著者らは、手術法の支援のためにAdrianeモズレーとLibuse Jerabek（スタンフォード）に感謝したいと思います。

すべての動物実験は、実験動物の管理と使用に関するUCLA​​の動物管理使用委員会のガイドラインと健康ガイドの国立研究所に従って行った。以下の手順の持続時間は、最初から最後まで約45〜60分である。 1。手術野の調製<ol><li>きれいな動物の手術室での手順を実行します。 </li><li>設備：イソフルラン気化、加熱パッド付きGaymer Tポンプ。 </li><li>無菌ツール：2湾曲したピンセット、細いハサミ、針ホルダ。 </li><li>滅菌手袋は全体の手順の間に使用されなければならない。 </li></ol> 2。動物の作製<ol><li>同じケージに同じ遺伝的背景から、同じような重さと大きさで、2雄または雌のマウスを配置し、調和のとれた共存を保証するために、少なくとも2週間を監視。雌マウスは、それらのより少なく積極的な行動のために好ましい。 </li><li>することにより、動物を麻酔イソフルラン気化器を使用する。ポジ·シール誘導チャンバ内でマウスをイソフルラン気化器（4-5％のV / V）に接続されている。麻酔が誘発されると、毛皮のシェービングエリアに動物を転送し、イソフルラン（1.5から2パーセントのV / V）に接続されているノーズコーンを通して作業中は麻酔を維持。ドライアイを防ぐために、Q-ティップで眼軟膏を適用します。 </li><li>仰臥位で動物を置きます。完全に右が少なくとも約1cmの1センチメートル膝下肘の上に載置された出発マウスの左右側に配置されたマウスの左側を剃る。 </li><li>無菌的に徹底的にアルコール綿続いベタジンに浸したワイプで（2〜3倍）を拭いて剃毛エリアを用意。無菌パッドでカバー加熱パッドの上にマウスを置きます。 </li><li>鎮痛のため、それぞれ10 mg / kgの0.1ミリグラム/ kgの用量で腹腔内または皮下にカルプロフェンおよびブプレノルフィンを管理します。 </li><li>で、背中合わせに、彼らの側に動物を配置上に向け剃毛分野をdjacent。手術領域の汚染を避けるため、操作領域を露出無菌ドレープでマウスを覆う。手術を行う際に、滅菌滞在する小さなドレープ開口部を作成します。私たちは、ビデオ撮影時に見やすくを持つようにドレープウィンドウを大きく。 </li></ol> 3。並体結合<ol><li>鋭いはさみを使用して、肘0.5cm上で膝関節下の0.5センチメートル（ 図1）へのすべての道を開始し、各動物の剃毛側面に縦方向の皮膚切開を行う。切開後、緩やかに湾曲鉗子で皮膚をかざして、皮​​下筋膜から皮膚をデタッチし、自由な皮膚の0.5cmに作成するために二対の筋膜を分離する。全体の切開に沿って、この分離を行う。 </li><li>他の右肘に1動物の左肘を取り付けることにより、接合を開始します。 olecranonsと膝関節の両方が目以下のはっきりと区別することができるEの皮膚切開。入社を容易にするために、最初のマウスの肘を曲げ肘頭下の非吸収性3-0縫合糸の針を渡します。同様に、第二のマウスの肘を曲げ、その下に、同じ縫合糸を通す。ダブル外科結びでしっかりとジョイントを取り付けます。 </li><li>同様の手順で膝関節を接続します。 </li><li>ジョイントの取り付けに続いて、ひざに向かって肘から腹始まる連続吸収5-0ビクリル縫合糸により2動物の皮膚に接続します。皮膚の破裂や剥離を防止する肘と膝の周りの領域における皮膚のタイトな縫合閉鎖を行う。腹側皮膚添付ファイルが完了すると、二重の手術結び目を実行します。腹臥位にマウスを置いて、二重の外科結びで終わる縫合背を続ける。縫合糸の連続性を確認して、開口部がないことを確認する。 </li><li> dehyを防ぐために、各マウスの皮下に0.9％NaClを0.5ミリリットルの管理dration。 </li></ol> 4。術後の回復<ol><li>回復するまで加熱されたパッドの上に動物を保管してください。 </li><li>回復後、鎮痛剤のカルプロフェンとブプレノルフィンを提供しています。 （ステップ2.5）は、上記と同じ投与量で48時間、それぞれ、腹腔内または皮下に、24および12時間後に繰り返します。このような揺れなどの痛みや苦痛の徴候のために動物を監視し、尾の無気力、咀嚼は、 などグルーミングの欠如は、2週間毎日、バックアーチ。 </li><li>予防的に、細菌感染を防ぐために、10日間ミリリットル/トリムミリグラム2mgのサルファ剤/ mlの0.4彼らのウォーターボトルにスルファメトキサゾール/トリメトプリム経口懸濁液をマウスに扱う。 </li><li>家の寝具材料の吸引を防ぐために、モノリシックの寝具材料（ 例えば 、紙タオルや吸収性の滅菌パッド）できれいにケージの各並体結合ペア。彼らは彼らの頭部を上にして胸骨横臥位を維持することができるとき、寝具いっぱいケージに動物を返します。歪みを最小限に抑えるために、並体結合の存在に調整しながら食品に手を伸ばすの、ケージの床に湿らせた食物ペレットを配置します。ネスティング材料を提供する。 1〜2週間で並体結合マウスは、外科的に対になっ前後と後肢で正常に歩き回るする能力を持っている。 </li><li>血液キメラ現象は、手術後10〜14日に行われます。 </li></ol> 5。確認と逆の手順<ol><li> 2週間の血液キメラ現象（ 図3）を確認するために、フローサイトメトリー分析のために各マウスの尾静脈から血液を引き出す後</li><li>フローサイトメトリー分析のための血液処理：10 mMのEDTAを500μlの血液を2〜3滴を希釈。 （PBSで希釈）の2％デキストランの500μl加え、完全に混合し、30分間37℃でインキュベートする。 、新しいチューブに上清を移し、5分間1,200 rpmでスピンし、フローサイトメトリー解析のために、PBS中のペレット化した細胞（または他のバッファ）懸濁します。 </li><li>実験設計に応じて、並体結合対は分離することができる後の時点でトン。我々の経験では、合併症のない最長9ヶ月間parabiosedペアを維持している。 </li><li>無菌外科表面に逆の手順を実行します。イソフルラン気化器（4-5％）に接続された導入室に配置することによってマウスを麻酔し（ステップ2.2）上記のように1.5〜2％イソフルランで麻酔を維持する。 </li><li> （2.3、2.4ステップ）初期縫合糸を周囲から髪を削除して、無菌的に上記のように剃らエリアを用意。 </li><li>鎮痛のために、それぞれ、10 mg / kgの0.1ミリグラム/ kgの用量で腹腔内または皮下カルプロフェン及びブプレノルフィンを投与する。 </li><li> （ステップ2.6）、上記のように、滅菌ドレープと手術領域をカバーしています。 </li><li>鋭いハサミを使用して、横方向の縫合糸に沿って縦切開を通してマウスを分離し、緩やかに湾曲鉗子のペアで2マウスとの間、新たに形成された筋膜を切り離す。関節を分離するために、縫合糸の結び目をカットそれらを接続する。滑らかなエッジを実現し、吸収性、連続5-0コーティングされたビクリル縫合糸で皮膚を再接続するために切開に沿って皮を切り落とす。 </li><li>脱水症を防ぐために、各マウスに0.9％のNaCl、皮下0.5mlのを投与。回復するまで加熱されたパッドの上に動物を保管してください。 </li><li>回復後、鎮痛剤を投与することを繰り返して、カルプロフェン（10 mg / kg）を24時間毎とブプレノルフィン（0.1 mg / kg）を48時間ごとに12時間。 </li><li>細菌感染を防止するために10日間、彼らのウォーターボトルに（2mgのサルファ剤/ mlの0.4 mg / mlのトリム）スルファメトキサゾール/トリメトプリム経口懸濁液をマウスに扱う。 </li></ol>

<ol>
	<li>Bert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exp&#233;riences+et+consid&#233;rations+sur+la+greffe+animale.">Exp&#233;riences et consid&#233;rations sur la greffe animale.</a> Journal de l'Anatomie et de la Physiologie. 1, 69-87 .</li><li>Bunster, E., Meyer, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+of+parabiosis.">Improved methods of parabiosis.</a> Anat. Rec. 57, 339-380 (1933).</li><li>Waskow, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+parabiotic+mice+for+the+study+of+DC+and+DC+precursor+circulation.">Generation of parabiotic mice for the study of DC and DC precursor circulation.</a> Methods Mol. Biol. 595, 413-428 (2010).</li><li>Finerty, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parabiosis+in+physiological+studies.">Parabiosis in physiological studies.</a> Physiol. Rev. 32, 277-302 (1952).</li><li>Aicher, A., Heeschen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonbone+marrow-derived+endothelial+progenitor+cells:+what+is+their+exact+location.">Nonbone marrow-derived endothelial progenitor cells: what is their exact location.</a> Circ. Res. 101, e102 (2007).</li><li>Abe, S., Boyer, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cells+derived+from+the+circulation+contribute+to+the+repair+of+lung+injury.">Cells derived from the circulation contribute to the repair of lung injury.</a> Am. J. Respir. Crit. Care Med. 170, 1158-1163 (2004).</li><li>Donskoy, E., Goldschneider, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymocytopoiesis+is+maintained+by+blood-borne+precursors+throughout+postnatal+life.+A+study+in+parabiotic+mice.">Thymocytopoiesis is maintained by blood-borne precursors throughout postnatal life. A study in parabiotic mice.</a> J. Immunol. 148, 1604-1612 (1992).</li><li>Powell, A. E., Anderson, E. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusion+between+Intestinal+epithelial+cells+and+macrophages+in+a+cancer+context+results+in+nuclear+reprogramming.">Fusion between Intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming.</a> Cancer Res. 71, 1497-1505 (2011).</li><li>Duyverman, A. M., Kohno, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transient+parabiosis+skin+transplantation+model+in+mice.">A transient parabiosis skin transplantation model in mice.</a> Nat. Protoc. 7, 763-770 (2012).</li><li>Ajami, B., Bennett, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+self-renewal+can+sustain+CNS+microglia+maintenance+and+function+throughout+adult+life.">Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.</a> Nat. Neurosci. 10, 1538-1543 (2007).</li><li>Weissman, I. L., Jerabek, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+and+the+H-Y+antigen:+Requirement+for+male+T+cells,+but+not+B+cells,+to+induce+tolerance+in+neonatal+female+mice.">Tolerance and the H-Y antigen: Requirement for male T cells, but not B cells, to induce tolerance in neonatal female mice.</a> Transplantation. 37, 3-6 (1984).</li></ol>

著者らは、競合する金融利害関係はありません。

ここで説明並体結合の方法は、低死亡率の最低限の技術的な問題と結果を示す。膝や肘の関節の取り付けはBunsterとマイヤー技術の大幅な改善である。しかし、手順はこのように、手術を通して無菌状態の維持が不可欠である侵襲性のままになります。さらに、手術部位の感染を防止するためには、parabiosed動物は抗生物質の組み合わせを受信し、定期的に監視することが重要である。しっか?...

並体結合、2生物の外科的接合は、最初に共有循環系を研究するためのモデルを開発するための方法として、ポール·バートによって1864に記載され、2匹のラット1の皮膚や筋肉の壁の接合で構成された。並体結合は、炎症3部位での微小血管の形成を促進し、このような下垂体および生殖腺だけでなく、高血圧4における腎臓の役割の間のホルモン通信などの生理学的研究にはいくつかのアプリケーションを、持っていた。さらに、血管新生5、造血幹細胞6の移動、およびリンパ球輸送7、ならびに腫瘍転移8,9における炎症性または幹細胞を循環させる役割と動態に前駆細胞の動員および組込みを調べるために使用されているおよび神経変性疾患10。 並体結合の嘘の一つの重要な利点その中で提携した動物は、免疫学的反応を誘発することなく、細胞移動および血管新生を可能にする一般的な循環抗原を共有しています。重要なことには、ワイズマンらは、雄と雌のマウス間の並体結合は、抗HY抗体11の形成をもたらさない示した。 ポール·バートによって記述、元のプロトコルでは、2匹の動物は、皮膚と筋肉の壁1の接続を介して接合した。しかしながら、この方法は、動物に重大な歪みを引き起こし、傷口の感染による高い死亡率となった。それ以来、並体結合技術が最も優勢が1933 2でBunsterとマイヤーによって提案されたプロトコルであることをいくつかのグループにより改訂されました。彼らの方法は、動物のためのより良いサポートが少なく、痛みを許可する、肩甲骨の関節、体腔と皮膚の接合含まれています。同時に、新しい方法は、最小限の術後ケアとsignifiの結果cantlyは死亡率を減少させた。本明細書に記載されたプロトコルは、低侵襲性であり、強固な接合が可能とBunsterマイヤー法の改良法である。すなわち、マウスは肘と膝関節、並びに皮膚を介して接続されている。したがって、これは皮膚のを防止拡張子を結合し、痛みが少なく、合併症を引き起こす。ここでは、マウスを表現する構成的GFPに野生型（WT）成体マウスの接合について説明します。私たちは、手術後2週間は、我々が共有循環系を作成するために、この外科処置の有効性を実証する血液キメラの50％を達成できることを示している。

<table border="1">
	<tbody>
		<tr>
			<td>Name of equipment</td>
			<td>Company</td>
			<td>Catalog number</td>
		</tr>
		<tr>
			<td>Isoflurane-Phoenix</td>
			<td>Clipper</td>
			<td>NDC 57319-559-06</td>
		</tr>
		<tr>
			<td>Posi-Seal Induction Chamber</td>
			<td>Molecular Imaging Products</td>
			<td>AS-01-0530-SM</td>
		</tr>
		<tr>
			<td>Portable Anesthesia System</td>
			<td>Molecular Imaging Products</td>
			<td>AS-01-0007</td>
		</tr>
		<tr>
			<td>Gaymer T Pump</td>
			<td>Gaymar Industries, Inc</td>
			<td>TP650</td>
		</tr>
		<tr>
			<td>Warming Blanket (Heating pad)</td>
			<td>Kent Scientific Corp</td>
			<td>TP-22G</td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Roboz</td>
			<td>RS-5101</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools (FST)</td>
			<td>FST 14063-09</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>FST</td>
			<td>FST 12501-13</td>
		</tr>
		<tr>
			<td>Electrical shaver</td>
			<td>Oster</td>
			<td>Golden A5</td>
		</tr>
	</tbody>
</table>

parabiosis in mice: a detailed protocol

並体結合は、血液循環の共有を可能にする2つの生物の外科的和集合である。 2動物の皮膚を取り付けることは、炎症部位での微小血管の形成を促進する。並体結合パートナーは、循環抗原を共有するため、有害な免疫反応の自由である。まず1864年1でポール·バートによって記述、並体結合手術は動物の生存2を改善するために1933年にBunsterとマイヤーによって精製した。現在のプロトコルでは、2匹のマウスを外科Bunsterとマイヤー法の改良法以下の接合されている。動物は縫合し、皮膚に負担を防ぐ強固なサポートを可能にする皮膚の取り付けに続いて、肘と膝関節を介して接続されている。ここで、我々は詳細に野生型（WT）マウスにマウスを表現するユビキタスGFPの合流並体結合を記述します。血液循環液の循環に二週間処置後、ペアを分離し、GFP陽性細胞をフローサイトメトリー分析によって検出することができるWTマウスのイオン。血液キメラ現象は、一方が他方にある1動物からの循環細胞の寄与を調べることができます。

2の生物の並体結合の予想結果は、一般的な血液循環（ 図2）に、それぞれの動物の循環系の等しい貢献である。一つは、簡単にフローサイトメトリー分析によってparabiosed WT及びGFP陽性マウスの血液の正常な平衡を確認することができます。ここでは、静脈血を、手術後2週間目に両方parabiontsの尾から得られ、（赤血球を除くために）、末梢血細胞を分画した。分画された造血由?...

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Parabiosis in Mice: A Detailed Protocol

2つの生物の結合並体結合は、共有循環系の開発につながる。このプロトコルでは、野生型マウスと恒常的GFP発現マウスとの間の並体結合接続を形成するための手術ステップを記載している。

マウスの並体結合：詳細なプロトコル

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

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73.7K ビュー.  University of California, Los Angeles.  2つの生物の結合並体結合は、共有循環系の開発につながる。このプロトコルでは、野生型マウスと恒常的GFP発現マウスとの間の並体結合接続を形成するための手術ステップを記載している。 

ビデオ: Parabiosis in Mice: A Detailed Protocol

The Trier Social Stress Test Protocol for Inducing Psychological Stress

This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1 Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2 In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3 These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3 Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3 Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4 In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1 Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1

This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.

心理的ストレスを誘導するためのトリーアの社会ストレステストプロトコル

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

医学

Protocol for Long Duration Whole Body Hyperthermia in Mice

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body temperature that is observed during fever. The use of hyperthermia as an adjuvant has emerged as a promising procedure for tumor regression in the field of cancer biology. For this purpose, the most important requirement is to have reliable and uniform heating protocols. We have developed a protocol for hyperthermia (whole body) in mice. In this protocol, animals are exposed to cycles of hyperthermia for 90 min followed by a rest period of 15 min. During this period mice have easy access to food and water. High body temperature spikes in the mice during first few hyperthermia exposure cycles are prevented by immobilizing the animal. Additionally, normal saline is administered in first few cycles to minimize the effects of dehydration. This protocol can simulate fever like conditions in mice up to 12-24 hr. We have used 8-12 weeks old BALB/Cj female mice to demonstrate the protocol.

This paper describes a protocol for whole body hyperthermia in mice that can stimulate fever like conditions up to 12-24 hr.

マウスの長い持続時間の全身温熱療法のためのプロトコル

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

ヒト膵臓の収集プロトコル

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

マウスの簡単な重要なサイズの大腿欠陥モデル

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Heterotopic Renal Autotransplantation in a Porcine Model: A Step-by-Step Protocol

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality of life when compared to dialysis. Although the last few decades have seen major improvements in patient outcomes following kidney transplantation, the increasing shortage of available organs represents a severe problem worldwide. To expand the donor pool, marginal kidney grafts recovered from extended criteria donors (ECD) or donated after circulatory death (DCD) are now accepted for transplantation. To further improve the postoperative outcome of these marginal grafts, research must focus on new therapeutic approaches such as alternative preservation techniques, immunomodulation, gene transfer, and stem cell administration.
Experimental studies in animal models are the final step before newly developed techniques can be translated into clinical practice. Porcine kidney transplantation is an excellent model of human transplantation and allows investigation of novel approaches. The major advantage of the porcine model is its anatomical and physiological similarity to the human body, which facilitates the rapid translation of new findings to clinical trials. This article offers a surgical step-by-step protocol for an autotransplantation model and highlights key factors to ensure experimental success. Adequate pre- and postoperative housing, attentive anesthesia, and consistent surgical techniques result in favorable postoperative outcomes. Resection of the contralateral native kidney provides the opportunity to assess post-transplant graft function. The placement of venous and urinary catheters and the use of metabolic cages allow further detailed evaluation. For long-term follow-up studies and investigation of alternative graft preservation techniques, autotransplantation models are superior to allotransplantation models, as they avoid the confounding bias posed by rejection and immunosuppressive medication.

Porcine models of organ transplantation provide an important platform to study mechanisms of organ preservation. This article describes a heterotopic porcine renal autotransplantation model, which allows investigating new approaches to improve the outcome of transplantation using marginal kidney grafts.

ブタモデルにおける異自家腎移植：ステップバイステップのプロトコル

Kidney transplantation is the treatment of choice for patients suffering from end-stage renal disease. It offers better life expectancy and higher quality ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

ウサギにおける網膜下手術のためのステッププロトコルによってステップ

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Creation of Abdominal Adhesions in Mice

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or intraabdominal infection. The precise mechanisms underlying adhesion formation are poorly understood. Many compounds and physical barriers have been tested for their ability to prevent adhesions after surgery with varying levels of success. The mouse and rat are important models for the study of abdominal adhesions. Several different techniques for the creation of adhesions in the mouse and rat exist in the literature. Here we describe a protocol utilizing abrasion of the cecum with sandpaper and sutures placed in the right abdominal sidewall. The mouse is anesthetized and the abdomen is prepped. A midline laparotomy is created and the cecum is identified. Sandpaper is used to gently abrade the surface of the cecum. Next, several figure-of-eight sutures are placed into the peritoneum of the right abdominal sidewall. The abdominal cavity is irrigated, a small amount of starch is applied, and the incision is closed. We have found that this technique produces the most consistent adhesions with the lowest mortality rate.

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

マウスの腹部癒着の作成

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

汗は小説、小型、ワイヤレスデバイスを使用した監視するための詳細なプロトコル

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

A Detailed Protocol for Physiological Parameters Acquisition and Analysis in Neurosurgical Critical Patients

Intracranial pressure (ICP) monitoring is now widely used in neurosurgical critical patients. Besides mean ICP value, the ICP derived parameters such as ICP waveform, amplitude of pulse (AMP), the correlation of ICP amplitude and ICP mean (RAP), pressure reactivity index (PRx), ICP and arterial blood pressure (ABP) wave amplitude correlation (IAAC), and so on, can reflect intracranial status, predict prognosis, and can also be used as guidance of proper treatment. However, most of the clinicians focus only on the mean ICP value while ignoring these parameters because of the limitations of the current devices. We have recently developed a multimodality monitoring system to address these drawbacks. This portable, user-friendly system will use a data collecting and storing device to continuously acquire patients&#39; physiological parameters first, i.e., ABP, ICP, and oxygen saturation, and then analyze these physiological parameters. We hope that the multimodality monitoring system will be accepted as a key measure to monitor physiological parameters, to analyze the current clinical status, and to predict the prognosis of the neurosurgical critical patients.

Recently, we have developed a portable multimodality monitoring system for the monitoring of various physiological parameters in neurosurgical critical patients. Detailed protocols on how to use this multimodality monitoring system are presented here.

生理学的パラメーターの詳細なプロトコル獲得と脳神経外科の重要な患者の分析

Intracranial pressure (ICP) monitoring is now widely used in neurosurgical critical patients. Besides mean ICP value, the ICP derived parameters such as ...