。 注：これは気管（ 図1）にカテーテルを演出容易にするであろう。 </li><li>カニューレの最後で縁を下に滑らかにするために細かいサンドペーパーを使用してください。 </li><li&gt;血管カテーテルチューブに鈍化楽器を返します。チューブは鈍化針より少し短いことを確認してください。 </li></ol> 2.手術前の手順<ol><li>動作領域にわたって10倍の倍率に設定されて手術用顕微鏡を配置することによって、手続きのためにサイトを設定します。 </li><li>動作領域での温暖化パッドを配置し、クリーンな吸収パッドでカバーしています。 </li><li>手術野の近くに心拍数及び血液酸素モニタをセットアップする。 </li><li>重量ベースの薬剤の服用（ 例えば、術後の鎮痛剤、抗生物質、またはリンパ球枯渇抗体）のためにラットを秤量する。 注： - 450gの典型的な成体ラットは、350の間で重量を量る。 </li><li>誘導チャンバーを用いて気化し、3％イソフルランO 2（3 L /分）と落ち着いたラットは、活性炭を含有する廃棄麻酔掃気システムにベント。気管内挿管のために必要な深い麻酔を提供するために、麻酔誘導のために5分を許可する。 </li><li>挿管や手術を進める前に皮下ラットにカルプロフェンまたはMeloxicane（4.4ミリグラム/ kg）を管理します。 </li><li>挿管前に麻酔したラットの首と胸を剃るために電気バリカンを使用してください。 </li></ol> 3.挿管<ol><li>挿管カニューレの先端に潤滑少量（ 例えば、KYゲル）を擦って挿管のセットアップを準備します。コネクタチューブを通してイソフルランガスの流れを開始するために人工呼吸器の電源をオンにします。 </li><li>挿管装置にラットを移し、その上側切歯（ 図2A）によって金属棒にラットを一時停止する。 メモ：一部の挿管装置は、オンラインで購入することができます。ここに示したモデルは、ブタンのトーチを用いて2×4インチの腰部片の切片の周囲に成形アクリル板を使用した自家製であり、補強材をクロロホルムで取り付けられている。バー（又は金属ワイヤ）はsの上に配置されたマスキングテープの上に穴を穿孔することによって取り付けられている分裂やクラックを防止するためのアクリルのIDE。 </li><li>首（ 図2B）の腹側表面2からセンチメートル-柔軟な高輝度光源1を配置することによって、ラットの首をトランスは、照らす。 </li><li>優しく上向きと下の歯の側に舌を引っ張って生徒標準パターン鉗子のペアを使用してください。片手の親指と人差し指の間に把持舌を舌の下側端部に対して鉗子の一方の側の平坦な内側面を配置している。を押して、腹側に喉頭（ 図2C）の喉頭蓋と開口部を露出させる。 </li><li>声帯を視覚化し、血管カテーテルのハブが切歯に触れるまで気管内にオープン声門を通して前方に挿管カニューレの上向き、平滑化終了を導く。 </li><li>金属スタイレットを取り外し、イソフルランで換気を開始するために血管カテーテル口に麻酔チューブを取り付けます。 注：典型的な酸素FLO人工呼吸器のワット率は3％イソフルラン3 L /分である。 <ol><li> 14 mmHgの - 〜12の圧力を達成し、ボリューム制御人工呼吸器モードで60呼吸/分の速度で人工呼吸器を設定します。 3cmのH 2 Oの正の呼気終末圧（PEEP）を使用</li></ol></li><li>気管内チューブの適切な配置を確実にするために人工呼吸器に同期してバイラテラル胸壁の拡張を観察する。 </li><li>麻酔下ながら乾燥を防ぐために、ラットの目に獣医師推奨の眼軟膏を適用します。 </li><li>麻酔下ながら乾燥を防ぐために、ラットの目に獣医軟膏を適用します。 </li><li>布テープのストリップでラットの頭部に気管内チューブを固定します。気管内チューブと麻酔薬の流入チューブの間にしっかりと接続を確認してください。 </li><li>ラットの足への血液の酸素と心拍数のモニターを接続し、監視を開始します。への反応がありません確認することにより、適切な麻酔を確認電子ピンチ。 </li></ol> 4.開胸と胸腺摘出<ol><li>手順の全体のための標準的な無菌操作に従ってください。 <ol><li>作業領域と手術台を清掃し、70％エタノール溶液で消毒する。 </li><li>手順中の無菌手術用手袋を使用して、手順の間使用されるすべての機器と材料をオートクレーブ。 </li><li>全体胸の部分に - イオジンを適用し、それを乾燥させます。その後、ガーゼに70％エタノールで皮膚の表面をきれいに。無菌手術野を露出するように穴をカットする透明なプラスチックのラップでラットをカバーしています。 </li></ol></li><li>上部の胸部に胸骨上切痕を識別します。胸骨上切痕上記3ミリメートルと鈍先端がシェイはさみを使って胸骨正中線に沿って遠位延長 - 2の起動皮膚を通して2cmの正中切開を行います。 </li><li>鈍先端がシェイはさみを使用して再度胸骨上切痕から1.5センチメートル胸骨正中切開を行います。 Kただゆっくり胸骨、事前の下にはさみの下端をEEP。 </li><li>前気管ストラップの筋肉（胸骨舌骨と胸骨甲状筋）を明らかにすることだけ分離胸骨とオープンの下に小さなアルムリトラクターを挿入します。ブラントグレーフェ鉗子を用いて予備気管ストラップの筋肉を分離してください。注：この時点で、気管を見ることができ、挿管チューブが気管の内側に視覚化されるべきである。 </li><li>分離ストラップの筋肉と胸骨の下に小さなアルムリトラクタの突起を配置します。胸腺の優れた面を露出させるために開創器を開きます。 </li><li>胸腺組織の側縁を解放し、下胸腺ローブを公開する細かいデュモンの鉗子を使用してください。 </li><li>上大静脈、鎖骨と頸動脈血管との接触を避けるように注意しながら、オープンな切開部位に優しく上方に胸腺を引き、胸腺、肺の間の微妙な胸膜ライニングの中断を最小限に抑え。 </li><li> Tと彼は血管が解剖された胸腺と明らかにし、それらを分割するために、マイクロはさみを使用しています。必要に応じて止血するための容器に圧力を保持するために綿棒を使用してください。 </li><li>切開部に下胸腺のローブを提供し、その後急激に後部アタッチメントを溶解。そのまま胸腺を取り外し、慎重に不足しているセクションの削除胸腺を検査。 注：小胸リンパ節、胸腺を可視化し、多くの場合、周囲の胸腺組織から区別することが困難であることができる。これらは、胸腺組織に似た外観を持つ個別の丸いノードになりますが、胸腺との連続性になりません。 </li><li>アルムリトラクターを取り外し、2 5-0マクソン縫合糸を中断して胸骨とsternothyoid筋肉を閉じます。 </li><li>陽圧換気下縦隔を密封するために、縫合糸両端の外科用グレードのシアノアクリレート組織接着剤の2滴を適用する。 注：これは気胸や血腫の発生率を減少させる。 </li><li> STを閉じる2とのernumは、切削針に4-0絹縫合糸を中断。下層の筋層を避けるために、ちょうど胸骨の下に針を案内するように注意しながら、リブ間の隙間を通って針を挿入します。 </li><li>実行中の4-0ナイロン縫合糸で皮膚層を閉じます。術後の麻酔の回復期間を短縮するために、この時点でイソフルランを中止する。 </li><li>生理食塩水で湿らせたガーゼで切開部位と周囲の皮膚を清掃してください。 </li><li>切開部をシールするために切開を介してシアノアクリレート接着剤の2滴 - 1、続いて局所麻酔用ブピバカイン（0.25％）数滴を適用する。 </li><li>ラットは独立した呼吸努力の兆候を示しているし、その四肢を動かし始めるまで換気を続けます。ラットを抜管し、それが温暖化パッド上に置かケージに近い観察下に回復することができます。 </li><li>ブプレノルフィンの管理（0.01から0.05 mg / kgを）皮下ラットにすぐに手術後および48時間のPOごとに8〜12時間を繰り返すST-動作可能（通常は5合計用量）。手術後24および48時間で、カルプロフェンまたはMeloxicane（4.4 mg / kgを）皮下（術前の投与量を含む3総投与量）を管理。 </li><li>ラットがケージの周りを移動されるまで監視し続ける。呼吸困難の兆候では、再挿管と胸の再探索を行う。 <ol><li>それは胸骨横臥位を維持するのに十分な意識を取り戻したまでは無人の動物を放置しないでください。 </li><li>完全に回復するまで、他の動物の会社に手術を受けた動物を返さないでください。 </li></ol></li><li>スキン層の縫合糸は1週間手術後に削除します。 </li></ol>

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The authors have nothing to disclose.

完全な胸腺摘出のた​​めの本プロトコルは、合併症を最小限に抑えるように設計外科グルー·アプリケーションとの3層切開の閉鎖と低侵襲性アプローチを提供します。胸腺の完全な除去は、ナイーブT細胞の喪失によってサイトケラチンための縦隔リンパ系組織のIHC染色によって実証された。 、胸膜と胸腺の下葉の密接な関係に起因する心とそのほとんどが周術期の合...

1960年代初頭以来、胸腺は、中央免疫寛容の開発におけるその重要な役割のために認識されてきた。げっ歯類の胸腺摘出は、リンパ球分化、自己寛容、および同種移植及び腫瘍転移の設定で免疫寛容における胸腺の役割を定義する上で不可欠な手順であることが証明された。ラットの胸腺の除去は、T細胞の枯渇またはネイティブナイーブT細胞の再出現することなく定義されたT細胞集団の養子移入を含む研究を可能にする。 新生児げっ歯類におけるThymectomiesは、信頼性の高い成果1で吸引技術を使用して達成することができる。成体ラットにおいて、この技術は、おおよそ20％の死亡率と関連しており、しばしば不完全な胸腺2になる。一貫して、成体ラットにおける完全な胸腺摘出を達成するために、胸骨正中切開を介して縦隔のオープン露光が必要とされる。しかし、この6％ の2- 4 -手順1.5の範囲の全体的な死亡率につながる気管損傷、出血、および気胸を含む合併症に関連している。 最後の20年間胸腺摘出技術の改善は、周術期合併症を減少しており、生存率が改善している。陽圧換気を可能に気管内挿管は、気胸率5軽減されている。挿管の方法は、以前に直接声帯の可視化を使用して、より侵襲性の低い方法に気管に開いた露出の範囲を説明した。挿管手順に関連する合併症は心臓穿刺または上大静脈の裂傷から生じる気管損傷、声帯破裂、意図しない食道挿管、および出血があります。また、胸膜ライニングに下胸腺ローブのすぐ近くには、気胸になることができます。 ここでは、技術を説明平滑末端エンド血管カテーテルおよび経皮的気管照明を用いて気管内挿管の簡単な方法で以下の低侵襲性2cmの皮膚切開を通して胸腺。胸腺摘出1.5センチ胸骨と縦隔を密封し、出血、呼吸器合併症の発生率を最小限にする手術用接着剤の用途に三層の閉鎖を伴う。胸腺及びIHC染色に胸腺組織の欠如後のCD4 +及びCD8 +ナイーブT細胞の消失によって証明されるように、この方法では、確実に完全な胸腺になる。手術時間とペリ手続き死亡率は最小限に抑えている。

<table border="0" cellpadding="0" cellspacing="0" width="718">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Name of Reagent/ Equipment</td>
			<td style="width:117px;">Company</td>
			<td style="width:152px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">2&#34; 14-gauge angiocatheter</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Operating microscope</td>
			<td style="width:117px;">Zeiss</td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Warming pad</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:283px;">Heart rate and blood oxygenation monitor for rodents with foot sensors</td>
			<td style="width:117px;">Harvard Apparatus</td>
			<td style="width:152px;">ST1 72-8010, ST1 72-8098 (Rat foot sensor)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Intubation apparatus (plastic with metal bar at the top)</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td style="width:167px;">See Figure 2&#160;</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:283px;">Small animal anesthesia system with induction box, isoflurane tank and O2 tank</td>
			<td style="width:117px;">Harvard Apparatus</td>
			<td style="width:152px;">ST1 72-6420</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:283px;">Small animal ventilator with tubing</td>
			<td style="width:117px;">CWE</td>
			<td style="width:152px;">12-02000 (ventilator)and 12-04000 (external valve assembly for mice/rats)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">High-intensity Fiber-Optic Illuminator</td>
			<td style="width:117px;">Dolan Jenner</td>
			<td style="width:152px;">EEG 2823M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Student standard pattern forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">91100-16</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Fine straight scissors</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">14060-09</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Blunt-tipped Shea scissors</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">14105-12</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Small Alm retractor (for sternum)</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">17008-07</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Blunt Graefe forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">11050-10</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Fine Dumont forceps</td>
			<td style="width:117px;">Fine Science Tools</td>
			<td style="width:152px;">11254-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">5-0 Maxon sutures</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">4-0 Silk sutures (with cutting needle)</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">6-0 Nylon suture</td>
			<td style="width:117px;">Ethicon</td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Cyanoacrylate glue (Endermil)</td>
			<td style="width:117px;"></td>
			<td style="width:152px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Lubrication gel</td>
			<td style="width:117px;">Akorn Animal Health</td>
			<td style="width:152px;">NDC 17478-162-35</td>
			<td></td>
		</tr>
	</tbody>
</table>

complete thymectomy in adult rats with non-invasive endotracheal intubation

新生児げっ歯類における胸腺摘出は、免疫学的研究のために設立され、信頼性のある手順です。しかし、成体ラットに、胸膜破壊からの出血と気胸の合併症は、かなりの死亡率をもたらすことができる。このプロトコルは、ミニ胸骨切開と気管内挿管を利用したラット胸腺摘出の簡単な方法です。挿管は、非侵襲的かつ容易に再現可能な方法で達成し、気胸を防ぐために陽圧換気及び胸膜の中断を最小限にするように注意胸腺切開するために十分な時間を可能にする制御された気道を可能にしている。まだ胸腺の完全な可視化を提供しながら、1.5センチメートル胸骨切開は、縦隔船舶や胸膜との接触を減少させる。縦隔の露光に続いて、胸腺の倍率で鈍的切開によって除去される。胸膜腔は、次に手術用接着剤の塗布に続いてプレ気管筋の縫合閉鎖によって封止されている。ザ·胸部は、その後皮膚の縫合閉鎖が続く胸骨の縫合閉鎖、によって閉鎖される。免疫組織化学（IHC）隔組織の染色、およびフローサイトメトリーによるナイーブT細胞の欠如によって証明されるように、すべてのthymectomiesが完了した、と手順96％の生存率であった。最小限の合併症を伴う完全な胸腺は、胸腺欠損成体ラットにおけるさらなる免疫学的研究のために所望される場合に適している。

この手順は、成体のルイスラット（n = 26）で行った。平均動作時間が15±3分であった。術中死亡はなかった。平均挿管時間 - 挿管装置にラットの配置から換気の確立には、 - 45±5秒であった。 24匹のラットは、術後の日（POD）POD 4 14. Oneラット開発呼吸困難を経て呼吸困難や出血の証拠のない操作から完全に回復を持っていたし、縦隔を探索するために再手術を受けた。ラットは、肺の虚脱ロ?...

Watch this Scientific Journal Video about Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation at JoVE.com

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

非侵襲的気管挿管と成体ラットにおける完全な胸腺摘出

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

complete-thymectomy-adult-rats-with-non-invasive-endotracheal

Research

JoVE Journal

Immunology and Infection

16.4K ビュー.  Duke University Medical Center. Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described. 

ビデオ: Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白血球ホーミングと移行の非侵襲的イメージング in vivoで</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

免疫・感染学

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

ゼブラフィッシュ幼生における播種性カンジダ症の非侵襲的イメージング

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

マウスのリンパ脈管構造の非侵襲的光イメージング

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

新生児と大人の中枢神経系から培養ミクログリア

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

神経病原の非侵襲的モデル<em&gt;エシェリヒア·コリ</em&gt;新生ラットにおける感染

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

のゼブラフィッシュ幼生モデルにおける先天性免疫応答の非侵襲的イメージング<em&gt;ストレプトコッカスイニエ</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

マウスにおける実験的細菌性肺炎の誘導と表現型のための非侵襲的および技術的に非集中的な方法

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

非侵襲的<em&gt;インビボ</em&gt;活性化可能な蛍光イメージング剤を用いた炎症性MMP活性の蛍光イメージング

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

使用してリンパ球血管外漏出の分析<em&gt;インビトロ</em&gt;ヒト血液脳関門のモデル

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

結膜から細胞を単離して表現する非侵襲的方法

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...