この研究は、国立衛生研究所(DK097335&T32AI007260)とアイオワ大学ステッド家族小児科を通じて部分的に支援されました。

すべての動物の手順は、アイオワ大学の制度的動物の世話と使用委員会によって承認されました (プロトコル#8041401).すべての動物は、アイオワ大学でラボ動物ケアの評価と認定協会(AALAC)承認されたビバリウムに収容されました。全てのマウスは野生型株C57Bl/6Jであった。
1. 妊娠中マウスにおけるFEMIの確立
<ol><li>LPSの準備<ol>
<li>大腸菌O55:B5(ストック濃度2mg/mL)に由来するLPSを使用してください。</li>
		<li>LPSストック濃度1:100、滅菌生理食塩水を使用して、作業濃度20μg/mLを使用します。</li>
	</ol>
</li>
	<li>母体LPS注射<ol>
<li>妊娠日e15で妊娠中のダムを注入します。この時点は、マウスの妊娠を通じて約75%であり、このモデルは、絨毛膜炎による早産の大部分が発生するヒト妊娠の第3学期の初期に発達的に似ています。</li>
		<li>注射の直前に妊娠中のマウスの体重を量り、適切なLPS投与を決定する。</li>
		<li>100 μg/kg の LPS の総投与量の 5 μL x グラム体重 (gbw) を使用して、作業濃度の線量を計算します。コントロール動物の場合は、通常の生理液剤の同等の量を注射に使用します。</li>
		<li>各注射の前に高い15秒間の渦LPS溶液を3回。</li>
		<li>LPSのボリュームを1mLのシリンジに引き上げる。</li>
		<li>擦り傷の技術で妊娠中のマウスを拘束します。後回しの不用な位置に保持し、注射を行います.<ol>
<li>30ゲージ8mmの針を、腹部の右下象限(膀胱および腹部の血管を避けるために)上に30\u201240°の角度で挿入します。針の長さを約1/4~1/2に挿入します。</li>
			<li>注射器プランジャーを引き戻し、注入する前に負圧を確保します。負圧が存在する場合は、射出を続行します。</li>
			<li>注射後、マウスを約30分間監視し、残りの妊娠のためにケージに戻します。</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 子孫の出産とケア、腸の収穫
<ol><li>e20で膣分娩を介して正常に子犬を送達する。 注:このモデルは 、図1 で見ることができる予想用量依存性胎児の損失率を有し、以下の結果で議論されている。</li>
	<li>子犬が母親と一緒に残り、アドリビタムフィードを受け取ることを許可します。</li>
	<li>収穫日には、通常、出生後14日目(P14)、制度的動物ケアと使用委員会のプロトコルに従って子宮頸部脱臼を介して子犬を安楽死させます。</li>
	<li>はさみと鉗子を使用して、腹部の全長に対して、皮膚と腹腹を通して、腹部の正中線を垂直に切開する。胃からセカムに小腸をはさみで切除し、鉗子で腸間腸を取り除きます。</li>
	<li>小腸の遠位1/3(ヒトの回腸を代表するセクション)を分離して保持し、近位小腸、盲腸、結腸を捨てる。</li>
	<li>はさみを使用して、回腸部分を半分に分割します。</li>
	<li>後でRNA定量を行うため、RNA安定化液に近位半分を入れる。</li>
	<li>スライド準備のために10%中性緩衝ホルマリンに遠位半分を入れます。</li>
</ol>3. 腸の損傷スコアリング
<ol><li>パラフィン埋め込まれた組織を5μmの厚いスライスに入れ、ガラススライドに取り付けます。 注:パラフィンの埋め込み、切り離し、スライドへの取り付けのために、標本をヒストロジーコアに送ります。</li>
	<li>標準的な手順に従ってスライドを分離します。</li>
	<li>標準的な手順に従ってヘマトキシリンおよびエオシンの染色切片。</li>
	<li>前述の42、43のように腸の損傷のための3ポイントスケールでセクションをスコア。<ol>
<li>軽顕微鏡を用いて、2人の別々の盲検研究者による一般化された腸損傷を評価し、villus完全性および基原膜43 からの分離を評価する3ポイントスケールで(補足図1)。腸の損傷は、20倍の倍率および数値開口0.50で最もよく評価される。</li>
		<li>通常の粘膜を表す0のスコアを割り当てます。</li>
		<li>1のスコアを割り当てると、上皮下グルエンハーゲンの空間、空胞化または上皮下持ち上げの発達を包含する軽度の傷害を説明する。</li>
		<li>上皮持ち上げおよび空胞化が絨毛の半分以上、絨毛の歪み、または粘膜潰瘍および層の崩壊によって示される重篤な傷害を記述するために2のスコアを割り当てる。</li>
	</ol>
</li>
</ol>4. パネス細胞とゴブレット細胞の定量化
<ol><li>脱パラフィン後、ステップ2.8からの組織切片の染色スライドは、アルシアンブルー/周期酸シフ染色を用いて、前述の44,45のゴブレットおよびパネス細胞の両方を示す。 注:アルシアンブルー/周期酸シフ染色はパネス細胞またはゴブレット細胞に固有ではありませんが、私たちの経験では、盲目の経験豊富な研究者は、この染色を使用して、細胞標的抗体と比較して同等の細胞定量を有し、バックグラウンド染色が大幅に少ない46.</li>
	<li>脱パラフィン、染色、脱水スライドは以下の通りである。<ol>
<li>10分間、キシレンにスライドを2回沈めます。 注意:キシレンはヒュームフードに使用する必要があります。</li>
		<li>100%EtOHでリンス。</li>
		<li>3分間100%EtOHでスライドを水没させ、その後90%のEtOHで3分間、次いで3分間70%のEtOHを、最後に3分間50%のEtOHでスライドを水没させます。</li>
		<li>水道水を流して5分間洗います。 注意:組織サンプルの損失を防ぐために、流水から離れてセクションを向けます。</li>
		<li>標準的なコーヒーフィルターとフィルターアルシアンブルー染色液。</li>
		<li>アルシアブルーのステインで15分間滑り、水道水を流して2分間洗います。</li>
		<li>200mLの二重蒸留水で1mgの周期酸を希釈する。このソリューションのスライドを5分間水没させます。その後、水道水を流して1分間洗います。</li>
		<li>シフの試薬を10分間染色する。水道水を流して5分間洗います。</li>
		<li>スライドをヘマトキシリンで1分間染色し、水道水を流して2分間洗います。</li>
		<li>酸性アルコール(塩酸1mLを99mLで70%EtOHに混合)に1分間沈める。</li>
		<li>スコットの水道水(水道水中のNaHCO3 濃度0.1%)に1分間水没し、水道水を流して1分間洗います。</li>
		<li>スライドを退避します。<ol>
<li>各スライドを70%のEtOHに10回浸し、その後90%のEtOHで10回、100%EtOHで10回浸します。</li>
			<li>100%EtOHで10分間滑液を沈め、続いてフレッシュキシレンに2回3分間沈水します。</li>
		</ol>
</li>
		<li>取り付け培地を試料に一滴置き、その上にカバースリップを置きます。</li>
	</ol>
</li>
	<li>ゴブレットセルカウント<ol>
<li>光顕微鏡を使用して、ゴブレット細胞をカウントする(補足図2)。腸組織の各部分について、ゴブレット細胞と500の上皮細胞の数を数え、100上皮細胞あたりの比率としてゴブレット細胞比を発現する。ゴブレット細胞は、20倍の倍率と開口数0.5で最もよくカウントされます。</li>
	</ol>
</li>
	<li>パネス セルの棚卸<ol>
<li>光顕微鏡を使用して、パネス細胞を数える(補足図2)。腸組織の各部分について、腸内窩当たりのパネス細胞の比率として表現する。腸組織の各部分ごとに100腸の納骨堂を数えます。パネスセルは、20x-60倍の倍率と数値絞り0.50-1.30で最もよくカウントされます。</li>
	</ol>
</li>
</ol>

著者らは開示するものは何もない。

絨毛膜炎は、2\u20124%の期間および25\u201230%の早産8,9に影響を及ぼす。しかしながら、絨毛膜炎の影響は、胎児および新生児10、11、12、13、14、15、16に有意な影響を及ぼすことを示しているので、過去の出生を長く延長することができる。<sup class="x...

絨毛膜は哺乳類の妊娠に不可欠な役割を果たす。これらは、複数の機能を提供する絨毛と羊膜が含まれています。それらは胎児を取り囲んで保護し、母体と胎児の区画1間のパラクリンシグナル伝達を促進し、そして分化の開始に関与し得る絨毛膜内に局所的なフィードバックループを作り出す。膜の現在の理解は、羊膜が構造的なバリア機能を提供することを示し、そして絨毛膜は、主に母親の免疫系2から発達中の胎児を保護するために免疫学的緩衝液を提供する。これらの膜の炎症は、絨毛膜炎として知られています。歴史的に、臨床絨毛膜炎の診断は、母体熱に加えて1つ以上の胎児または母親の臨床所見3,4の存在に続いて行われた。しかし、この定義は臨床的に有用であるが、その精度の欠如は、絨毛膜炎の研究を困難にしている。2015年、診断を明らかにしようとして、ユーニス・ケネディ・シュリバー国立小児保健人間開発研究所による専門家パネルワークショップは、子宮内炎症、または感染症、またはその両方(トリプルI)3として絨毛膜炎を定義した。微生物誘発感染は子宮/羊膜炎症の重要な原因であるが、滅菌子宮/羊膜炎症5、6、7よりも一般的に起こらないので、この明確化は重要である。全体として、絨毛膜炎は、定期出産の2\u20124%および早産期期の25\u201230%8,9に見られるように、依然として重大な公衆衛生上の問題である。
絨毛膜炎は胎児および新生児に重大な影響を及ぼす可能性がある。絨毛膜炎は、気管支肺異形成10、脳白器損傷11、脳内出血12、前産児13の網膜症、および早期発症新生児敗血症14、15を含む多くの未熟児罹患率のリスクの増加に関連することが文献に十分に文書化されている。未熟腸管の損傷や修復機構に関心を持つため、絨毛膜炎は壊死性腸炎(NEC)15,16の後の発症にも関連していることに注意することが重要である。NECは早産児の壊滅的な胃腸疾患であり、炎症およびその後の腸壊死に対する調節不全宿主応答をもたらす。毎年、NECは米国で4000人以上の乳児に影響を及ぼし、これらの乳児の3分の1までは18の病気で死亡する。NECの病因は、腸の未熟さ、未熟免疫系の調節不変性、腸内炎症、細菌転位19の組み合わせを含み、腸壊死の最終的な共通経路で最高潮に達する可能性が高い。重要なことに、NECの発症は出生後数週間および絨毛膜炎への潜在的な暴露によってしばしば起こり、絨毛膜炎とその後のNECの発症との間の機械論的なつながりを20に不明瞭にする。絨毛膜炎がNECの病態生理学に寄与する可能性のあるメカニズムの1つは、母親の免疫系のアップレギュレーションを通じて、その後、正常な胎児発達パターン21、22、23を破壊する可能性のある強い胎児炎症反応を生じさせる。
絨毛膜炎の複数の哺乳類モデルは、げっ歯類および羊24、25、26、27、28、29、30、31、32に存在する。しかし、絨毛膜炎誘発性胎児の母体炎症(FEMI)への初期の新生児期を超えて腸管の発達に関するデータはほとんど存在しない。FEMIとそれに伴う未熟腸管の損傷の発症との関係を探るため、我々はリポ多糖(LPS)誘導型FEMIモデルを適応させました。リポ多糖は、グラム陰性細菌上の細胞外表面の主要な成分であり、ヒト33を含む複数の真核生物種の自然免疫系の強力な刺激剤である。母体LPS注射は、生きた細菌の交感作用を伴わない無菌炎症カスケードをもたらし、早産34の誘導のための確立されたモデルであり、また、最も重篤な形態の絨毛膜炎24、35である急性絨毛膜炎および胎児炎症反応症候群(FIRS)のモデルである。また、羊モデル36とマウスモデル37、38、39、40の脳白と灰色物質の両方の傷害を誘発することが示されている。しかし、我々の知る限りでは、この絨毛膜炎とFEMIのモデルを使用して、過去の出生時の胃腸管の発達への影響を調べるとともに、絨毛膜炎と後にNEC41,42の発症との間の可能な機械化の関連を調査する。

<table>
	<tbody>
		<tr>
			<td>10% neutral buffered formalin</td>
			<td>Sigma</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian blue stain</td>
			<td>Newcomer supply</td>
			<td>1003A</td>
			<td></td>
		</tr>
		<tr>
			<td>C57Bl6/J mice</td>
			<td>Jackson Laboratories</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon labs</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Sigma</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin stain</td>
			<td>Leica</td>
			<td>381562</td>
			<td></td>
		</tr>
		<tr>
			<td>LPS</td>
			<td>Sigma</td>
			<td>L2880</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ni-U Microscope</td>
			<td>Nikon</td>
			<td>2CE-MQVJ-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Periodic Acid</td>
			<td>ACROS</td>
			<td>H5106</td>
			<td>CAS# 10450-59-9</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Thermofisher</td>
			<td>Am7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Schiff&#39;s reagent</td>
			<td>Sigma</td>
			<td>S5133</td>
			<td></td>
		</tr>
		<tr>
			<td>Secor Imager 2400</td>
			<td>Meso Scale Discovery (MSD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>V-Plex Assay</td>
			<td>Meso Scale Discovery (MSD)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma</td>
			<td>534056</td>
			<td></td>
		</tr>
	</tbody>
</table>

a murine model of fetal exposure to maternal inflammation to study the effects of acute chorioamnionitis on newborn intestinal development

絨毛膜炎は早産の一般的な沈殿物であり、壊死性腸炎(NEC)を含む多くの未熟児の罹患率に関連している。しかし、これら2つの条件間の機械的なリンクはまだ発見されていない。我々は、リポ多糖(LPS)誘発胎児の母体炎症(FEMI)を含む絨毛膜炎のマウスモデルを採用した。FEMIのこのモデルは、生殖不能、胎盤、および胎児の炎症性カスケードを誘発し、臨床絨毛膜炎の多くの症例にも存在する。生きた細菌を利用し、より正確に絨毛膜炎をもたらす上昇感染の病態生理を模倣するモデルが存在するが、これらの方法は、未熟な腸管および関連する開発マイクロバイオームの発達に間接的な影響を及ぼす可能性がある。このプロトコルを用いて、LPS誘導FEMIが妊娠喪失および早産の用量依存的増加、ならびに子孫における正常な腸の発達の中断をもたらすことを実証した。さらに、FEMIは子孫の腸損傷および血清サイトカインを有意に増加させる一方で、腸の炎症に対する先天性免疫の第1行を提供するゴブレットおよびパネス細胞を減少させることが実証された。LPS誘導FEMIの同様のモデルは、絨毛膜炎とその後の中枢神経系の異常との関連をモデル化するために使用されてきたが、このプロトコルは、絨毛膜炎と後の腸内発達の間の機械化リンクを発育する最初の試みである。

胚性15日目にFEMIに曝露すると、用量依存性の妊娠喪失と早産率の用量依存率(図1)42に至る。実験では、妊娠の喪失と未熟児(未熟児と子宮内胎児の終焉の間の50%の損失)を最小限に抑えながら、有意な炎症性侮辱にさらすために100μg/kgのLPS用量を使用することを選択しました。
このアプローチを用いて、次に、FEMIが子孫のその後の傷...

Watch this Scientific Journal Video about A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development at JoVE.com

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

生きた生物の合併症を伴わない胎児の母体炎症(FEMI)をシミュレートする絨毛膜炎のモデルを開発し、子孫の腸管の発達に及ぼすFEMIの影響を調べた。これは、絨毛膜炎後の腸の損傷の発症のための機械化原因の研究を可能にする。

新生児腸管の発達に及ぼす急性絨毛膜炎の影響を研究する母体炎症への胎児暴露のマウスモデル

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

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3.7K ビュー.  University of Iowa. 生きた生物の合併症を伴わない胎児の母体炎症(FEMI)をシミュレートする絨毛膜炎のモデルを開発し、子孫の腸管の発達に及ぼすFEMIの影響を調べた。これは、絨毛膜炎後の腸の損傷の発症のための機械化原因の研究を可能にする。

ビデオ: A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

同所性大動脈移植：慢性血管障害の発生研究のラットモデル

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

医学

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

胎児性アルコール暴露のゼブラフィッシュモデルの催奇形性変化を評価する

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

胎児心エコー検査と子宮内発育制限のウサギモデルにおけるパルス波ドップラー超音波

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

ネズミ角膜移植：固体臓器移植の最も一般的な形態を研究するためのモデル

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

A Murine Model of Irreversible and Reversible Unilateral Ureteric Obstruction

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive nephropathy induced by unilateral ureteric obstruction (UUO), which can either be irreversible (UUO) or reversible (R-UUO). In the irreversible UUO model, the ureter may be obstructed for variable periods of time in order to induce increasingly severe renal inflammation and interstitial fibrotic scarring. In the reversible R-UUO model the ureter is obstructed to induce hydronephrosis, tubular dilation and inflammation. After a suitable period of time the ureteric obstruction is then surgically reversed by anastomosis of the severed previously obstructed ureter to the bladder in order to allow complete decompression of the kidney and restoration of urinary flow to the bladder. The irreversible UUO model has been used to investigate various aspects of renal inflammation and scarring including the pathogenesis of disease and the testing of potential anti-inflammatory or anti-fibrotic therapies. The more challenging model of R-UUO has been used by some investigators and does offer significant research potential as it allows the study of inflammatory and immune processes and tissue remodeling in an injured and scarred kidney following the removal of the injurious stimulus. As a result, the R-UUO model offers investigators the opportunity to explore the resolution of kidney inflammation together with key aspects of tissue repair. These experimental models are of relevance to human disease as patients often present with obstruction of the renal tract that requires decompression and are commonly left with significant residual kidney impairment that has no current treatment options and may lead to eventual end stage kidney failure.

The murine model of irreversible unilateral ureteric obstruction (UUO) is presented together with the model of reversible UUO in which the ureteric obstruction is relieved by anastomosis of the severed ureter into the bladder. These models enable the study of renal inflammation and scarring as well as tissue remodeling.

不可逆とリバーシブル片側尿管閉塞のマウスモデル

Obstruction of the kidney may affect native or transplanted kidneys and results in kidney injury and scarring. Presented here is a model of obstructive ...

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

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

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

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

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

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

胎盤におけるオートファジーの制御の母性的な肥満に関連付けられている炎症の影響を検討する主なひと胎盤絨毛細胞モデル

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

嚢胞性線維症気道マイクロバイオームに対する酸素付加の効果を研究するモデルの設計と開発

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...

ラットの変形性膝関節症に対する標準化されたTuinaマニピュレーション

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

著者スポットライト:ラットにおけるTuina操作のメカニズムの理解 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

変形性膝関節症ラットの炎症と軟骨喪失を軽減するためのTuina操作

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...