この作品は、（HPL）は、NIH助成金R01 GM087609、（MTL）は（HPL）のアンソニー·シュウ、NIHのグラントU01のHL099776に敬意を表しイングリッド·レイとビル·シュウからの贈り物、Hagey研究のための助成金によって部分的にサポートされていました小児再生医療と（MTLとHPLへ）オーク財団。 GGWはスタンフォード大学医学部、スタンフォード医学者研修プログラム、及びNIGMS訓練助成金GM07365によってサポートされていました。 MSHはCIRM臨床フェロー研修グラントTG2-01159によってサポートされていました。 WXHはサーノフ循環器財団からの資金によってサポートされていました。

注：この論文で説明されているすべての手順は、実験動物管理上のスタンフォードの紛争処理パネル（APLAC）によって確立されたガイドラインに従って実施されている。 1.時限妊娠 - 過排卵技術（図1） 注：正確にE16.5とE18.5での胎児手術のためのマウス胚の妊娠期間のタイミングは非常に重要である。このセクションの我々の詳細には過剰排卵を誘発するために妊娠馬血清（PMS）およびヒト絨毛性ゴナドトロピン（HCG）注射を用いて、マウスの妊娠のタイミングのために私たちのプロトコル。 <ol><li> 1日目のために一時と午後3時間100μlのPBSの容量でPMSの3.0から5.0国際単位（IU）でメスのマウス（&lt;ケージ当たり5）を腹腔内（IP）を注入します。 </li><li> 12：00 3日目（四〇から五PMSの注射後時間〜47）の2:00 PMの間に、100μlのPBSの容量でHCGの3.0から5.0 IUとメスのマウスIPを注入する。 注：HCG注射を誘導<html>排卵の約12時間後に注射。 </li><li>直後にHCGの注射後、8-16週齢の雄と雌交尾。 注：我々は、典型的には、個々の男性のケージに2人の女性を置く。 </li><li>胎児の年齢E0.5として - （10:00 AM 6時00分）とレコード4日目の朝に、男性とは別の雌。 注：ワギナプラグは、この時点で確認することができます。しかし、膣のプラグの観察は、妊娠を保証するものではありませんし、何プラグが観察されない時に女性が妊娠することができます。妊娠が妊娠年齢のE16.5及びE18.5での目視検査および/または触診によって一般的に観察可能であることを考えると、4日目の朝に膣栓をチェックすると、必ずしも必要ではありません。我々の経験では、歪みに応じて、超排卵の雌の約30から50%はここに記載されている技術を使用して妊娠。 </li></ol>pgの &quot;/&gt; 過排卵技術については、図1の回路図。マウスでの過剰排卵時限妊娠の回路図を示す手順。 <a href="https://www.jove.com/files/ftp_upload/52297/52297fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> E16.5およびE18.5胚2.マウス胎児手術（背傷つけ処理）（図2） <ol><li>手続きの前に、70％のイソプロピルアルコールで手術室や機器の全ての表面をきれいに。また、それらをオートクレーブ処理により手順で使用されるすべての手術用品や楽器を殺菌。いくつかの機関は、ホットビーズ滅菌のその後の使用を可能にすることができる。操作ごとに、ガーゼや手術器具を含む無菌パックを使用しています。 </li><li>毎分1リットルでのメンテナンス麻酔に続いて、毎分2リットルで2.5％イソフルラン/酸素混合物の下で妊娠中の母親（胎児の年齢E16.5またはE18.5）で麻酔を誘導する。 </li><li> CONへしっかりと適切な麻酔、マウスの深いペダル反射が抑制されていることを確認し、腹臥位でマウスを置く。 </li><li>手術中眼刺激や乾燥を防ぐためにそのようなPuralubeとして獣医眼軟膏を適用します。 </li><li> 30秒（ 図2A）より、もはや用脱毛クリームの光アプリケーションを投与することにより、腹部を準備します。 </li><li>ポビドンヨードとアルコール（ 図2Bおよび2C）との無菌手術のために腹部を準備します。 </li><li>顕微はさみ（ 図2Dおよび2E）を用いて顕微鏡下で正中開腹を行います。 </li><li>静かに手術（ 図2F）用に選択子宮や胎児を公開します。 </li><li>鈍い先端の針を用いて温かい（38℃）リン酸緩衝生理食塩水（PBS）で手術野を洗浄 注：One慎重大口径の針の先端を折り曲げて作製することができる。 （ 図2Gおよび2H）。 </li><li>許可するようにした胎児を置き背にフルアクセスだ。 </li><li>意図された背側創傷（ 図2I）のサイトの上にある子宮を通して7-0ナイロン縫合糸を使用した巾着ステッチを渡します。脊髄の左または右へ、および大血管を欠く子宮壁の領域における背の領域にわたって位置巾着。 </li><li>巾着（ 図2J）の中央に子宮壁と羊膜嚢を通じて3mmの切開を行います。 </li><li>温かい（38℃）PBSで切開部位を洗浄する。 </li><li>顕微はさみを使用して、胎児の背に、単一の全層切除傷、長さ約1ミリメートルをカット。 </li><li>そっとブロット切開部位は、綿チップアプリケーターで乾燥させます。 </li><li>傷（ 図2K）の場所をマークするために、創傷部位に3μlのボリューム墨汁の皮下に注入します。 </li><li>インクが創傷部位内に保持されていることを確認するために温かい（38℃）PBSで灌漑。 </li><li>外科助手の私を持っているnject巾着など羊膜嚢に鈍い先端10のG注射器を通して温かい（38℃）、PBS（ 図2L）を閉じている。巾着の閉鎖が完了します（ 図2M）を近づくと注射器を撤回。 </li><li>静かに腹腔（ 図2N）に子宮を返す。 </li><li>エバートの皮膚や腹膜。 </li><li>温かい（38℃）PBSで腹腔を外科助手を灌漑している。 </li><li>皮膚や腹膜ステープルすばやく閉じる腹部が（ 図2O）閉じた。標準的な閉鎖は二層で行われる。腹膜および第2層における一方の層、皮下組織および皮膚の腹筋。即時の犠牲と胎児の収穫のために、私たちのデモが1層で閉鎖を示している。 </li><li> 30分間または動物が胸骨横臥位を維持するのに十分な意識を取り戻すまで、37℃に設定した暖かいインキュベーター内で観察下に動物を置きます。 </li><li> retにしないでくださいそれは完全にプロシージャから回復するまで、他の動物の会社に動物をURN。 </li><li>麻酔からとその後の48時間の間に覚醒時には、ブプレノルフィンの皮下注射（0.05 mg / kgを）疼痛評価に基づいて、必要に応じて鎮痛のために12時間ごとに管理する。必要に応じて追加の術後疼痛緩和のために皮下注射を介してカルプロフェン（5ミリグラム/ kg）を管理します。 </li><li>ケージに動物を返し、食物と水を自由にそれらを提供する。 </li><li>痛みの症状のために密接に監視します。 </li><li> 48時間後に手術、イソフルランと収穫負傷胎児の過剰摂取で妊娠中の母親を生け贄に捧げる。これを行うためには、5％以上にイソフルラン濃度を調整し、呼吸停止後に1分間曝露を維持する。頸椎脱臼で安楽死を確認してください。年齢をマッチさせたコントロールのための無傷胚を収穫。後期胚は、次のような動物実験委員会の勧告、と一貫安楽死の別のメソッドを持っている必要がありますデ人頭、頸椎脱臼、または化学薬品注入。 </li></ol><img alt="図2" src="/files/ftp_upload/52297/52297fig2highres.jpg" /> 図2.回路図マウス胎児外科。E16.5及びE18.5マウス胚で負傷背部するための一般的な手順。（A）マウスの腹部の脱毛。（BとC）マウスの腹部の調製。（D）顕微鏡外科に使用子宮を通じて巾着縫合の手順。（E）ミッドライン開腹。（F）子宮の露出を。鈍先端針の（G）の作成 ​​。温かい生理食塩水で子宮の（H）灌漑。（I）の作成​​。（J）切開壁と1ミリメートルフル層切除創傷世代。（K）墨汁の皮下注射。（LとM）巾着縫合の閉鎖。（N及びO）腹部の閉鎖。 <a href="https://www.jove.com/files/ftp_upload/52297/52297fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

<ol>
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The authors have nothing to disclose.

ここで紹介するの外科的プロトコルは、最初に私たちの研究室10が2006年に出版さ胎児マウスの瘢痕治癒の切除モデルについて説明します。切除創傷11の他の確立されたモデルに加えて、胎児マウス瘢痕治癒の切開モデルが同様に存在する12,13。サル、子羊、ウサギ、オポッサム、ラットで胎児の瘢痕創傷治癒の調査は、14〜17を報告されている。しかし、マウス...

胎児皮膚創傷は、妊娠後期1まで急速かつscarlessly癒す。胎児の瘢痕創傷修復は、正常な組織構造および機能の再生によって特徴付けられる。瘢痕化の表現型への瘢痕からの移行は、ヒトにおけるマウス2,3における胎生18日（E18）の周りに妊娠後期に起こる。成人と比較して、胎児の創傷修復を迅速上皮、結合組織の堆積、および線維芽細胞の遊走によって特徴付けられる。 多くの研究は、初期の胎児の発達中の瘢痕創傷治癒の現象の可能な説明を提供してきました。炎症は、大人の創傷修復の基本的なコンポーネントです。しかし、胎児の創傷は、急性炎症4の欠如によって特徴づけられる。これは胎児の段階で免疫系の機能的な未熟さの結果であるかどうかは不明なままである。最近の研究では、豊富に違い、マットと示唆urity、およびE18胎児の皮膚対E15における肥満細胞の機能は、少なくともマウス3において、瘢痕の表現型への移行に関与し得る。他の研究では、プロパティと胎児および成人の創傷マクロファージの存在量の違いが胎児の創傷修復5中に、通常の細胞外マトリックス（ECM）の改革のために責任があることを断定。 胎児および成人の発達中の環境要因の違いはまた、創傷の修復に影響する場合があります。 Longakerらは、胎児から創傷液が成人の創傷液6でなしに比べ、ヒアルロン酸刺激活性の高いレベルを持っていることを示した。従って、胎児の創傷環境におけるヒアルロン酸の高いレベル、細胞運動性および増殖につながる微小環境を促進するグリコサミノグリカンは、初期の胎児の発育中に見られる瘢痕の表現型の原因である可能性がある。フェタチーズはその事実に証拠ポイントのその他のラインLの創傷環境は、比較的低酸素および成長因子7に富む無菌羊水中に浸漬される。しかし、決定的な答えは、線維性の修復に瘢痕再生への移行をトリガー胚形成の間の重要なイベントや要因のために提供されていない。 胎児に瘢痕治癒の責任のメカニズムを理解することは、正確で再現性のあるモデルを必要とする。ここでは詳細E16.5（瘢痕）とE18.5（瘢痕化）マウス胚の背で胎児の瘢痕創傷治癒の再現可能なモデル。さらに、このモデルの小さな変化は、胎児の創傷及び皮膚8,9の遺伝子発現解析など、さらなる研究の数を実行するために利用することができる。正確なタイミング妊娠がこの胎児の瘢痕創傷治癒モデルの成功した要約のために重要であることを考えると、私たちは過剰排卵時限妊娠についても詳細私たちのプロトコル。

<table border="0" cellpadding="0" cellspacing="0" width="665">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:219px;">Name of Material/Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:108px;">Catalog Number</td>
			<td style="width:155px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7-O MONOSOF Suture</td>
			<td>eSuture</td>
			<td>SN-1647G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Surgical Forceps</td>
			<td>Kent Scientific</td>
			<td>INS650916</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro-scissors</td>
			<td>Kent Scientific</td>
			<td>INS600127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Autoclip 9mm</td>
			<td>Texas Scientific Instruments</td>
			<td>205060</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin Syringe</td>
			<td>Thermo Fisher Scientific</td>
			<td>22-272-382</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Black Pigment</td>
			<td>AIMS</td>
			<td>242</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BD Safety-Lok 3ml Syringe</td>
			<td>BD Biosciences</td>
			<td>309596</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>10010-049</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OPMI-MD Surgical Microscope</td>
			<td>Carl Zeiss Surgical Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pregnant Mares Serum (PMS)</td>
			<td>Millipore</td>
			<td>367222</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Chorionic Gonadotropin (HCG)</td>
			<td>Sigma-Aldrich</td>
			<td>CG10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Povidone Iodine Prep Solution</td>
			<td>Dynarex</td>
			<td>1415</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nair (depilatory cream)</td>
			<td>Church and Dwight Co.</td>
			<td>22600267058</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mouse fetal skin model of scarless wound repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

組織学的分析のために、背側の皮膚E16.5及びE18.5における皮膚創傷マウス胚を4％PFAで固定し48時間後創傷、収穫されるべきであり、パラフィン包埋した。蛍光トランスジェニックモデルでは、10月との凍結保存が適切であり得る。細胞および結合組織の構造を可視化するために使用できるいくつかの汚れがある。ヘマトキシリンとエオシン、核青と好酸球の構造（ すなわち 、細胞質と?...

Watch this Scientific Journal Video about A Mouse Fetal Skin Model of Scarless Wound Repair at JoVE.com

A Mouse Fetal Skin Model of Scarless Wound Repair

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

瘢痕創傷修復のマウス胎児皮膚モデル

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

a-mouse-fetal-skin-model-of-scarless-wound-repair

Research

JoVE Journal

Medicine

12.2K ビュー.  Stanford University School of Medicine. During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

ビデオ: A Mouse Fetal Skin Model of Scarless Wound Repair

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

全血刺激のためのアッセイを用いて炎症性サイトカインIL -1βの正確かつ簡単に測定

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

医学

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

無限の地平線インパクターを用いた片側頚髄損傷の打撲のモデル

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

虚血性創傷モデルを作製するための簡略化手法

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

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, ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

胎児のマウスモデルにおける生物活性剤の気管内投与のための新しい手術アプローチ

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

創傷治癒のマウスモデル

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

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 ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

血管内大動脈瘤の修復の新しいマウスモデル

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation of the animal for surgery, opening the thoracic body wall, exposing the desired rib from the surrounding intercostal muscles, excising the desired section of rib without inducing a pneumothorax, and closing the incisions. Compared to the bones of the appendicular skeleton, the ribs are highly accessible. In addition, no internal or external fixator is necessary since the adjacent ribs provide a natural fixation. The surgery uses commercially available supplies, is straightforward to learn, and well-tolerated by the animal. The procedure can be carried out with or without removing the surrounding periosteum, and therefore the contribution of the periosteum to repair can be assessed. Results indicate that if the periosteum is retained, robust repair occurs in 1 - 2 months. We expect that use of this protocol will stimulate research into rib repair and that the findings will facilitate the development of new ways to stimulate bone repair in other locations around the body.

The overall goal of this procedure is to successfully resect a portion of bone from the rib of a mouse. The procedure was developed as a model to study large-scale long bone repair.

マウスリブを切除するための外科的手順：大規模ロング骨修復のためのモデル

This protocol introduces researchers to a new model for large-scale bone repair utilizing the mouse rib. The procedure details the following: preparation ...

Demonstration of the Rat Ischemic Skin Wound Model

The propensity for chronic wounds in humans increases with ageing, disease conditions such as diabetes and impaired cardiovascular function, and unrelieved pressure due to immobility. Animal models have been developed that attempt to mimic these conditions for the purpose of furthering our understanding of the complexity of chronic wounds. The model described herein is a rat ischemic skin flap model that permits a prolonged reduction of blood flow resulting in wounds that become ischemic and resemble a chronic wound phenotype (reduced vascularization, increased inflammation and delayed wound closure). It consists of a bipedicled dorsal flap with 2 ischemic wounds placed centrally and 2 non-ischemic wounds lateral to the flap as controls. A novel addition to this ischemic skin flap model is the placement of a silicone sheet beneath the flap that functions as a barrier and a splint to prevent revascularization and reduce contraction as the wounds heal. Despite the debate of using rats for wound healing studies due to their quite distinct anatomic and physiologic differences compared to humans (i.e., the presence of a panniculus carnosus muscle, short life-span, increased number of hair follicles, and their ability to heal infected wounds) the modifications employed in this model make it a valuable alternative to previously developed ischemic skin flap models.

The rat, due to its size, availability, and rather docile behavior, has been utilized as a research model for many years. The goal of this protocol is to utilize the rat as an ischemic skin wound healing model to provide valuable insight into the pathophysiology of chronic wounds.