We thank members of the Mariani lab for critical reading of the manuscript. Our funding sources were: the Baxter Medical Scholar Research Fellowship (to M.K.S.), USC undergraduate fellowships and the Provost, Dean Joan M. Schaeffer, and Rose Hills Fellowships, (to M.K.S.). We also acknowledge a CIRM BRIDGES fellowship through Pasadena City College (to T.T.T). and the James H. Zumberge Research and Innovation Fund, the USC Regenerative Medicine Initiative, and the NIAMS NIH under Award Number R21AR064462 (to F.V.M).

注：すべての手順は、南カリフォルニア大学の施設内動物管理使用委員会（IACUC）によって承認された動物のプロトコルに従っている。 外科1.準備<ol><li>必要な手術器具や消耗品（滅菌、アイテム1 - 材料のリストの18）をレイアウトし、フード内やダウンドラフトテーブルの上に解剖顕微鏡および光ファイバトランスイルミを手配。 </li><li>麻酔を誘導するために、4％イソフルランで誘導チャンバー内に（3ヶ月 - - 30グラム、1〜20）性的に成熟したマウスを置きます。 </li><li>足および/またはテールピンチテストで麻酔を確認してください。 </li><li>手術中に目の乾燥を避けるために、マウスの目に軟膏を適用します。 </li><li>顕微鏡ステージにマウスを移動します。メンテナンスホースのノーズコーンにマウスを置き、横方向に配置する。体温を維持するために動物に隣接した温暖化ポーチを提供します。 NOTE：マウスの​​位置は個人的な好み、および/または外科医の利き手に依存している、左または右のいずれかの側は問題ありません。 </li><li>メンテナンスのため、3％、および呼吸数を見て - 2にイソフルランを調整します。 </li><li> 25 G針を有する手術後の痛みのためにブプレノルフィン（0.05 mg / kgを）皮下（脚の横脇腹）を注入します。脚部は正常な反応であるけいれんがあります。 </li></ol> 2.オープニング切開<ol><li>触診が希望するリブを検出し、シェーバーで毛のエリアをクリア。 注：リブ1-7が肺に近いと11-13がかなり短いので、リブ8-10を推奨します。肋骨を数えるのは数1のような最も吻側リブで始める必要があります。 </li><li> （3Xは、多くの場合、動物使用プロトコルによって推奨）ポビドンヨードとイソプロパノール（70％）スワブを交互にエリアを準備します。 </li><li>真上中小マイクロサージェリーのはさみで希望の肋骨に平行、皮膚を介して2cmの切開をカット。 Incis下の筋肉と脂肪の層を介して電子。 </li><li>切開部のサイズを最小限に抑えながら、手術領域を露出させるために開創器にすべての3つの層（皮膚、筋肉、脂肪）を配置します。 </li></ol> 3.リブを切除<ol><li> 5.0ミリメートルのメスでリブの所望の部分の上にある肋間筋を切断。リブとして非常に湾曲していない場合に、このようなchondrocostal共同の5 mmの近位の周りのこの切開がある。慎重に先端の細いピンセットで骨から筋肉を分離する。 </li><li>動物に骨膜を保持しながら、切除を作成するために、5.0ミリメートルメスとリブの長さに沿って、骨膜を切断。慎重に先端の細い鉗子で横方向に下にある骨から骨膜を分離する。骨膜は非常にデリケートですし、ゼラチン状の一貫性を持っていたように、注意して進みます。 </li><li>次に細かいマイクロハサミで一方の端部に骨の断面を作る。必要に応じて、との切除を測定顕微鏡や定規タイプのゲージでレチクル。その後、慎重に骨膜から骨を持ち上げ、もう一方の端をカット。 注：慎重に進みます。これは胸膜を引き裂くと気胸になります下の肋骨を切除せずに引き上げなど、ほとんどの繊細なステップ、である。胸膜が破れた場合胸腔の内部が見えるようになるので、これは容易に明らかであろう。 </li><li>出血を止めるために5秒 - 出血が骨の切断の結果として発生した場合、4用綿棒との切断端に圧力をかける。 注：出血が続くことをまれに、それはかなりの血液損失が回復が損なわれるように動物を停止し、安楽死が必要な場合があり（流体を交換することなく、安全に失われることができる最大血液量は、全血液容量の10％であるまたは7.7 - 。）を200μl9 - 8μL/ gの25グラムのマウスの場合、これは約180に相当します。 </li><li>すぐにに削除されたリブを配置今後の分析のために4％PFA。 注：骨と骨膜の両方の切除を行うべきである場合、骨膜切開分離（ ステップ3.2）を省略する。骨膜は非常に緊密胸膜に取り付けられているのでティアリングを回避するために、ピンセットで離れて胸膜から肋骨をからかって、慎重に進んでください。 </li></ol> 4.切開を閉じる<ol><li> 9-0縫合糸（2縫合糸は通常十分です）と残りの骨膜スリーブの上に肋間筋を縫合。手術の場所のための指標として機能するように、直接、リブの切断端の上に縫合糸を配置します。 </li><li>開創器を取り外します。 （ - 4縫合通常は十分である3）9-0縫合糸で覆っている筋肉と脂肪を縫合。筋肉および/ ​​または脂肪の特に厚い層を有するマウスでは、個別の層を縫合（ 例えば、筋肉層の縫合糸の1層および脂肪層のための縫合糸の1層）。 </li><li>閉じ（ - 5縫合糸、通常は十分であり、4）は7-0縫合糸で皮膚。 </li><li>大鉗子で一緒に端をつまんで、縫合糸接着剤で切開を固定します。 </li><li>ゆっくりと最初の数分間、1％に調整することで、オフイソフルランマウスを離乳してからオフにしてください。 </li><li> 10分 - 、5加熱ランプ下にマウスを置き、意識が回復されるまで残す。意識が回復した後、治癒期間中、マウスが移動し、正常に歩き回ると苦痛の兆候を示さないはずである。 </li></ol> 5.回復と分析<ol><li> 0.5ミリグラムの経口ゼラチン状のブプレノルフィンの投与を含む術後疼痛管理を提供/ 48時間ごとに12時間をキロ。経口投与は、胸部に痛みを引き起こす可能性が動物を拘束する必要はありません。 </li><li>治癒期間中に食物と水を自由にケージにマウスを維持します。切開が乾燥した後、雌の動物は、雄ながら同時収容することができるsは戦いを防ぐために分離したままにします。 </li><li>治癒期間の後、安楽死のために承認された手順に従ってください。固定および分析のために胸郭を削除します。 </li></ol>

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The authors have nothing to disclose

最初のこのプロトコルを学習するとき、どこに挑戦することができます最初の切開を見つけるには、決定する。しかし、安楽死させたマウスでの練習は、外科医が最初の切開を置き、切​​除されるべき所望のリブを公開する場所を学​​ぶことができます。死体に作業することも骨膜の有無にかかわらずリブ部分を除去するために必要な細かい運動能力を向上させます。また、この手順に?...

骨格傷害、慢性変形性関節症を衰弱させる、と再建手術に関連した重大な問題は、経済生産性、家族の幸福、そして生活の質に影響を与える。小休憩と病変がかなりよく癒すことができますが、人間は大きな欠陥を修復することができないため、構造と機能を回復するために再建手続きに頼らなければならない。復興は、同種またはheterogeneic移植片、morcellized骨、移植された足場、または伸延骨形成を伴ってもよい。残念ながら、だけでなく、これらの治療法はなく、修復骨の本来の強さはほとんど達成されていないに関連した永続的な罹患率の要因があります。このため、新たな臨床アプローチが必要とされている。 分節欠損を治療するための革新的な方法を開発する一方向は、大規模な修復が天然に存在する状況を研究することである。哺乳類が目に限定されると考えている間に両生類は有名、骨格要素を再生成することができます能力である。しかし、20 世紀の初頭以来、人間のリブにおける再生の報告はほとんど人間が1-4そのように限定されないかもしれないことを示唆して発表されている。現在、この現象は、最高の顎、顔、耳再構成のために隔壁材料を使用する外科医に知られているが、それは、より広く5が認識されない。より詳細には、この修復を研究するために、我々は、マウスを使用して、外科的モデルを開発した。このプロトコルを使用して、研究者は、関連する生来の因子を特定し、他の場所で骨治癒を促進するためにこの情報を使用することができる。 骨修復を研究するためのモデルとしてのリブを使用することには多くの利点がある。 （大腿骨6,7の切除と比較して）まず、周囲のリブは自然な固定器を提供する。これは、内部​​と外部固定の罹患リスクを減少させ、手術手順を簡素化します。胸のWAの第二に、薄い筋肉層LL頭蓋冠切除8の利便性の検定に匹敵する簡単なアクセスと優れた視認性を提供する。第三に、膜内骨化によってその形、軟骨内骨化によってリブ形と中央骨幹のいずれかの端部に位置する成長板に拡張を介して長さに成長する頭蓋冠とは対照的である。そのため、リブの修理は、四肢骨格の長骨の修理により匹敵し得る。さらに、我々は、大腿骨と比べて、リブの骨膜が厚く、より簡単に操作することができることを見出した。このように、骨膜を研究または細胞治療、薬理学的薬剤、および/または組織足場をテストする目的で、骨修復を検定したい研究者らは、この外科的モデルが役に立つでしょう。要約すると、このリブ切除モデルは、一般的に使用されるそのようなモデルは、現在存在しないように、哺乳動物において天然の大規模骨修復を研究するためにその中にコンテキストを提供する。

<table border="0" cellpadding="0" cellspacing="0" width="896">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Name of Reagent/ Equipment</td>
			<td style="width:160px;">Company</td>
			<td style="width:157px;">Catalog Number</td>
			<td style="width:304px;">Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:275px;">Medium sized micro-dissection scissors (Vannas-T&#252;bingen Spring Scissors 5 mm)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Fine micro-dissection scissors (Vannas Spring Scissors - 2mm Cutting Edge)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">15000-04</td>
			<td>curved tip is beneficial</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Micro-scalpel 5.0 mm</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>10315-12</td>
			<td>other fine scalpels can be substituted</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Dumont 55 forceps</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">11295-51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Retractor&#160;</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td>17004-05</td>
			<td>adjustability is convenient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Micro-needle holders</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">12060-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">9.0 nylon sutures (Ethilon), taper point best</td>
			<td style="width:160px;">Ethicon</td>
			<td>2819G or similar</td>
			<td>taper point best but reverse cutting is also good</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">7.0 prolene sutures (Prolene)</td>
			<td style="width:160px;">Ethicon</td>
			<td>8700H or similar</td>
			<td>6-0 can be used too, needle point can vary</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Large forceps (Adson Forceps)</td>
			<td style="width:160px;">Fine Science Tools</td>
			<td style="width:157px;">11006-12</td>
			<td>other brands are fine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Lubricant Eye Ointment (Akwa Tears)</td>
			<td style="width:160px;">Akorn</td>
			<td style="width:157px;">17478-062-35</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:275px;">Suture glue (GLUture Topical Tissue Adhesive)</td>
			<td style="width:160px;">Abbot</td>
			<td style="width:157px;">32046-01</td>
			<td>has excellent working time</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Shaver</td>
			<td style="width:160px;">Wahl</td>
			<td>9918-6171 or similar</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Clamp lamp</td>
			<td>Zoo Med</td>
			<td style="width:157px;">LF-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Infrared Bulb, 75W</td>
			<td style="width:160px;">Zoo Med</td>
			<td>RS-75</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:275px;">RC2 Rodent Anesthesia System</td>
			<td style="width:160px;">VetEquip&#160;</td>
			<td style="width:157px;">922100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">IsoFlo (Isoflurane)</td>
			<td style="width:160px;">Abbot</td>
			<td style="width:157px;">05260-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Buprenorphine (Buprenex)</td>
			<td style="width:160px;">Reckitt Benckiser</td>
			<td style="width:157px;">12496-0757-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Betadine</td>
			<td style="width:160px;">Purdue Frederick</td>
			<td>67618015017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:275px;">Flavored Gelatin, raspberry</td>
			<td style="width:160px;">Jell-O</td>
			<td>B000E1FYL0</td>
			<td>made up firm, to the consistency of &#39;jigglers&#39;</td>
		</tr>
	</tbody>
</table>

a surgical procedure for resecting the mouse rib: a model for large-scale long bone repair

このプロトコルは、マウスのリブを利用した大規模な骨修復のための新しいモデルに研究者を紹介しています。 、周囲の肋間筋から目的の肋骨を露出させ、胸部体壁を開いて気胸を誘発することなく、リブの所望の部分を切除し、切開部を閉じる、手術のための動物の準備：手順は、以下の詳細を示します。四肢骨格の骨と比較すると、リブは非常にアクセス可能です。また、全く内部または外部固定器は、隣接するリブは自然な固定を提供するので必要ありません。手術は、市販品を使用して、学ぶことは簡単であり、そして動物によって十分に許容さ。手順は、または周囲の骨膜を除去することなく行うことができるので、修理の骨膜の寄与を評価することができる。 2ヶ月 - 結果は、骨膜が保持されている場合、強固な修復が1で発生したことを示している。私たちは、このプロトコルの使用が期待リブの補修に及び所見は体の周りの他の場所で骨修復を刺激するための新しい方法の開発を容易にするような研究を刺激する。

動物は、一般的に切開がうまく癒す、このプロシージャからすぐに回復し、動物は正常な挙動を示す。手順は、市販品を使用して練習した後、1時間の下で完了させることができる。実験データを生成するために、この手順を使用する前に、任意の大きい骨片を切除ゾーン内に残っているかどうかを判断するために、0日目の時点で切除を分析することが重要である。これは、典型的には、特?...

Watch this Scientific Journal Video about A Surgical Procedure for Resecting the Mouse Rib: A Model for Large-Scale Long Bone Repair at JoVE.com

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

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 ...

a-surgical-procedure-for-resecting-mouse-rib-model-for-large-scale

Research

JoVE Journal

Medicine

13.0K ビュー.  Keck School of Medicine, University of Southern California. 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.

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

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 ...

医学

Surgical Technique for Spinal Cord Delivery of Therapies: Demonstration of Procedure in Gottingen Minipigs

This is a compact visual description of a combination of surgical technique and device for the delivery of (gene and cell) therapies into the spinal cord. While the technique is demonstrated in the animal, the procedure is FDA-approved and currently being used for stem cell transplantation into the spinal cords of patients with ALS. While the FDA has recognized proof-of-principle data on therapeutic efficacy in highly characterized rodent models, the use of large animals is considered critical for validating the combination of a surgical procedure, a device, and the safety of a final therapy for human use. The size, anatomy, and general vulnerability of the spine and spinal cord of the swine are recognized to better model the human. Moreover, the surgical process of exposing and manipulating the spinal cord as well as closing the wound in the pig is virtually indistinguishable from the human. We believe that the healthy pig model represents a critical first step in the study of procedural safety.

Short visual description of the surgical technique and device used for the delivery of (gene and cell) therapies into the spinal cord. The technique is demonstrated in the animal but is entirely translatable and currently being used for human application.

セラピーの脊髄配達のための外科テクニック：ゲッティンゲンミニブタの手順のデモンストレーション

This is a  compact visual description of a combination of surgical technique and device  for the  delivery of (gene and cell) therapies into the spinal ...

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.

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 ...

The Mouse Round-window Approach for Ototoxic Agent Delivery: A Rapid and Reliable Technique for Inducing Cochlear Cell Degeneration

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research involving the cochlea and its associated neural pathways entails exposure of model cochleae to a variety of ototoxic agents. This allows investigators to study the effects of targeted damage to cochlear structures, and in some cases, the self-repair or regeneration of those structures. Various techniques exist for delivery of ototoxic agents to the cochlea. When selecting a particular technique, investigators must consider a number of factors, including the induction of inadvertent systemic toxicity, the amount of cochlear damage produced by the surgical procedure itself, the type of lesion desired, animal survivability, and reproducibility/reliability of results. Currently established techniques include parenteral injection, intra-peritoneal injection, trans-tympanic injection, endolymphatic sac injection, and cochleostomy with perilymphatic perfusion. Each of these methods has been successfully utilized and is well described in the literature; yet, each has various shortcomings. Here, we present a technique for topical application of ototoxic agents directly to the round window niche. This technique is non-invasive to inner ear structures, produces rapid onset of reliably targeted lesions, avoids systemic toxicity, and allows for an intra-animal control (the contra-lateral ear). Results stemming from this approach have helped deeper understanding of auditory pathophysiology, cochlear cell degeneration, and regenerative capacity in response to an acute injury. Future investigations may use this method to conduct interventional studies involving gene therapy and stem cell transplantation to combat hearing loss.

Various methods exist for introducing ototoxic agents to the cochleae of animal models. Presented is a surgical protocol for delivery of ototoxic agents to the round window niche. The procedure is reliable, creates targeted intra-cochlear lesions, and avoids mechanical damage to the microarchitecture. Examination of cochlear self-repair/regeneration is possible.

蝸牛細胞の変性を誘導するための迅速かつ信頼性の高いテクニック：耳毒性剤送達のためのマウスラウンドウィンドウアプローチ

Investigators have utilized a wide array of animal models and investigative techniques to study the mammalian auditory system. Much of the basic research ...

A Novel Murine Model of Arteriovenous Fistula Failure: The Surgical Procedure in Detail

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the exact pathophysiology remains unknown. In order to unravel the pathophysiology a murine model of AVF-failure was developed in which the configuration of the anastomosis resembles the preferred situation in the clinical setting. A model was described in which an AVF is created by connecting the venous end of the branch of the external jugular vein to the side of the common carotid artery using interrupted sutures. At a histological level, we observed progressive stenotic intimal lesions in the venous outflow tract that is also seen in failed human AVFs. Although this procedure can be technically challenging due to the small dimensions of the animal, we were able to achieve a surgical success rate of 97% after sufficient training. The key advantage of a murine model is the availability of transgenic animals. In view of the different proposed mechanisms that are responsible for AVF failure, disabling genes that might play a role in vascular remodeling can help us to unravel the complex pathophysiology of AVF failure.

Here we present a murine model of arteriovenous fistula (AVF) failure in which a clinically relevant anastomotic configuration is incorporated. This model can be used to study the pathophysiology and to test possible therapeutic interventions.

動静脈瘻の障害の新規マウスモデル：詳細の外科的手順

The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient remodeling and intimal hyperplasia from which the ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

豚脛骨移植外科的血管新生: 新しい大型動物の骨血管複合移植モデル

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Evaluating the Procedure for Performing Awake Cystometry in a Mouse Model

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among laboratories. The goal of this study was to describe the microsurgical procedure used to implant an intravesical tube and the experimental technique for recording urinary bladder pressure in an awake, freely moving mouse. In addition, experimental data is presented to show how surgery, as well as tubing type and size, affect lower urinary tract function and recording sensitivity. The effect of tube diameter on pressure recording was assessed in both polyethylene and polyurethane tubing with different internal diameters. Subsequently, the best performing tube from both materials was surgically implanted into the dome of the urinary bladder of male C57BL/6 mice. Twelve-hour, overnight micturition frequency was recorded in healthy, intact animals and animals 2, 3, 5, and 7 days post-surgery. At harvest, bladders were assessed for signs of swelling using gross observation and were subsequently processed for pathological analysis. The greatest extent of bladder swelling was observed on day 2 and 3, which correlated with behavioral voiding data showing significantly impaired bladder function. By day 5, bladder histology and voiding frequency had normalized. Based on the literature and evidence provided by our studies, we propose the following steps for in vivo recording of intravesical pressure and voided volume in an awake mouse: 1) Perform the surgery using an operating microscope and microsurgical tools, 2) Use polyethylene-10 tubing to minimize movement artifacts, and 3) Perform cystometry on post-operative day 5, when bladder swelling resolves.

This study describes the surgical procedures and experimental techniques for performing awake cystometry in a freely moving mouse. In addition, it provides experimental evidence to support its optimization and standardization.

マウスモデルにおける覚醒時膀胱内圧測定の実施手順の評価

Awake filling cystometry has been used for a long time to evaluate bladder function in freely moving mice, however, the specific methods used, vary among ...

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset (Callithrix jacchus)

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset (Callithrix jacchus) is a small, easy-to-handle New World monkey which has a similar anatomy of the temporal bone, including the middle ear ossicular chains and inner ear to humans, than in comparison with that of rodents. Here, we report a reproducible, safe, and rational surgical approach to the cochlear round window niche for the drug delivery to the inner ear of the common marmoset. We adopted posterior tympanotomy, a procedure used clinically in human surgery, to avoid manipulation of the tympanic membrane that may cause conductive hearing loss. This surgical procedure did not lead to any significant hearing loss. This approach was possible due to the large bulla structure of the common marmoset, although the lateral semicircular canal and vertical portion of the facial nerve should be carefully considered. This surgical method allows us to perform the safe and accurate administration of drugs without hearing loss, which is of great importance in obtaining pre-clinical proof of concept for translational research.

A Surgical Procedure for the Administration of Drugs to the Inner Ear in a Non-Human Primate Common Marmoset Callithrix jacchus

We report a surgical method to administrate drugs to the inner ear of a non-human primate, the common marmoset (Callithrix jacchus), via the round window membrane.

ヒト以外の霊長類コモンマーモ (用いた) で内耳に薬の管理術

Hearing research has long been facilitated by rodent models, although in some diseases, human symptoms cannot be recapitulated. The common marmoset ...

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 ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

外科用マウスモデルにおける変形性関節症の標準化組織形態評価

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...