Name: establishment of deep hypothermic circulatory arrest in rats
Uploaded: 2022-12-16T12:30:00
Description: Watch this Scientific Journal Video about Establishment of Deep Hypothermic Circulatory Arrest in Rats at JoVE.com

著者らは、実験中にビデオデータを収集するのを手伝ってくれたLiang Zhangに感謝している。この研究は、中国国家自然科学基金会(助成金番号:82070479)および中央大学基礎研究基金(助成金番号:3332022128)の支援を受けました。

プロトコルは制度的レビューを受け、中国医学科学院の不破病院の施設動物管理および使用委員会から承認を受けました(FW-2021-0005)。すべての実験手順は、国立衛生研究所によって発行された実験動物の世話と使用のためのガイドに従って実行されました。
注:オスのSprague-Dawleyラット(体重:500〜600 g、年齢:12〜14週間)は、餌と水に自由にアクセスできる標準的な実験室条?...

<ol>
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(133), e56986 (2018).</li><li>Ha, J. Y., Kim, J. S., Kim, S. E., Son, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+activation+of+mitophagy+and+autophagy+by+staurosporine+protects+against+dopaminergic+neuronal+cell+death.">Simultaneous activation of mitophagy and autophagy by staurosporine protects against dopaminergic neuronal cell death.</a> Neuroscience Letters. 561, 101-106 (2014).</li><li>Yamamoto, A., Yue, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+its+normal+and+pathogenic+states+in+the+brain.">Autophagy and its normal and pathogenic states in the brain.</a> Annual Review of Neuroscience. 37, 55-78 (2014).</li><li>You, X. 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=Rat+cardiopulmonary+bypass+model:+Application+of+a+miniature+extracorporeal+circuit+composed+of+asanguinous+prime.">Rat cardiopulmonary bypass model: Application of a miniature extracorporeal circuit composed of asanguinous prime.</a> Journal of Extra-Corporeal Technology. 37 (1), 60-65 (2005).</li><li>Chen, Q., Lei, Y. Q., Liu, J. F., Wang, Z. C., Cao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beneficial+effects+of+chlorogenic+acid+treatment+on+neuroinflammation+after+deep+hypothermic+circulatory+arrest+may+be+mediated+through+CYLD/NF-&#954;B+signaling.">Beneficial effects of chlorogenic acid treatment on neuroinflammation after deep hypothermic circulatory arrest may be mediated through CYLD/NF-&#954;B signaling.</a> Brain Research. 1767, 147572 (2021).</li><li>Li, Y. 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=Differential+expression+profiles+of+circular+RNAs+in+the+rat+hippocampus+after+deep+hypothermic+circulatory+arrest.">Differential expression profiles of circular RNAs in the rat hippocampus after deep hypothermic circulatory arrest.</a> Artificial Organs. 45 (8), 866-880 (2021).</li><li>Linardi, D., 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=Slow+versus+fast+rewarming+after+hypothermic+circulatory+arrest:+effects+on+neuroinflammation+and+cerebral+oedema.">Slow versus fast rewarming after hypothermic circulatory arrest: effects on neuroinflammation and cerebral oedema.</a> European Journal of Cardiothoracic Surgery. 58 (4), 792-780 (2020).</li><li>Engelman, 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=The+Society+of+Thoracic+Surgeons,+The+Society+of+Cardiovascular+Anesthesiologists,+and+The+American+Society+of+ExtraCorporeal+Technology:+Clinical+practice+guidelines+for+cardiopulmonary+bypass--Temperature+management+during+cardiopulmonary+bypass.">The Society of Thoracic Surgeons, The Society of Cardiovascular Anesthesiologists, and The American Society of ExtraCorporeal Technology: Clinical practice guidelines for cardiopulmonary bypass--Temperature management during cardiopulmonary bypass.</a> Annals of Thoracic Surgery. 100 (2), 748-757 (2015).</li><li>Jenke, 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=AdipoRon+attenuates+inflammation+and+impairment+of+cardiac+function+associated+with+cardiopulmonary+bypass-induced+systemic+inflammatory+response+syndrome.">AdipoRon attenuates inflammation and impairment of cardiac function associated with cardiopulmonary bypass-induced systemic inflammatory response syndrome.</a> Journal of the American Heart Association. 10 (6), 018097 (2021).</li></ol>

カニューレ挿入は、ラットにDHCAを確立するための最も基本的な手順です。カニューレ挿入の前に、動脈を0.5 mLの2%リドカインに浸すと、カニューレ挿入が容易になります。カニューレ挿入後、微小血栓形成を避けるために、外頸静脈を介した500IU / kgヘパリンによるヘパリン化が必要です17。我々は、この用量のヘパリンの活性化凝固時間(ACT)>480秒の目標を達成できる?...

深部低体温循環停止(DHCA)は、1953年以来心臓外科で使用されています1。DHCAには、体への血流を世界的に遮断する前に、患者の深部温度を深刻な低体温レベル(15〜22°C)に下げることが含まれます2。循環停止は、比較的無血の手術野を提供することができます。深部低体温は、特に脳および心筋における代謝を低下させ、これは虚血に対する効果的な保護方法である3。DHCAは、複雑な先天性心疾患、大動脈弓疾患、さらには大静脈血栓を伴う腎腫瘍または副腎腫瘍の手術中に一般的に適用されます4,5。したがって、DHCA動物モデルの確立は、手順の改良と臨床現場での合併症の予防のための重要な参考資料を提供します。
イヌ6、ウサギ7、その他の動物でモデルを樹立することができるが、操作性や低コストからラットを用いることが好ましい。DHCAラットモデルは、2006年にJungwirthらによって初めて記載された8。循環停止の持続時間が神経学的転帰に影響を与えることがわかった。それ以来、DHCAラットモデルは広く調査されてきました。DHCAが全身性炎症反応症候群(SIRS)9を誘発することが明らかになっています。その後の研究では、薬理学者は、SIRSによって誘発されるDHCA関連の神経炎症がレスベラトロール10およびトリプトリド11によって弱める可能性があることを発見しました。私たちのチームはまた、DHCA関連の神経炎症が、低温誘導性RNA結合タンパク質12を阻害することによって軽減できることを発見しました。心血管系では、スーパーオキシドジスムターゼはDHCA13中の虚血/再灌流(I / R)損傷に対して心臓保護効果があります。これらの結果は、DHCA関連の病態生理学的プロセスの理解を拡大し、DHCAの転帰を改善するための新しい方向性を提供しました。しかし、DHCA後のエンドトキシン血症、酸化ストレス、オートファジーに関する結果は決定的ではありません。DHCAは心肺バイパス(CPB)14と同じ運用技術を使用していますが、その管理戦略は異なり、DHCAを生成する手順はチームによって異なります8、9、10、11。本研究は、ラットにおけるDHCA手順を確立するための方法を提供することを目的とする。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Heat Exchanger</td>
			<td>Xi&#8217;an Xijing Medical Appliance Co., Ltd</td>
			<td>Animal-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Membrane Oxygenator</td>
			<td>Dongguan Kewei Medical Instrument Co., Ltd.</td>
			<td>Micro-M</td>
			<td></td>
		</tr>
		<tr>
			<td>Monitor</td>
			<td>Chengdu Techman Co., Ltd</td>
			<td>BL-420s</td>
			<td></td>
		</tr>
		<tr>
			<td>Roller Pump</td>
			<td>Changzhou Prefluid Technology Co.,Ltd</td>
			<td>BL100</td>
			<td></td>
		</tr>
		<tr>
			<td>SD Rat</td>
			<td>HFK Bioscience Co.,Ltd.</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Maruishi Pharmaceutical Co. Ltd</td>
			<td>H20150020</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaver</td>
			<td>Hangzhou Huayuan Pet Products Co.,Ltd.</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer</td>
			<td>SPACECABS</td>
			<td>/</td>
			<td></td>
		</tr>
		<tr>
			<td>Ventilator</td>
			<td>Shanghai Alcott Biotech Co., Ltd</td>
			<td>ALC-V8S</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Tank</td>
			<td>Maquet Critical Care AB</td>
			<td>Jostra HCU20-600</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of deep hypothermic circulatory arrest in rats

深部低体温循環停止(DHCA)は、複雑な先天性心疾患および大動脈弓疾患の手術中に日常的に適用されます。本研究は、ラットにおいてDHCAを確立する方法を提供することを目的とする。バイタルサインに対するDHCAプロセスの影響を評価するために、循環停止のない常温心肺バイパス(CPB)ラットモデルを対照として使用しました。予想通り、DHCAは体温と平均動脈血圧の有意な低下をもたらしました。血液ガス分析の結果,DHCAは乳酸濃度を上昇させたが,血中pHおよびヘモグロビン,ヘマトクリット,Na+,Cl-,K+,グルコース濃度には影響しなかった。さらに、常温CPBラットと比較して、透過型電子顕微鏡観察の結果、DHCAラットにおける海馬オートファゴソームの軽度の増加を示した。

対照群として、循環停止のない正常温度CPB(NtCPB)ラットは、処置全体を通して安定した平均動脈血圧(MAP)および体温を示したが、DHCAラットのMAPは心停止中に減少した(p < 0.01、 図3A)。DHCAラットの温度は冷却段階で急速に低下し、再加温段階で徐々に回復しました。DHCA回路からラットを離乳させると、DHCAラットの温度は正常に戻った(図3B)。...

Watch this Scientific Journal Video about Establishment of Deep Hypothermic Circulatory Arrest in Rats at JoVE.com

Establishment of Deep Hypothermic Circulatory Arrest in Rats

このプロトコルは、ラットにおける深部低体温循環停止の確立を示しており、全身性炎症反応症候群、虚血/再灌流障害、酸化ストレス、神経炎症などの調査に適用できます。

ラットにおける深部低体温循環停止の確立

Deep hypothermic circulatory arrest (DHCA) is routinely applied during surgeries for complex congenital heart disease and aortic arch disease. The present ...

ラットにおける深部低体温循環停止の確立。深部低体温循環停止であるDHCAは、複雑な先天性心疾患および大動脈弓疾患の手術中に日常的に適用されます。本研究は、ラットにおいてDHCAを確立する方法を提供することを目的とする。プロトコルは治験審査を受け、中国医学科学院不破病院の施設動物管理および使用委員会から承認を得ました。準備作業。CPB回路に、モルフドロッパー、ローラーポンプ、熱交換器、膜型酸素供給器、接続チューブ、および回路を接続した水タンクから変更されたリザーバーが含まれていることを確認し、1ミリリットルのヘパリンナトリウムと1ミリリットルの生理食塩水で12ミリリットルのヒドロキシデンプンを作りますローラーポンプを穏やかに回転させて40ミリリットルのプライミング溶液で回路をプライミングします。第二に、麻酔とカニューレ挿入。チューブを人工呼吸器とサイドパラメータに接続します。電気加熱毛布と右側を置き、テープで右向きにします。眼科用軟膏を目に塗り、乾燥を防ぎます。シェーバーで右鼠径部、右頸部、尾部の毛を剃ります。その後、アルデヒドとアルコールで皮膚を消毒します。皮膚と左鼠径部を切断し、筋肉と組織の軟骨を解剖して左大腿静脈と動脈を露出させます。動脈を慎重に分離します。22ゲージの静脈内カテーテルを左大腿動脈にカニューレ挿入します。絹で結紮された動脈とカテーテル。凝固を避けるためにカニューレを洗い流すためにヘパリンを含む生理食塩水を使用してください。カテーテルをプレスセンサーに接続して血圧を監視します。尾の皮膚を切り取り、メスを使って尾動脈の表在性を切断し、術野の中央にある尾動脈を露出させます。22ゲージの静脈内カテーテルで尾動脈をカニューレ挿入します。絹で動脈とカテーテルを結睭します。凝固を避けるためにカテーテルを洗い流すためにヘパリンを含む生理食塩水を使用してください。静脈の右頸静脈の皮膚を切り取り、筋肉と組織を分離して静脈を露出させます。16ゲージの自家製マルチエイジ静脈カテーテルを静脈の右外頸静脈に挿入し、右下大静脈または右心房に慎重に入れます。汚染を避けるために、右外静脈が湿ったボールで各カニューレ領域を覆う場所にヘパリンナトリウムを投与します。3つ目は、回路を尾動脈のカテーテルに接続し、ポンプを毎分1〜2ミリリットルの低レートに保つことです。次に、リザーバーを右外頸静脈のカテーテルに接続します。リザーバーには常に約1センチメートルの血中濃度があることを確認してください。水タンクの電源を入れ、最初に水温を摂氏37度に設定します。血圧が安定したら、ポンプを毎分80〜100ミリリットルまで静かに増やします。4、冷却。室温を摂氏20度前後に設定します。この使い捨て手袋に角氷を入れてから、右の頭と側面に置きます。タンクの温度は、米の直腸温度に応じてリアルタイムで提案されました。血液ガス分析の結果に応じて、人工呼吸器の関連パラメータを適切に変更してください。5、深い低体温循環停止。その後、直腸温は摂氏15〜20度に下がります。アイスパックを交換し、手袋を廃棄して、循環停止中の深い低体温症を確実に維持します。ローラーポンプを停止します。リザーバーを環境と接触させ、外頸静脈からリザーバーにゆっくりと血液を排出します。血圧改革に注意してください。血圧と心拍がなくなったら、排水を停止し、貯水池を閉じたままにします。人工呼吸器をオフにします。6、ウォームアップと再灌流。使い捨て手袋をすべて取り外し、室温を摂氏25度に上げます。静脈ドレナージチューブのクリッピングを維持しながら、メモリ酸素インターベンチレーションを復元します。ローラーポンプをオンにして、リザーバー内の血液がゆっくりと正しい体に戻ることを確認します。貯水池の血中濃度が1センチメートルのままになったら、人工呼吸器の電源を入れます。ドレナージチューブを使用して、右心房からリザーバーに血液をゆっくりと排出します。加熱ランプ、加熱パッド、水タンクをオンにします。水タンクの温度を摂氏25度に設定します。次に、タイムリーに左の温度に調整します。直腸温度が摂氏34度に戻ったら、加熱ランプを取り外します。7、CPBから離乳します。ポンプ流量のローラーをゆっくりと徐々に減らし、大静脈属の速度を毎分1ミリリットルに減らす流量に調整します。貯水池を環境と接触させてください。残りの血液を毎分1ミリリットルの流量で回路に注入します。メモリー酸素化とローラーポンプを停止します。機械的換気と深い麻酔の期間の後に安楽死させた。代表的な結果。対照群としては、常温CPB、NtCPB、ラットが挙げられる。循環停止なしでは、処置全体を通して安定した平均動脈血圧MAPおよび体温を示した。一方、DHCAラットのMAPは心停止中に減少した。DHCAラットの温度は冷却段階で急速に低下し、回転段階で徐々に回復しました。DHCA回路の離乳により、DHCAラットの温度は正常に戻った。上記のデータは、DHCA手順が診療所の患者の実際の体温とMAPの変化を模倣していることを示しました。DHCA処理装置および上昇の影響を血液ガス分析により検討した。一次溶液との全血接触後、ヘモグロビンの濃度は両方の群でデシリットル当たり6グラムより高かった。DHCA表面からの離乳により、CPB回路内の残りの血液が右側に再注入されたため、デシリットルあたり9グラムに増加しました。ヘマトクリット値HCTはHBと同様の傾向を示した。DHCA再灌流後、乳酸酸は急速に増加し、DHCA群でより顕著であった。DHCA手順後にpHが低下しましたが、これは乳酸の蓄積の結果である可能性が最も高いです。実験全体を通して、ナトリウム、塩化物、カリウムイオン、およびグルコースの濃度は、各10点で有意差を示さなかった。この結果から,DHCAは乳酸への増加を誘導しただけで,血中pHやヘモグロビン,ヘマトクリット,ナトリウムイオン,塩化物イオン,カリウムイオン,グルコース濃度には影響しないことが示唆された。神経細胞のオートファジーに対するDHCAの影響を評価するために、透過型電気顕微鏡を使用し、驚くべきことに、DHCAラットの海馬でオートファゴソームの数を発見し、増加させました。上記のデータは、DHCAモデルが診療所の患者の実際の体温とMAPの変化を模倣していることを示しました。このモデルは、全身性炎症反応症候群、神経炎症などを調査するために適用することができる。

establishment-of-deep-hypothermic-circulatory-arrest-in-rats

Research

JoVE Journal

Immunology and Infection

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

ラット同所性後肢移植

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

免疫・感染学

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

実験用ラットとマウスにおけるエクト - とEndoparasitesの診断

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Epstein - Barrウイルスの成長形質転換リンパ芽球様細胞株の樹立

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

設立<em&gt;インビトロ</em&gt;システムの細胞内挙動を研究するために、<em&gt;カンジダ·グラブラタ</em&gt;人間のTHP-1マクロファージにおける

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

非侵襲的気管挿管と成体ラットにおける完全な胸腺摘出

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

熱の予防によりラットに悪影響をストレス<em&gt;枯草菌</em&gt;ひずみ

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

下大静脈狭窄: 深部静脈血栓症のマウスモデル

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

高周波超音波による監視マウスのうっ滞による深部静脈血栓症

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

深い皮膚注入として、モデルカンジダ皮膚感染症病理組織学的解析のための

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

ウイルス感染とキイロショウジョウバエのホスト ウイルスの相互作用の解析

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...