Name: a preclinical model of exertional heat stroke in mice
Uploaded: 2021-07-01T14:30:00
Description: Watch this Scientific Journal Video about A Preclinical Model of Exertional Heat Stroke in Mice at JoVE.com

この研究は、国防総省W81XWH-15-2-0038(TLC)とBA180078(TLC)とBKとベティ・スティーブンス基金(TLC)によって資金提供されました。JMAはサウジアラビア王国からの資金援助によって支えられた。ミシェル・キングは、この研究が行われた時点でフロリダ大学にいました.現在は、ペプシコ研究開発部門のガトラーデ・スポーツ・サイエンス・インスティテュートに勤務しています。

すべての手順は、フロリダ大学IACUCによって見直され、承認されています.C57BL/6J雄マウスまたは雌マウスは、生後〜4ヶ月、それぞれ27〜34gおよび20〜25gの範囲内で重量を量り、研究に使用される。
1. 遠隔測定温度監視システムの外科的移植
<ol><li>ベンダーから到着すると、動物は、輸送のストレスを最小限に抑えるために、手術前に少なくとも1週間のビバリウムで休む?...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+prior+illness+on+exertional+heat+stroke+presentation+and+outcome.">Influence of prior illness on exertional heat stroke presentation and outcome.</a> PLOS One. 14 (8), 0221329 (2019).</li><li>Carter, R., 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=Epidemiology+of+hospitalizations+and+deaths+from+heat+illness+in+soldiers.">Epidemiology of hospitalizations and deaths from heat illness in soldiers.</a> Medicine and Science in Sports and Exercise. 37 (8), 1338-1344 (2005).</li><li>Howe, A. S., Boden, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-related+illness+in+athletes.">Heat-related illness in athletes.</a> The American Journal of Sports Medicine. 35 (8), 1384-1395 (2007).</li><li>Wallace, R. F., Kriebel, D., Punnett, L., Wegman, D. H., Amoroso, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prior+heat+illness+hospitalization+and+risk+of+early+death.">Prior heat illness hospitalization and risk of early death.</a> Environmental Research. 104 (2), 290-295 (2007).</li><li>Wang, J. -. 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=The+association+between+heat+stroke+and+subsequent+cardiovascular+diseases.">The association between heat stroke and subsequent cardiovascular diseases.</a> PLOS One. 14 (2), 0211386 (2019).</li><li>Leon, L. R., Blaha, M. D., DuBose, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+cytokine,+corticosterone,+and+tissue+injury+responses+in+mice+during+heat+strain+recovery.">Time course of cytokine, corticosterone, and tissue injury responses in mice during heat strain recovery.</a> Journal of Applied Physiology. 100 (4), 1400-1409 (2006).</li><li>Leon, L. R., DuBose, D. A., Mason, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+stress+induces+a+biphasic+thermoregulatory+response+in+mice.">Heat stress induces a biphasic thermoregulatory response in mice.</a> American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 288 (1), 197-204 (2005).</li><li>Leon, L. R., Gordon, C. J., Helwig, B. G., Rufolo, D. M., Blaha, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoregulatory,+behavioral,+and+metabolic+responses+to+heatstroke+in+a+conscious+mouse+model.">Thermoregulatory, behavioral, and metabolic responses to heatstroke in a conscious mouse model.</a> American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 299 (1), 241-248 (2010).</li><li>King, M. A., Leon, L. R., Morse, D. A., Clanton, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+cytokine+and+chemokine+responses+to+exertional+heat+stroke+in+mice.">Unique cytokine and chemokine responses to exertional heat stroke in mice.</a> Journal of Applied Physiology. 122 (2), 296-306 (2016).</li><li>Costa, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=l-Arginine+supplementation+prevents+increases+in+intestinal+permeability+and+bacterial+translocation+in+Male+Swiss+mice+subjected+to+physical+exercise+under+environmental+heat+stress.">l-Arginine supplementation prevents increases in intestinal permeability and bacterial translocation in Male Swiss mice subjected to physical exercise under environmental heat stress.</a> The Journal of Nutrition. 144 (2), 218-223 (2014).</li><li>Hubbard, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+exercise+in+the+heat+on+predisposition+to+heatstroke.">Effects of exercise in the heat on predisposition to heatstroke.</a> Medicine and Science in Sports. 11 (1), 66-71 (1979).</li><li>Hubbard, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+model+of+acute+heatstroke+mortality.">Rat model of acute heatstroke mortality.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 42 (6), 809-816 (1977).</li><li>Hubbard, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+significance+of+selected+serum+enzymes+in+a+rat+heatstroke+model.">Diagnostic significance of selected serum enzymes in a rat heatstroke model.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 46 (2), 334-339 (1979).</li><li>Hubbard, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+physical+effort+in+the+etiology+of+rat+heatstroke+injury+and+mortality.">Role of physical effort in the etiology of rat heatstroke injury and mortality.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 45 (3), 463-468 (1978).</li><li>Garcia, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-dependent+responses+to+exertional+heat+stroke+in+mice.">Sex-dependent responses to exertional heat stroke in mice.</a> Journal of Applied Physiology. 125 (3), 841-849 (2018).</li><li>Garcia, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Ibuprofen+during+Exertional+Heat+Stroke+in+Mice.">Effects of Ibuprofen during Exertional Heat Stroke in Mice.</a> Medicine and Science in Sports and Exercise. 52 (9), 1870-1878 (2020).</li><li>King, M. A., Leon, L. R., Mustico, D. L., Haines, J. M., Clanton, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+multi-organ+injury+in+a+pre-clinical+model+of+exertional+heat+stroke.">Biomarkers of multi-organ injury in a pre-clinical model of exertional heat stroke.</a> Journal of Applied Physiology. 118 (10), (2015).</li><li>Murray, K. O., 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=Exertional+heat+stroke+leads+to+concurrent+long-term+epigenetic+memory,+immunosuppression+and+altered+heat+shock+response+in+female+mice.">Exertional heat stroke leads to concurrent long-term epigenetic memory, immunosuppression and altered heat shock response in female mice.</a> The Journal of Physiology. 599 (1), 119-141 (2021).</li><li>Leon, L. R., DuBose, D. A., Mason, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+stress+induces+a+biphasic+thermoregulatory+response+in+mice.">Heat stress induces a biphasic thermoregulatory response in mice.</a> American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 288, 197-204 (2005).</li><li>Laitano, O., 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=Delayed+metabolic+dysfunction+in+myocardium+following+exertional+heat+stroke+in+mice.">Delayed metabolic dysfunction in myocardium following exertional heat stroke in mice.</a> The Journal of Physiology. 598 (5), 967-985 (2020).</li><li>Iwaniec, J., 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=Acute+phase+response+to+exertional+heat+stroke+in+mice.">Acute phase response to exertional heat stroke in mice.</a> Experimental Physiology. 106 (1), 222-232 (2020).</li><li>He, S. -. X., 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=Optimization+of+a+rhabdomyolysis+model+in+mice+with+exertional+heat+stroke+mouse+model+of+EHS-rhabdomyolysis.">Optimization of a rhabdomyolysis model in mice with exertional heat stroke mouse model of EHS-rhabdomyolysis.</a> Frontiers in Physiology. 11, (2020).</li><li>Lopez, J. R., Kaura, V., Diggle, C. P., Hopkins, P. M., Allen, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+hyperthermia,+environmental+heat+stress,+and+intracellular+calcium+dysregulation+in+a+mouse+model+expressing+the+p.G2435R+variant+of+RYR1.">Malignant hyperthermia, environmental heat stress, and intracellular calcium dysregulation in a mouse model expressing the p.G2435R variant of RYR1.</a> British Journal of Anaesthesia. 121 (4), 953-961 (2018).</li><li>Laitano, O., Murray, K. O., Leon, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+mechanisms+of+exertional+heat+stroke+and+malignant+hyperthermia:+evidence+vs.+conjecture.">Overlapping mechanisms of exertional heat stroke and malignant hyperthermia: evidence vs. conjecture.</a> Sports Medicine. 50 (9), 115-123 (2020).</li><li>Casa, D. J., Armstrong, L. E., Kenny, G. 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この技術レビューは、マウスにおけるEHSの前臨床モデルのパフォーマンスに関するガイドラインを提供することを目的としています。可変的な大等性の再現可能なEHSエピソードの実行に必要な詳細なステップと材料が提供される。重要なことに、このモデルは、ヒトEHSの犠牲者11、19で観察される徴候、症状、および多臓器機能不全を主に模倣する?...

熱中症は、中枢神経系機能障害およびその後の高体温被験者における臓器損傷を特徴とする。熱中症には2つの症状があります。古典的な熱中症(CHS)は、暑い夏の日に太陽にさらされた車に残された熱波や子供の間に主に高齢者の集団に影響を与えます1.運動熱中症(EHS)は、身体運動中に十分に熱調節することができない場合に生じ、通常、常に、神経症状、温熱療法、およびそれに続く多臓器機能不全および損傷をもたらす高い周囲温度の下で2。EHSは、レクリエーションやエリートアスリートだけでなく、軍人や労働者の中で、付随する脱水3、4で発生します。実際、EHSは身体活動中のアスリートの死亡率の第3位の原因である5.エピソードは致命的であるか、長期的な否定的な健康結果につながる可能性がありますので、人間のEHSを研究することは非常に困難です6,7.したがって、EHSの信頼性の高い前臨床モデルは、ヒトEHSの犠牲者における遡及的および連想的臨床観察の限界を克服するための貴重なツールとして役立つ可能性がある。げっ歯類や豚のCHSの前臨床モデルは、8、9、10でよく特徴付けられています。しかし、CHSの前臨床モデルは、体温調節プロファイルおよび自然免疫応答11に対する身体運動の独特の効果のために、EHS病態生理学に直接変換しない。さらに、げっ歯類における前臨床EHSモデルの開発に関する以前の試みは、感電による過重応力刺激、直腸プローブの挿入、および高い死亡率12、13、14、15、16の事前定義された最高コア体温を含む、重要な制限を提起した 現在の疫学的データと一致しない。これらは、データ解釈を混乱させ、信頼性の低いバイオマーカーインデックスを提供する可能性のある重大な制限を表しています。したがって、このプロトコルは、上記の制限からほとんど自由であるマウスにおけるEHSの標準化された、非常に再現性の高い、翻訳可能な前臨床モデルのステップを特徴付け、記述することを目的としている。中等度から致命的な熱中症の結果を段階的に生じ得るモデルの調整について説明する。著者らの知る限りでは、これは、このような特性を持つEHSの唯一の前臨床モデルであり、仮説主導的な方法で関連するEHS研究を追求することを可能にする11、17、18。

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a preclinical model of exertional heat stroke in mice

熱中症は、熱関連疾患の最も深刻な症状です。従来の熱中症(CHS)は、受動的な熱中症としても知られ、安静時に起こるのに対し、運動熱中症(EHS)は身体活動中に起こる。EHSは、多臓器機能障害の病因、臨床プレゼンテーション、および後遺症におけるCHSとは異なる。最近まで、CHSのモデルだけが確立されています。このプロトコルは、麻酔、拘束、直腸プローブ、感電などの主要な制限要因から解放されたEHSの洗練された前臨床マウスモデルのガイドラインを提供することを目的としています。雄および雌のC57Bl/6マウスは、コア温度(Tc)テレメトリプローブを用いたこのモデルに利用された。ランニングモードに慣れるには、マウスは自主的な走輪と強制走行ホイールの両方を使用して3週間のトレーニングを受けます。その後、マウスは、42.1〜42.5°CのTcで症状の制限(意識喪失など)を表示するまで、37.5°Cおよび40%-50%相対湿度(RH)に設定された気候室内の強制ホイールで走るが、34.5〜39.5°Cと湿度30%90°Cの間のチャンバー温度で適切な結果を得ることができる。所望の重症度に応じて、マウスは周囲温度の回復のためにすぐにチャンバーから取り出されるか、またはより長い期間加熱された部屋に留まり、より重度の暴露および死亡率のより高い発生率を誘発する。結果は、シャムマッチエクササイズコントロール(EXC)および/またはナイーブコントロール(NC)と比較されます。このモデルは、意識喪失、重度の温熱療法、多臓器損傷、炎症性サイトカイン放出、免疫系の急性期応答など、ヒトEHSで観察された病態生理学的転帰の多くを反映している。このモデルは、EHSの発症を遅らせたり、この症状を特徴づける多臓器損傷を減らしたりする予防および治療戦略をテストするための仮説主導の研究に最適です。

EHS プロトコル全体およびマウスの早期回復の間の典型的な体温調節プロファイルを 図 1Aに示します。このプロファイルは、チャンバー加熱ステージとして定義できる4つの異なる相、インクリメンタル運動段階、定常運動段階、および急速冷却(R)または重度(S)法17のいずれかによる回復段階を含む。主な体温調節結果は、達成された最大Tc(Tc,max)とTc,max?...

Watch this Scientific Journal Video about A Preclinical Model of Exertional Heat Stroke in Mice at JoVE.com

A Preclinical Model of Exertional Heat Stroke in Mice

このプロトコルは、感電などの有害な外部刺激を含まないマウスにおける運動熱中症(EHS)の標準化された、反復可能な前臨床モデルの開発を記述する。このモデルは、機械治療、予防、および治療研究のためのプラットフォームを提供します。

マウスにおける運動熱中症の前臨床モデル

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, ...

これまでは、熱中症の受動的モデルのみが確立されていました。この運動熱中症のモデルは、大きな制限から解放され、より密接に人間の病理生理学を模倣しています。命にかかわる状態であるため、人間の熱中症を効果的に研究することはできません。この前臨床モデルは、ヒト運動熱中症の臨床的特徴を保持する。この技術は、運動熱中症からの回復に対する冷却速度の影響、薬物摂取からの併存疾患、ならびに熱中症暴露の長期的な影響をテストするのに役立ちます。この手順のデモンストレーションは、クラントン博士の研究室の上級博士課程の学生、ジャマル・アルザラニです。マウスを麻酔した後、獣医軟膏などの目の潤滑油を使用して、手術中の損傷や怪我から動物の目を保護します。手術部位を準備するには、小さな動物の毛バリや市販のヘアリムーバーで下腹部を剃ります。1キログラムブプレノルフィンあたり皮下0.1ミリグラムの最初の用量を投与する。ポビドンヨウ素またはクロルヘキシジンの3回の洗浄で領域をスクラブし、続いて70%イソプロピルアルコールリンス、次にマウスを手術領域に移し、接着剤ドレープを使用してマウス上の外科部位を分離する。無菌技術で無菌器具を使用して、肋間部から約0.5センチメートルのリネアアルバに沿って正線に約1センチメートルの切開を行い、皮膚を筋肉層から分離し、リネアアルバの少し小さな切開を行い、腸や内臓に損傷を与えないように注意する。筋肉層が開いたら、生殖管内の空洞に無菌テレメーターを置き、口蓋内腔と消化器官に後ろ線を入れ、自由に浮かべるようにします。無菌5-O吸収性縫合糸で腹部開口部を閉じ、5-Oプロリン縫合糸で簡単に中断されたステッチを使用して皮膚を閉じます。ケージの下に寄り添うマイクロ波加熱パッドで、きれいなケージにマウスを置きます。麻酔からの回復の最初の時間の間に15分ごとにマウスを監視し、動物の住宅施設に戻ります。運動熱中症のプロトコルの前夜、室温で環境室にマウスを置き、チャンバーに順応させる。連続コア温度またはTCを一晩で30秒間隔で平均収集するために、データ取得システムを使用します。運動熱中症のプロトコルの朝、マウスが温度を上げる前に、マウスが36〜37.5°Cの日次温度の正常範囲以下であることを確認して、マウスが発熱を起こさないようにするか、またはこの期間中に過度のストレスを経験していないことを確認します。マウスが安定し、通常の安静時コア温度の範囲内で、食品と水を除去し、動物の重量を量る。チャンバーのドアを閉め、チャンバー温度を摂氏37.5度、相対湿度40~50%または所望の環境温度と湿度に上げます。温度と湿度を校正したモニターでチャンバーの温度と湿度を確認します。プロトコル中に光と妨害を最小限に抑えるために、遮光カーテンでチャンバーを囲みます。リモートIR照明カメラを介してプロトコル中にマウスを継続的に監視します。2台目のカメラを、走行ホイールの近くに設置した温度と湿度モニターに焦点を合わせます。環境チャンバセットポイントのコントローラを調整して、動物の近くの正確な温度測定値を確保します。チャンバーが温度モニターの2番目のカメラで測定した目標温度に達したら、すぐにチャンバードアを開け、強制的な走行ホイールにマウスを置きます。強制走行ホイールプロトコルを毎分2.5メートルの速度で開始し、マウスが摂氏41度のコア温度に達するまで10分ごとに毎分0.3メートルの速度を上げます。コア温度が達成されると、意識の明らかな喪失、後方転倒または失神、および車輪に対する走り続けや保持を続けることができないことを特徴とする症状の制限が発生するまで、速度を一定に保ちます。マウスが物理的な応答の兆候なしにホイール上で 3 回後方回転を行う時点を確認します。または、ローカルの IACUC ルールに従って人道的エンドポイントを特定し、プロトコルを停止するタイミングを決定します。このエンドポイントは、本質的にすべてのマウスにおける症状の制限をわずかに上回る。急速冷却プロトコルを実行するには、マウスが症状の制限に達したらホイールを停止し、強制実行ホイールからマウスをすぐに取り外します。マウスの重量を量り、室温で回復するためにホームケージに戻します。この間、チャンバードアを開けて室温に戻し、チャンバーが急速に冷却できるようにして、99%以上の長期生存を実現します。より重度の運動熱打撃暴露を行うためには、運動熱打撃プロトコルの間に37.5°Cのチャンバーの中に動物のケージを保つ。動物が症状の限界に達すると、遠隔カメラで観察された意識に戻るまでランニングホイールに残り、走っている車輪からマウスを素早く取り外し、事前に暖めたケージに直接戻して、はるかに遅い冷却プロファイルを生じさせ、本質的に運動熱ストローク低相性位を排除します。この間、ケージからフィルタートップを取り外してチャンバとの平衡を改善します。室温に事前冷却された回復ケージは、生存率が100%の低体温相を抑制し、重度の運動熱度プロトコルで低体温相を抑制し、回復中にマウスを注意深く監視し、回復中の正常な動きを含む人道的エンドポイントを継続的にチェックするために、より深刻な代替手順を実行するために使用できます。 正常な呼吸、そして舐める。この間、コア温度を監視します。マウスは、回復段階でコア温度が方向を逆転し、最終的に摂氏40度を超えると回復する可能性は低い。このような場合は、実験を終了し、標準的な人道的エンドポイントのマウスの評価を続行します。マウスの早期回収における運動熱打撃プロトコルの全体の間の典型的な体温調節プロファイルは、チャンバー加熱ステージ、インクリメンタル運動段階、定常状態運動段階、および迅速または低速冷却法による回復段階として定義できる4つの異なる相から成る。重症モデルの場合、回復温度プロファイルは、コア温度が2時間の回復期間を通じて摂氏37度を超え続けたことを示した。雌マウスは、このモデルでは熱中症に対してより耐性があり、雄のマウスよりもほぼ2倍の長い距離を走った。2つのモデルで39.5°Cに回復するために必要な時間に違いは認められなかった。しかし、37.5°Cの環境温度まで冷却する時間は大幅に長引きました。最も重要なことは、手術後および運動後熱中症の常時動物の注意深い監視である。この方法は、数ヶ月までの熱中症暴露の長期的な影響を研究するのに役立ち、また、非ステロイド性抗炎症薬やエピジェネティクスの摂取などの熱中症の追加要因の影響を測定することができました。

a-preclinical-model-of-exertional-heat-stroke-in-mice

Research

JoVE Journal

Medicine

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

マウスにおけるモデリングのストローク - フィラメントモデルと中大脳動脈閉塞

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

医学

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

前臨床モデルにおける虚血性心不全のための遺伝子導入

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

マウスにおけるレーザードップラー流量計の指針と虚血性脳卒中に対する腔内中大脳動脈閉塞（MCAO）モデル

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

くも膜下出血のマウスモデル

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脳Microslicesの興奮毒性刺激<em&gt;インビトロ</em&gt;脳卒中のモデル

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

肝転移の前臨床マウスモデル

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

マウスにおけるモデリングのストローク：遠位中大脳動脈の永久的な凝固

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

一過性脳低酸素性虚血に基づく血栓性脳卒中モデル

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

マウスの中大脳動脈閉塞再灌流誘発性の脳卒中のための外科的アプローチ

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and role of certain neuroimmune interactions in stroke. Specifically, Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of activated microglia and peripheral myeloid-lineage cells, provides a means to detect and track neuroinflammation in vivo. Here, we present a method to accurately quantify neuroinflammation using [11C]N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide ([11C]DPA-713), a promising second generation TSPO-PET radiotracer, in distal middle cerebral artery occlusion (dMCAO) compared to sham-operated mice. MRI was performed 2 days post-dMCAO surgery to confirm stroke and define the infarct location and volume. PET/Computed Tomography (CT) imaging was carried out 6 days post-dMCAO to capture the peak increase in TSPO levels following stroke. Quantitation of PET images was conducted to assess the uptake of [11C]DPA-713 in the brain and spleen of dMCAO and sham mice to assess central and peripheral levels of inflammation. In vivo [11C]DPA-713 brain uptake was confirmed using ex vivo autoradiography.

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation in a mouse model of ischemic stroke.

虚血性脳卒中のマウス モデルで [11C] DPA-713 Neuroinflammation のイメージングをペットします。

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...