著者には、任意の受信確認がありません。

方法と術後のケアと同様に、麻酔科の期間を含む動物のすべてのプロシージャが獣医に相談し、制度的ケアおよび使用委員会 (IACUC)、によって承認された、所属機関。本稿では、すべての手順を行った 15 週齢の c57bl/6 男性マウスに 8 とプロトコル コロラド大学で IACUC によって承認されたアニマル ・ ケアのガイドラインに従います。
 1 チューブ準備 注: 接種を植え付ける、針はポリエチレン (PE) 管 ( 図 1 a) または小さなゲージに接続されている (&#60; 24 G) 血管カテーテルは使用される (をすることができます。 図 1)。血管カテーテルの使用には、70% エタノール (エタノール) と殺菌以外の準備は不要です。接種が尿路に注入/注射/堆積物質です。これは細菌、ウイルス、または化学物質が含まれているソリューションをすることができます。これは、前の時間と研究プロトコルごとに用意しています。たとえば、研究は下部尿路の 大腸菌 感染の影響を評価することを目的、研究者が接種/下部尿路に注入することができます細菌を含む接種材料を準備する必要が。
<ol><li>PE チューブに取り付けられた針の作製は簡単で、次の構成: 標的臓器と動物の年齢に基づいて PE チューブのサイズを選択します。12 週間より若い動物用 PE 10 チューブ (外径 0.024 インチ、内径 0.011 インチ)。PE 10 チューブが接種前前立腺がん (AP) と精嚢 (SV) の (1) のプライマリ シードまたは 12 週齢より若い動物の膀胱で菌の (2) の主なシードのため適切であると示唆されました。サイズ (外径 0.038 インチまでと 0.023 インチ最大内径) PE 50 はより古い動物の膀胱で菌の主なシードに使用されることができますまでの大径 PE チューブ</li>。
	<li>PE チューブの約 2.5 インチをカットします</li>。
	<li>使用する選択した PE 径を合わせて針を選択します。27 G 針が PE 10 チューブの内腔に合うことができるし、22 G 針に適合 PE 50 チューブの内腔。
	<ol><li>市販油石針の鋭いエッジをこすることによって PE チューブ挿入前に針の準備 ( 図 1A -B) 鈍い、または市販の鈍を使用針します</li>。
	</ol></li> <li>PE チューブの内腔に鈍い針 ( 図 1) をご紹介します。内腔の中心部に針のままを確保することによって内腔または偽腔への参入の損傷を防ぐ。( 図 1 -E)。鈍い針の挿入時に PE チューブが破損した場合破損した端をカットし、プロセスを再起動します</li>。
	<li>は、70 %etoh の手順については、使用するまで滅菌針添付 PE 管を配置します。フラッシュは EtOH 針 PE チューブを通じて内腔を消毒し、ことを確認はありませんから PE チューブから 70% 損傷針挿入の過程です。漏れ、針 PE チューブを破棄されます 。
	注: PE チューブの先端は、カテーテル挿入前に滑らかなする必要があります。尿道に PE 管を導入することで問題を回避するには、は、カテーテル検査 ( 図 1 f) の直前に PE チューブの先端をカットすることをお勧めします。PE 管の推奨される最小の長さは 1.5 インチ、針の周辺部分が含まれている</li>。
</ol> 2。カテーテル検査手順 <ol><li>麻酔導入、前にマウスの膀胱を空にします。
	<ol><li>穏やかな圧力を適用し、マウスの下腹部にマッサージします。これらの演習は、通常自発的な排尿に します。任意の浸透させた菌希釈または即時漏れのフル膀胱があります 。
		注: マウスの空膀胱を確保するための追加オプションは尿道注入のプロシージャの開始前に少なくとも 1 時間は水の動物を奪うため。水の剥奪は、動物のプロトコルの一部として機関によって承認されるべき</li>。
	</ol></li> <li>麻酔器イソフルレン フロー (2%) を使用して麻酔。
	<ol><li>動物に麻酔がかかる、一度暖かい毛布の上に置き、継続的な麻酔の維持のためノーズ内鼻と青いパッドでそれをカバーします</li>。
		<li>ペダル反射 (つま先ピンチ) をチェックすることによって動物が麻酔下であることを確認します</li>。
	</ol></li> <li>ペダル反射が存在しないと、包皮を後退して続行します。
	<ol><li>プレス片手の人差し指や親指に陰部 ( 図 2 a と 2 b)、親指と他の手 (人差し指を使用して腺、陰茎を抜くと 図 2)。これが正しくできる、包皮、陰茎亀頭および陰茎体が露出したまま。( 図 2 D および 図 2 f)。これは組織の損傷になると腺ペニスを抜く鉗子を使用しないでください</li>。
	</ol></li> <li>カテーテルとカテーテルの挿入前に滅菌外科潤滑油を亀頭の先端に注油します。PE チューブの挿入を簡単になります 。
	注: 1 つの大規模な管ではなく、手術の潤滑剤の個別包装された正方形は無菌状態を維持するために各の使用に適しています</li>。
	<li>鈍い鉗子を使用して、陰茎の先端を優しく保持し、そっと開く尿道口を引き起こす亀頭を絞る</li>。
	<li>注射器針添付 PE チューブまたは PE チューブ自体を保持して尿道口から PE チューブを挿入します 。
	メモ: 注射器針添付 PE 管または管の親指、人差し指と中指を使用して PE の三脚グリップは安定性のため勧めします 。
	注: カテーテルのパスは、 図 3A および 図 3 b に示すように、陰茎亀頭と陰茎成人男性マウス体内に囲まれた尿道の自然の曲率による湾曲する必要があります。カテーテルの PE 管カテーテル挿入中に抵抗を満たすことができる、標的臓器にさらに PE チューブ上下ヘルプ事前にチューブを傾けることによって角度を調整するため、お勧め。必要な場合、PE 管は尿道の様々 なシャープなターンを操縦するように少し回転できます。あまり抵抗がある、チューブは引き戻されている必要があり、最小限の圧力を再び進めます。PE チューブを引っ張って完全より潤滑を使用してがあります</li>。
	<li>一度 PE チューブ (5-200 μ L のボリュームをお勧めします) 接種の望ましいボリュームを浸透させる針プランジャーを押すことで目的の臓器に達している接種の注入を実行します。
	、膀胱に、菌の注入を進める PE チューブの約 0.75 インチまで PE チューブのための <ol><li>が挿入されます ( 図 3 a). 
		注: の AP と SV は、一般的に、回で接種の注入前方の前立腺に開口部のポイントに達すれば、e 挿入チューブは抵抗を満たしています。上下にチューブを傾けることによって角度を調整して、PE チューブを進めるさらに PE チューブの約 1 インチまで ( 図 3 b) を挿入します</li>。
	</ol></li></ol> 3。生体内で下部尿路と腎臓のイメージング 注: 生体内 イメージング本稿の目的はある: 接種; の空間定位を確認するには (1) と (2) に接種の逆流の発生率を評価するために、腎臓。この目的のため麻酔下または安楽死させたマウスのいずれかを使用できます。
<ol><li>成人男性マウスの下部尿路を視覚化する膀胱、前立腺、精嚢を公開するより低い腹部正中開腹を行う。
	<ol><li>はさみを使用して剣状突起の真下に恥骨から正中の皮膚を切開します</li>。
		<li>皮下組織から皮膚を昇格します</li>。
		<li>正中線に位置する alba 無血管リネアを識別し、すべての臓器が腹膜腔を入力して切開します</li>。
		<li>ブルーの染料または別の適切な追跡エージェントの可視化による空間定位を確認します</li>。
	</ol>
	</li>
</ol>

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J., Bushman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+bacterial+infection+and+inflammation+incite+reactive+hyperplasia+in+a+mouse+model+of+chronic+prostatitis.">Chronic bacterial infection and inflammation incite reactive hyperplasia in a mouse model of chronic prostatitis.</a> Prostate. 67 (1), 14-21 (2007).</li><li>Boehm, B. J., Colopy, S. A., Jerde, T. J., Loftus, C. J., Bushman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+bacterial+inflammation+of+the+mouse+prostate.">Acute bacterial inflammation of the mouse prostate.</a> Prostate. 72 (3), 307-317 (2012).</li><li>Bjorling, D. E., Wang, Z. Y., Bushman, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+inflammation+of+the+lower+urinary+tract.">Models of inflammation of the lower urinary tract.</a> Neurourol Urodyn. 30 (5), 673-682 (2011).</li><li>Thai, K. H., Thathireddy, A., Hsieh, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transurethral+induction+of+mouse+urinary+tract+infection.">Transurethral induction of mouse urinary tract infection.</a> J Vis Exp. (42), (2010).</li><li>Bjorling, D. E., Wang, Z. 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著者が明らかに何もありません。

この原稿は、大人の男性マウスに経尿道的注入法を詳しく説明します。差分のプライマリ シード領域は、異径 PE 管を使用してアクセスできます。PE 50 PE 10 を使用して AP と SV (図 3 aB) に到達する大径ながら、膀胱内腔に正常に到達するため勧めします。PE チューブと接種の標的臓器のサイズの選択に加えて植え付け接種材料の量など、他の要因はまた終了?...

経尿道的注入アプローチ前の研究で膀胱の病態1,2,3,4の齧歯動物モデルを作成する方法の 1 つとして使用されています、評価に使用することができます、マウスでローカルに配送された治療法の有効性動物モデルできない人間の病理を要約して完全に、動物研究の基になるメカニズムの同定は、間質性膀胱炎/膀胱痛症候群、神経因性など人間の LUT 疾患の理解を深める膀胱炎、自己免疫性の膀胱炎と前立腺の炎症5。
大人の雄マウスに経尿道的注入術は大人の雌マウス6の膀胱内注入よりももっと技術的に挑戦的です。その小径と一緒に男性の尿道の自然に湾曲した解剖学は、尿道へのカテーテル挿入を達成するが技術的に難しくなっています。したがって、する詳しい手順を経尿道的誘導マウス尿路感染症6と LUT 炎症7,8,9,10,11,12,13,14,前述の雌マウスだけの膀胱に挿入されたカテーテルを介して15だった。この原稿は、雄マウスのビデオ クリップ、画像、イラストなどの物質の経尿道的注入の技術の段階的な説明を提供するために目指しています。経尿道的注入変数を持つ大人の男性マウスの手順を実行できますが類似する以前の筋電図記録などの追加のテクニックを吸収合併し、雌マウス7に記載されている再接種します。観た応答 (VMR)16。

<table>
	<tbody>
		<tr>
			<td>BD INTRAMEDIC Polyethylene Tubing/ PE 10</td>
			<td>BD Medical</td>
			<td>63019-004</td>
			<td></td>
		</tr>
		<tr>
			<td>BD INTRAMEDIC Polyethylene Tubing/ PE 50</td>
			<td>BD Medical</td>
			<td>63019-048</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Insyte-N Autoguard/ Angiocath</td>
			<td>BD Medical</td>
			<td>311411</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol (EtOH)</td>
			<td>Fisher Scientific</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Needle Only 22 Gauge (G) 1 inch</td>
			<td>Becton Dickson</td>
			<td>205155</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Needle Only 27 Gauge (G) 1/2 inch</td>
			<td>Becton Dickson</td>
			<td>305129</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer stub, 22 G X 0.5 in / Blunt Needle</td>
			<td>Instech</td>
			<td>LS22</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer stub, 27 G X 0.5 in / Blunt Needle</td>
			<td>Instech</td>
			<td>LS27</td>
			<td></td>
		</tr>
		<tr>
			<td>6&#34; Long x 2&#34; Wide x 1&#34; Thick, Aluminum Oxide Sharpening Stone / Oil Stone</td>
			<td>Norton</td>
			<td>40204034</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Lubricant Sterile Bacteriostatic Folilpac</td>
			<td>Surgilube</td>
			<td>281020543</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Fluriso</td>
			<td>13985-528-60</td>
			<td></td>
		</tr>
		<tr>
			<td>SomnoSuite Low-Flow Anesthesia System</td>
			<td>Kent Scientific</td>
			<td>SS-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Forceps, Curved</td>
			<td>vwr</td>
			<td>82027-392</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 1 ml Syringe</td>
			<td>BD Medical</td>
			<td>309626</td>
			<td></td>
		</tr>
		<tr>
			<td>India Ink Solution., 0.2% in PBS buffer</td>
			<td>Alfa Aesar</td>
			<td>J61007</td>
			<td></td>
		</tr>
		<tr>
			<td>8~12 weeks old male C57B/6 Mice</td>
			<td>Jackson Lab</td>
			<td>#000664</td>
			<td></td>
		</tr>
	</tbody>
</table>

transurethral instillation procedure in adult male mouse

尿道注入を使用する膀胱膀胱の病態モデル動物を誘導するかを評価するにローカル有効成分 (例えば薬剤、化学物質、細菌、およびウイルス) とさまざまなソリューションを提供することができます、膀胱内注入治療効果。下部尿路 (LUT) 病態の最も齧歯動物モデルは、女性の尿道を介して物質の膀胱内注入を容易にするため雌マウスに誘導されます。しかし、女性と男性の LUT で解剖学的な違いがあるため男性マウスに経尿道的注入は非常に挑戦的なプロシージャとみなされている、以前記載されていません。本稿では、雄マウスの尿道にその後挿入するためポリエチレン (PE) 管を準備する方法の詳細な説明を提供します。また、接種の目的のサイトによって使用される PE チューブの理想的な種類をについて説明します。さらに、ポイント ポイントで尿道の損傷を回避し、目的の場所へのソリューションの配信に成功した経尿道的投与の動物を準備する方法について述べる.手順は、包皮、尿道口の開口部を公開する亀頭の取り消しによって開始されます。次に、亀頭は、陰茎と PE チューブを安定させるために鈍的非破砕, 鉗子で把持です。PE チューブが尿道口動物の体に平行に挿入し、尿道の自然の曲率を次にそれを操縦するカテーテルを傾けることによって角度を調整します。この手法は、誘導膀胱病態モデルマウスを使用したり、男性マウスの膀胱内注入治療の有効性を評価します。

PE 50 チューブを介して青色色素の経尿道的注入はそれぞれ膀胱 (図 4 a) と SV と AP (図 4 b) のカラリゼーションにおける PE 10 チューブを介して色素の注入で起因しました。行う経尿道的教養を高める腎臓へ菌のインスタント逆流を発生は、またはないかどうかを評価するため、腎臓と尿管青い染料・蛍光染料 (<strong class="xfig"...

Watch this Scientific Journal Video about Transurethral Instillation Procedure in Adult Male Mouse at JoVE.com

Transurethral Instillation Procedure in Adult Male Mouse

経尿道的注入が困難なプロシージャであり、文献にも記載されていません。本稿の目的は、大人の男性マウスの前立腺や膀胱に活性物質と液体の膀胱配信するためカテーテルの尿道挿入の技術を記述するためです。

大人の雄のマウスに経尿道的注入手順

Transurethral instillation can be used to deliver different solutions with active ingredients (e.g., drugs, chemicals, bacteria, and viruses) locally into ...

transurethral-instillation-procedure-in-adult-male-mouse

Research

JoVE Journal

Immunology and Infection

15.4K ビュー.  University of Colorado School of Medicine. 経尿道的注入が困難なプロシージャであり、文献にも記載されていません。本稿の目的は、大人の男性マウスの前立腺や膀胱に活性物質と液体の膀胱配信するためカテーテルの尿道挿入の技術を記述するためです。

ビデオ: Transurethral Instillation Procedure in Adult Male Mouse

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37&deg;C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

マウス尿路感染症の経尿道的誘導

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

免疫・感染学

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

マウスT細胞におけるT細胞受容体のレトロウイルス導入

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, in particular, are known to migrate to the lung draining lymph nodes (LDLNs) where they present inhaled antigens to T cells initiating an 
appropriate immune response to a variety of immunogens1,2. To model interactions between the lungs and airborne antigens in mice, antigens can be 
administered intranasally1,3,4, intratracheally5 or as aerosols6. Delivery by each route involves distinct technical skills and limitations that need to be 
considered before designing an experiment. For example, intranasal and aerosolized exposure delivers antigens to both the lungs and the upper respiratory 
tract. Hence antigens can access the nasal associated lymphoid tissue (NALT)7, potentially complicating interpretation of the results. In addition, swallowing, 
sneezing and the breathing rate of the mouse may also lead to inconsistencies in the doses delivered. Although the involvement of the upper respiratory tract may 
be preferred for some studies, it can complicate experiments focusing on events specifically initiated in the lungs. In this setting, the intratracheal (i.t) 
route is preferable as it delivers test materials directly into the lungs and bypasses the NALT. Many i.t injection protocols involve either blind intubation of the 
trachea through the oral cavity or surgical exposure of the trachea to access the lungs. Herein, we describe a simple, consistent, non-surgical method for i.t 
instillation. The opening of the trachea is visualized using a laryngoscope and a bent gavage needle is then inserted directly into the trachea to deliver the 
innoculum. We also describe procedures for harvesting and processing of LDLNs and lungs for analysis of antigen trafficking by flow cytometry.

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

フローサイトメトリーによる肺および肺リンパ節の分析とマウスの非外科的気管内注入

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

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 ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

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 ...

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, which accurately reflect disease establishment and progression, permit dissection of host-pathogen interactions and generation of immunity to infection. In mice, intravesical instillation of uropathogenic E. coli, the causative agent in more than 85% of community acquired UTI, recapitulates many of the stages of infection observed in humans. Until recently, however, UTI could only be modeled in female animals. This limitation has hindered the study of sex-related differences in UTI, as well as other bladder pathologies, such as cancer. Here, we describe a method to instill male mice that allows direct comparison between female and male animals and provide a detailed protocol to assess bladder tissue by flow cytometry as a means to better understand host responses to infection. Together, these approaches will aid in the identification of host factors that contribute to sex biases observed in UTI and other bladder-associated diseases.

The established model of transurethral catheterization of mice allows the study of bladder pathologies, including urinary tract infection, but can only be performed in females. A new model of male transurethral instillation, presented here, will enable research in an area marked by strong clinical and epidemiological differences between the sexes.

小動物モデルにおける尿路感染症: 雌雄マウスの尿道カテーテル

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, ...

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the aquatic environment, where it interacts with a variety of higher aquatic species. Vertebrate fish are known to be an environmental host and are a potential V. cholerae reservoir in nature. Both V. cholerae and the teleost fish species Danio rerio, commonly known as zebrafish, originate from the Indian subcontinent, suggesting a long-standing interaction in aquatic environments. Zebrafish are an ideal model organism for studying many aspects of biology, including infectious diseases. Zebrafish can be easily and rapidly colonized by V. cholerae after exposure in water. Intestinal colonization by V. cholerae leads to the production of diarrhea and the excretion of replicated V. cholerae. These excreted bacteria can then go on to colonize new fish hosts. Here, we demonstrate how to assess V. cholerae-intestinal colonization in zebrafish and how to quantify V. cholerae-induced zebrafish diarrhea. The colonization model should be useful to researchers who are studying whether genes of interest may be important for host colonization and/or for environmental survival. The quantification of zebrafish diarrhea should be useful to researchers studying any intestinal pathogen who are interested in exploring zebrafish as a model system.

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Zebrafish are a natural Vibrio cholerae host and can be used to recapitulate and study the entire infectious cycle from colonization to transmission. Here, we demonstrate how to assess V. cholerae colonization levels and quantify diarrhea in zebrafish.

コレラの植民地化と大人のゼブラフィッシュ モデルで下痢を定量化

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

結核抗酸菌 marinum感染成人ゼブラフィッシュをモデル化

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

A Model of Self-limited Acute Lung Injury by Unilateral Intra-bronchial Acid Instillation

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic findings similar to human acute respiratory distress syndrome (ARDS). The resulting alveolar edema, alveolar-capillary barrier damage, and leukocyte infiltration predominantly affect the left lung, preserving the right lung as an uninjured control and allowing animals to survive. This model of self-limited acute lung injury enables investigation of tissue resolution mechanisms, such as macrophage efferocytosis of apoptotic neutrophils and restitution of alveolar-capillary barrier integrity. This model has helped identify important roles for resolution agonists, including specialized pro-resolving mediators (SPMs), providing a foundation for the development of new therapeutic approaches for patients with ARDS.

Selective intra-bronchial acid instillation to the left lung in mice results in unilateral and self-limited acute lung injury that models human acute respiratory distress syndrome (ARDS) induced by gastric acid aspiration.

一方的気管支内酸インスティレーションによる自己限定急性肺損傷のモデル

Selective intra-bronchial instillation of hydrochloric acid (HCl) to the murine left mainstem bronchus causes acute tissue injury with histopathologic ...

Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia selectively), when combined with IBA1 (which provides an exceptional visualization of their morphology), allows investigation of changes in density, distribution, and morphology. Quantifying these parameters is important in providing insights into the roles exerted by microglia, the resident macrophages of the brain. Under normal physiological conditions, microglia are regularly distributed in a mosaic-like pattern and present a small soma with ramified processes. Nevertheless, as a response to environmental factors (i.e., trauma, infection, disease, or injury), microglial density, distribution, and morphology are altered in various manners, depending on the insult. Additionally, the described double-staining method allows visualization of infiltrating macrophages in the brain based on their expression of IBA1 and without colocalization with TMEM119. This approach thus allows discrimination between microglia and infiltrating macrophages, which is required to provide functional insights into their distinct involvement in brain homeostasis across various contexts of health and disease. This protocol integrates the latest findings in neuroimmunology that pertain to the identification of selective markers. It also serves as a useful tool for both experienced neuroimmunologists and researchers seeking to integrate neuroimmunology into projects.

This protocol describes a step-by-step workflow for immunofluorescent costaining of IBA1 and TMEM119, in addition to analysis of microglial density, distribution, and morphology, as well as peripheral myeloid cell infiltration in mouse brain tissue.

大人の雄のマウスに経尿道的注入手順

要約

さらに動画を探す

この動画の章

マウス尿路感染症の経尿道的誘導

マウスT細胞におけるT細胞受容体のレトロウイルス導入

フローサイトメトリーによる肺および肺リンパ節の分析とマウスの非外科的気管内注入

新生児と大人の中枢神経系から培養ミクログリア

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

小動物モデルにおける尿路感染症: 雌雄マウスの尿道カテーテル

コレラの植民地化と大人のゼブラフィッシュモデルで下痢を定量化

結核抗酸菌 marinum感染成人ゼブラフィッシュをモデル化

一方的気管支内酸インスティレーションによる自己限定急性肺損傷のモデル

マウス脳における微小グリア密度、形態および末梢骨髄細胞浸潤解析のためのIBA1およびTMEM119を用いた免疫蛍光染色

大人の雄のマウスに経尿道的注入手順

要約

さらに動画を探す

この動画の章

マウス尿路感染症の経尿道的誘導

マウスT細胞におけるT細胞受容体のレトロウイルス導入

フローサイトメトリーによる肺および肺リンパ節の分析とマウスの非外科的気管内注入

新生児と大人の中枢神経系から培養ミクログリア

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

小動物モデルにおける尿路感染症: 雌雄マウスの尿道カテーテル

コレラの植民地化と大人のゼブラフィッシュ モデルで下痢を定量化

結核抗酸菌 marinum感染成人ゼブラフィッシュをモデル化

一方的気管支内酸インスティレーションによる自己限定急性肺損傷のモデル

マウス脳における微小グリア密度、形態および末梢骨髄細胞浸潤解析のためのIBA1およびTMEM119を用いた免疫蛍光染色

コレラの植民地化と大人のゼブラフィッシュモデルで下痢を定量化