本研究は、公共の福祉によって支えられた &#38; 安全研究プログラムを通じて、国立研究財団の韓国 (NRF) 文部省、科学および技術 (NRF 2014M3C8A5030612)、によって資金を供給、鄭陽、チャチャ ヤングによってサポートされて太陽、MD やチャンこんにちはチュ ユー Sa 記念基金します。

ここで説明したすべてのメソッドが出生後 6 週間国際動物ケアおよび使用委員会 (IACUC) の韓国大学大学の医学によって承認されている、実験者ができる enthansasia を誘導すると収集。皮膚、ラットの血清など様々 な試料。
 1 新生児カプサイシン処置によって広告の誘導 <ol><li>カプサイシン ソリューション 5 mg/mL を準備するには、トゥイーン 80、15 mL の円錐管の 100% エタノール 1 mL に 1 mL に 50 mg のカプサイシンを追加します。</li>。
	<li>は、円錐管に生理食塩水入りの 8 mL を追加します。表示しているレイヤー分離がなくなったまで精力的にチューブを振る</li> <li>正中線と新生児 Sprague-dawley ラットの首の裏側に、皮下組織にカプサイシン溶液 (10 μ L/g) を注入します。
	<ol><li>配信 (重大) 生後後 48 時間 12 h 間カプサイシン 。
		メモ: 注入がより数日遅れた場合皮膚炎は開発しません、それは数週間のためにのみ保持されます。注入が早すぎる場合、呼吸が激しい痛みのため発生する可能性が</li>。
		カプサイシン投与による呼吸停止から新生児ラットを防ぐために <li>を押して呼吸を助長するまたは呼吸になるノーマル。 以来、カプサイシン誘発の痛みすることができますまで酸素室おいて、新生児に新生児の胸部呼吸の阻止、おそらく呼吸困難を悪化させる全身麻酔を追加します。実験者がラットの生存を増加する anesthesics を使用しないでくださいしたがって、.</li>
	</ol></li></ol> 2。掻痒症と広告のモデルラットにおける皮膚炎の評価 <ol><li><ol><li>準備プラスチックの 自発の掻破行動を数えることによってそう痒症の測定 室 (20 cm × 30 cm × 20 cm) 完全なカバレッジを許可するミラーを装備閲覧と呼吸するラットを許可するように穴にします</li>。
		<li>設定して正しくミラー ビューおよびラットのフロント ビューを同時に記録するデジタル ビデオ カメラを調整します</li>。
		<li>商工会議所や開口部を防ぐために重いオブジェクトの部屋の天井をカバー プラスチック場所ラット</li>。
		<li>1 h のラットの自発行動を記録、ビデオ クリップを再生および動作の傷の数をカウントします</li>。
	</ol></li> <li>得点システムを使用して皮膚病変の評価 <ol><li>誘導室でラットを配置します</li>。
		<li>2 L/分の酸素流量計をオンに流量し、麻酔の 3% にイソフルラン気化器を調整します</li>。
		<li>程度とラットの他の部分と耳の皮膚炎の重症度を識別します</li>。
		<li>0.25 cm 2 と次 の表 1 による皮膚病変の重症度として皮膚病変の範囲の単位を定義する: 重症度; 度程度と重症度を乗じて皮膚炎スコア (皮膚炎のスコアを参照してください <強いクラス ="xfig"> 図 1B).</li>
	</ol></li></ol> 3。ガス FA の動物への暴露 <ol><li>吸入室の準備 <ol><li>アクリル ガラス ボックスを準備 (55 cm × 40 cm × 35 cm) 反対側に 2 つのアウトレットがある</li>。
		<li>注意。FA ガスの循環、入口より出口を配置し、入口よりも出口のサイズを大ききます</li>。
		<li>注意。、店舗別の側面の内部のガス濃度測定の閉じることができます 3 つの小さな通路をドリルします。</li>
		両方の店に <li>接続チューブ。循環フードまたはガスの放出のために建物の外排水チューブを置き、他のアウトレット チューブを医療用酸素レギュレータに接続します</li>。
		<li>アクリル ガラス ボックス内に従来のラットのおりを配置します</li>。
		<li>(2 時間/日、5 日/週) 5 週間出産後 1 週間からネズミをケージに入れています</li>。
		<li>シール アクリル ガラス ボックス ガス漏れを防ぐために可能な限り緊密 。
		注: タイトなシールの室蓋はゴムを含める必要があります。密封のために部屋をラップするラップを適用します</li>。
	</ol></li> <li>医療用酸素レギュレータを用いた気体 FA の世代 <ol><li>FA の 5 g を 1 L の水に溶解する</li>。
		<li>医療用酸素レギュレータの加湿器ボトルに入れ、0.5 %fa ソリューションです</li>。
		<li>レギュレータにインレット チューブを接続した後、レギュレータをオンにし 5 L/分の流量を設定</li>
		<li>溶液濃度とチャンバー内のガスの濃度を決定するための流量を調整します</li>。
	</ol>
	</li>
</ol>

<ol>
	<li>Geha, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergy+and+hypersensitivity.+Nature+versus+nurture+in+allergy+and+hypersensitivity.">Allergy and hypersensitivity. Nature versus nurture in allergy and hypersensitivity.</a> Curr Opin Immunol. 15 (6), 603-608 (2003).</li><li>Novak, N., Bieber, T., Leung, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+mechanisms+leading+to+atopic+dermatitis.">Immune mechanisms leading to atopic dermatitis.</a> The J Allergy Clin Immunol. 112 (Suppl 6), S128-S139 (2003).</li><li>Back, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronically+relapsing+pruritic+dermatitis+in+the+rats+treated+as+neonate+with+capsaicin;+a+potential+rat+model+of+human+atopic+dermatitis.">Chronically relapsing pruritic dermatitis in the rats treated as neonate with capsaicin; a potential rat model of human atopic dermatitis.</a> J Dermatol Sci. 67 (2), 111-119 (2012).</li><li>Jin, H., He, R., Oyoshi, M., Geha, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+atopic+dermatitis.">Animal models of atopic dermatitis.</a> J Invest Dermatol. 129 (1), 31-40 (2009).</li><li>Han, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde-Induced+Aggravation+of+Pruritus+and+Dermatitis+Is+Associated+with+the+Elevated+Expression+of+Th1+Cytokines+in+a+Rat+Model+of+Atopic+Dermatitis.">Formaldehyde-Induced Aggravation of Pruritus and Dermatitis Is Associated with the Elevated Expression of Th1 Cytokines in a Rat Model of Atopic Dermatitis.</a> PLOS ONE. 11 (12), e0168466 (2016).</li><li>Xu, B., Aoyama, K., Takeuchi, M., Matsushita, T., Takeuchi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+cytokine+mRNAs+in+mice+cutaneously+exposed+to+formaldehyde.">Expression of cytokine mRNAs in mice cutaneously exposed to formaldehyde.</a> Immunol Lett. 84 (1), 49-55 (2002).</li><li>Kim, J. Y., Jeong, M. S., Park, K. Y., Seo, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggravation+of+atopic+dermatitis-like+symptoms+by+consecutive+low+concentration+of+formaldehyde+exposure+in+NC/Nga+mice.">Aggravation of atopic dermatitis-like symptoms by consecutive low concentration of formaldehyde exposure in NC/Nga mice.</a> Exp Dermatol. 22 (3), 219-221 (2013).</li><li>Laske, N., Niggemann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+the+severity+of+atopic+dermatitis+correlate+with+serum+IgE+levels?.">Does the severity of atopic dermatitis correlate with serum IgE levels?.</a> Pediatr Allergy Immunol. 15 (1), 86-88 (2004).</li><li>Fujii, Y., Takeuchi, H., Sakuma, S., Sengoku, T., Takakura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+2,4-dinitrochlorobenzene-induced+chronic+dermatitis+model+in+rats.">Characterization of a 2,4-dinitrochlorobenzene-induced chronic dermatitis model in rats.</a> Skin Pharmacol Physiol. 22 (5), 240-247 (2009).</li><li>Sugimoto, T., Xiao, C., Ichikawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+primary+neuronal+death+induced+by+capsaicin+and+axotomy+involves+an+apoptotic+mechanism.">Neonatal primary neuronal death induced by capsaicin and axotomy involves an apoptotic mechanism.</a> Brain Res. 807 (1-2), 147-154 (1998).</li><li>Saito, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+skin+inflammation+induced+by+repeated+exposure+of+toluene,+xylene,+and+formaldehyde+in+mice.">Characterization of skin inflammation induced by repeated exposure of toluene, xylene, and formaldehyde in mice.</a> Environ Toxicol. 26 (3), 224-232 (2011).</li><li>Jung, W. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+exposure+induces+airway+inflammation+by+increasing+eosinophil+infiltrations+through+the+regulation+of+reactive+oxygen+species+production.">Formaldehyde exposure induces airway inflammation by increasing eosinophil infiltrations through the regulation of reactive oxygen species production.</a> Environ Toixcol Pharmacol. 24 (2), 174-182 (2007).</li></ol>

著者が明らかに何もありません。

ラットの広告のような症状の誘発のためここで説明されたプロトコルは、AD の病態を研究するために適応し、薬物検査のスクリーニング ツールとして使用できます。ガス FA のカプサイシン投与ラットへの暴露の楽器は、多様な動物モデルに及ぼす各種ガスへの暴露の評価に適用できます。
新生児カプサイシン処理広告モデル、DNCB 治療モデル9と皮膚感...

ホルムアルデヒド (FA) アトピー性皮膚炎 (AD) の進行の効果を調査するには、広告誘導プロトコルを設立し、FA の慢性露出のための従来の循環室を開発しました。
広告は、遺伝的素因と環境トリガー1,2間の複雑な相互作用に起因する慢性の炎症性皮膚疾患です。適切な動物モデルは臨床状態で広告を勉強する必要があります。これは、環境要因の影響の下で AD の病因を解明するとき最も重要です。また、広告進行に及ぼす環境的誘因をよく調べるためには、それは個々 の環境要因を分離し、暴露の程度を定量化することが欠かせません。
我々 は新生児カプサイシン処置による広告の新規ラットモデルを実証しました。この広告モデル3皮膚感作性, 遺伝子組み換え広告モデルおよび広告4の自発的なマウス ・ モデルによる広告モデルなど他の広告モデルにいくつかのメリットがあります。まず、このモデルは幼児広告に対応し、Nc/nga マウスなど広告の他の自発的なマウス モデルより前は 3 週間の時代に広告のような症状を開発しています。遺伝子組み換えの広告モデルとは異なり、いくつかのケースで広告モデルの症状は治まるし、自発的に 20 週間にわたって再発します。また、モデルの広告のような症状の誘導プロセスはより誘導プロセスの観点からアレルギー性の接触皮膚炎に似ている皮膚感作モデルより簡単です。
小児および思春期の期間に広告の開発の FA 吸入の効果を研究するアクリル ガラス室と従来の医療用酸素レギュレータ5を使用して FA 曝露動物モデルにおける新しいプロトコルを紹介しています。レギュレータを一定濃度ガス流量を調整することによって、溶液濃度に動物を公開できました。以前の研究は、動物モデルへの FA の暴露のいくつかの方法を報告しています。徐らは、どの FA ソリューション皮膚6へ適用した直接露光法を説明します。別の最近の研究は対処モデル 49127特殊揮発性溶剤の発電機を用いた FA ガス曝露プロトコルです。従来の方法と比較して、新しい方法がより簡単に装備されてし、FA に加えて他のソリューションの適用することができます。

<table width="776">
	<tbody>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Medical Oxygen Regulator</td>
			<td style="width:159px;">Yuyao Jiahua Medical</td>
			<td style="width:129px;"></td>
			<td style="width:257px;">manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Tube</td>
			<td style="width:159px;"></td>
			<td style="width:129px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Acrylic Chamber</td>
			<td style="width:159px;">Taejin Acryl</td>
			<td style="width:129px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Plastic Chamber</td>
			<td style="width:159px;">Taejin Acryl</td>
			<td style="width:129px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Paraformaldehyde, powder</td>
			<td style="width:159px;">Sigma</td>
			<td align="right">158127</td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Wrap filme</td>
			<td style="width:159px;">Uniwrap</td>
			<td></td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Capsaicin</td>
			<td style="width:159px;">Sigma</td>
			<td>M2028</td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Tween80</td>
			<td style="width:159px;">Sigma</td>
			<td>P4780</td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Digital Video Camera</td>
			<td style="width:159px;">Sony</td>
			<td>HDR-CX380y</td>
			<td>manufacturer will not affect results</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:231px;height:21px;">Isoflurane</td>
			<td style="width:159px;">JW pharmaceutical</td>
			<td style="width:129px;"></td>
			<td>manufacturer will not affect results</td>
		</tr>
	</tbody>
</table>

effects of exposure of formaldehyde to a rat model of atopic dermatitis induced by neonatal capsaicin treatment

アトピー性皮膚炎掻痒湿疹を慢性的な再発は、先進国を中心に世界中で勝ちます。遺伝・環境要因間の複雑な相互作用は、アトピー性皮膚炎の病態に重要な役割を再生する知られています。しかし、我々 はまだこの病気の病因の詳細な画像を欠いています。したがって、アトピー性皮膚炎の進行を解明するための適切な動物モデルの開発に重要なのです。また、アトピー性皮膚炎に及ぼす大気汚染物質などの環境要因を調査して病気の理解を広げます。ここでは、新生児カプサイシン処置ラットにおけるアトピー性皮膚炎とアトピー性皮膚炎小児と思春期の発達に及ぼす影響を明らかにするラットへの一定ホルムアルデヒド濃度の暴露のためのプロトコルを誘導する方法を述べる期間。これらのプロトコルはいくつかの実験に正常に適用されているし、他の物質に使用することができます。

7 週齢でカプサイシン処理広告ラット モデルの広告のような皮膚の炎症は、図 1 aに提示されます。広告ラットのカプサイシン処理モデルの堅牢な掻破行動をビデオ 1に示します。新生児カプサイシン処置が皮膚炎を誘発 3 週齢ラットの行動を傷を悪化し、広告のような症状が数週間 (図 1 bC) の永続...

Watch this Scientific Journal Video about Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment at JoVE.com

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

新生児カプサイシン処置ラットとアトピー性皮膚炎に対するホルムアルデヒド吸入の影響を調査するため気化したホルムアルデヒドの露出でアトピー性皮膚炎を誘発する方法をご紹介します。

新生児カプサイシン処置によるアトピー性皮膚炎のモデルラットにホルムアルデヒドの暴露の影響

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

effects-exposure-formaldehyde-to-rat-model-atopic-dermatitis-induced

Research

JoVE Journal

Immunology and Infection

9.5K ビュー.  Korea University. 新生児カプサイシン処置ラットとアトピー性皮膚炎に対するホルムアルデヒド吸入の影響を調査するため気化したホルムアルデヒドの露出でアトピー性皮膚炎を誘発する方法をご紹介します。

ビデオ: Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

高純度の取得トキソプラズマ</em不連続塩化セシウム勾配別>オーシスト

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

免疫・感染学

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In genetically predisposed individuals, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies and their direct deposition into small intestinal wall 1,2. The presence of such antibodies constitutes one of the major hallmarks of the celiac disease (CD). Epidermal transglutaminase (eTG) is another member of the transglutaminase family that can also function as an autoantigen in a small minority of CD patients. In these relatively rare cases, eTG triggers an autoimmune reaction (a skin rash) clinically known as dermatitis herpetiformis (DH). Although the exact mechanism of CD and DH pathogenesis is not well understood, it is known that tTG2 and eTG share antigenic epitopes that can be recognized by serum antibodies from both CD and DH patients 3,4.
In this study, the confocal microscopy examination of biopsy samples from skin lesions of two rhesus macaques (Macaca mulatta) with dermatitis (Table 1, Fig. 1 and 2) was used to study the affected tissues. In one animal (EM96) a spectral overlap of IgA and tTG2 antibodies (Fig. 3) was demonstrated. The presence of double-positive tTG2+IgA+ cells was focused in the deep epidermis, around the dermal papillae. This is consistent with lesions described in DH patients 3. When EM96 was placed on a gluten-free diet, the dermatitis, as well as tTG2+IgA+ deposits disappeared and were no longer detectable (Figs. 1-3). Dermatitis reappeared however, based on re-introduction of dietary gluten in EM96 (not shown). In other macaques including animal with unrelated dermatitis, the tTG2+IgA+ deposits were not detected. Gluten-free diet-dependent remission of dermatitis in EM96 together with presence of tTG2+IgA+ cells in its skin suggest an autoimmune, DH-like mechanism for the development of this condition. This is the first report of DH-like dermatitis in any non-human primate.

Recognition of Epidermal Transglutaminase by IgA and Tissue Transglutaminase 2 Antibodies in a Rare Case of Rhesus Dermatitis

Dermatitis herpetiformis (DH) is a chronic inflammatory condition characterized by an autoimmune reaction between IgA and epidermal transglutaminase (eTG). DH develops in a very small portion of gluten-sensitive and/or celiac patients. The results of this study indicate that DH can also develop in a rhesus monkey host with symptoms of idiopatic dermatitis.

の稀なケースでIgAおよび組織トランスグルタミナーゼ2抗体による表皮トランスグルタミナーゼの認識アカゲザル炎

Tissue transglutaminase 2 (tTG2) is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

アレルゲン誘発性喘息のマウスモデル

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

新生仔ラットの脳組織の混合グリア細胞培養物から主要なミクログリアの分離

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

A Microplate Assay to Assess Chemical Effects on RBL-2H3 Mast Cell Degranulation: Effects of Triclosan without Use of an Organic Solvent

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a process that results in the exocytosis of allergic mediators. Modulation of mast cell degranulation by drugs and toxicants may have positive or adverse effects on human health. Mast cell function has been dissected in detail with the use of rat basophilic leukemia mast cells (RBL-2H3), a widely accepted model of human mucosal mast cells3-5. Mast cell granule component and the allergic mediator &#946;-hexosaminidase, which is released linearly in tandem with histamine from mast cells6, can easily and reliably be measured through reaction with a fluorogenic substrate, yielding measurable fluorescence intensity in a microplate assay that is amenable to high-throughput studies1. Originally published by Naal et al.1, we have adapted this degranulation assay for the screening of drugs and toxicants and demonstrate its use here.
Triclosan is a broad-spectrum antibacterial agent that is present in many consumer products and has been found to be a therapeutic aid in human allergic skin disease7-11, although the mechanism for this effect is unknown. Here we demonstrate an assay for the effect of triclosan on mast cell degranulation. We recently showed that triclosan strongly affects mast cell function2. In an effort to avoid use of an organic solvent, triclosan is dissolved directly into aqueous buffer with heat and stirring, and resultant concentration is confirmed using UV-Vis spectrophotometry (using &#949;280 = 4,200 L/M/cm)12. This protocol has the potential to be used with a variety of chemicals to determine their effects on mast cell degranulation, and more broadly, their allergic potential.

Mast cell degranulation, the release of allergic mediators, is important in allergy, asthma, and parasite defense. Here we demonstrate techniques1 for assessing effects of drugs and toxicants on degranulation, methodology recently utilized to exhibit the powerful inhibitory effect of antibacterial agent triclosan2.

RBL-2H3マスト細胞脱顆粒に対する化学的影響を評価するために、マイクロプレートアッセイ：有機溶剤を使用せずに、トリクロサンの影響

Mast cells play important roles in allergic disease and immune defense against parasites. Once activated (e.g. by an allergen), they degranulate, a ...

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 ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10における粒子結合のBet V 1アレルゲンのフローサイトメトリー分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

A Controlled Mouse Model for Neonatal Polymicrobial Sepsis

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse polymicrobial sepsis can be induced by injecting cecal slurry intraperitoneally into day of life 7 mice and monitoring them for the following week. Presented here are the detailed steps necessary for the implementation of this neonatal sepsis model. This includes making a homogeneous cecal slurry stock, diluting it to a weight- and litter-adjusted dose, an outline of the monitoring schedule, and a definition of observed health categories used to define humane endpoints. The generation of a homogeneous cecal slurry stock from pooled donors allows for the administration into many litters over time, reducing the variation between donors, and preventing the use of potentially toxic glycerol. The monitoring strategy used allows for the anticipation of survival outcome and the identification of mice that would later progress to death, allowing for an earlier identification of the humane endpoint. Two main behavioral features are used to define the health scores, namely, the ability of the neonatal mice to right themselves when placed on their back and their level of mobility. These criteria could potentially be applied to address humane endpoints in other studies of neonatal disease in mice, as long as a pilot study is performed to confirm accuracy. In conclusion, this approach provides a standardized method to model newborn sepsis in mice, while providing resources to assess animal welfare used to define early humane endpoints for challenged animals.

This protocol provides the necessary steps to establish and evaluate neonatal sepsis in 7-day-old mice.

新生児モデル敗血症マウスの制御モデル

Neonatal sepsis remains a global burden. A preclinical model to screen effective prophylactic or therapeutic interventions is needed. Neonatal mouse ...

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

膣内HIV曝露とHIV感染治療に対するヒト化NOGマウス

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

グラム陰性細菌敗血症の新生児画像モデル

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...