Name: sodium taurocholate induced severe acute pancreatitis in c57bl/6 mice
Uploaded: 2021-06-28T13:00:00
Description: Watch this Scientific Journal Video about Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice at JoVE.com

著者はサンパウロ大学の医療クリニックでの卒業後の卒業プログラムに感謝します。クオルデナサン・デ・アペルフェイソアメント・デ・ペソアル・デ・ニーヴェル・スーペリア校(CAPES)とサンパウロ大学医学部(FMUSP)。

このプロトコルは、USP医学部の動物の使用のための倫理委員会によって承認されました, Num. プロジェクト: 1343/2019-CEUA: FMUSP.このプロトコルについては、C57BL/6マウス、6週間齢、2g±20個の重さを用いた(n=9/群)。
1. 腹腔内術
<ol><li>1 mLシリンジと13x0.45mm針26G 1/2を使用して、キシラジン(10mg/kg)およびケタミン溶液(体重0.1mL/10g)を使用して動物を麻酔する。つまんで十分な麻酔?...

<ol>
	<li>Li, 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=Significantly+different+clinical+features+between+hypertriglyceridemia+and+biliary+acute+pancreatitis:+A+retrospective+study+of+730+patients+from+a+tertiary+center.">Significantly different clinical features between hypertriglyceridemia and biliary acute pancreatitis: A retrospective study of 730 patients from a tertiary center.</a> BMC Gastroenterology. 18 (1), 1-8 (2018).</li><li>Rechreche, H., Abbes, A., Iovanna, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+antioxidant+mechanisms+in+lung+during+experimental+pancreatitis+in+rats.">Induction of antioxidant mechanisms in lung during experimental pancreatitis in rats.</a> Indian Journal of Experimental Biology. 58 (5), 297-305 (2020).</li><li>T, 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=Intraductal+infusion+of+taurocholate+followed+by+distal+common+bile+duct+ligation+leads+to+a+severe+necrotic+model+of+pancreatitis+in+mice.">Intraductal infusion of taurocholate followed by distal common bile duct ligation leads to a severe necrotic model of pancreatitis in mice.</a> Pancreas. 44 (3), (2015).</li><li>Botoi, G., Andercou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+17-prognostic+marker+of+severe+acute+pancreatitis.">Interleukin 17-prognostic marker of severe acute pancreatitis.</a> Chirurgia. 104 (4), 431-438 (2009).</li><li>Li, D., Li, J., Wang, L., Zhang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+IL-1beta,+IL-8,+and+IL-10+polymorphisms+and+risk+of+acute+pancreatitis.">Association between IL-1beta, IL-8, and IL-10 polymorphisms and risk of acute pancreatitis.</a> Genetics and Molecular Research. 14 (2), 6635-6641 (2015).</li><li>Feng, 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=Effect+of+peritoneal+lavage+with+ulinastatin+on+the+expression+of+NF-kappaB+and+TNF-alpha+in+multiple+organs+of+rats+with+severe+acute+pancreatitis.">Effect of peritoneal lavage with ulinastatin on the expression of NF-kappaB and TNF-alpha in multiple organs of rats with severe acute pancreatitis.</a> Experimental and Therapeutic Medicine. 10 (6), 2029-2034 (2015).</li><li>Fang, D. Z., 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+sildenafil+on+inflammatory+injury+of+the+lung+in+sodium+taurocholate-induced+severe+acute+pancreatitis+rats.">Effects of sildenafil on inflammatory injury of the lung in sodium taurocholate-induced severe acute pancreatitis rats.</a> International Immunopharmacology. 80, (2020).</li><li>Ceranowicz, P., Cieszkowski, J., Warzecha, Z., Dembinski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+acute+pancreatitis.">Experimental models of acute pancreatitis.</a> Post&#281;py Higieny i Medycyny Do&#347;wiadczalnej(Online). 69, 264-269 (2015).</li><li>Wan, M. H., 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=Review+of+experimental+animal+models+of+biliary+acute+pancreatitis+and+recent+advances+in+basic+research.">Review of experimental animal models of biliary acute pancreatitis and recent advances in basic research.</a> HPB (Oxford). 14 (2), 73-81 (2012).</li><li>Mayerle, J., Sendler, M., Lerch, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretagogue+(Caerulein)+induced+pancreatitis+in+rodents.">Secretagogue (Caerulein) induced pancreatitis in rodents.</a> Pancreapedia: The Exocrine Pancreas Knowledge Base. (1), (2013).</li><li>Wang, N., 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=Resveratrol+protects+against+L-arginine-induced+acute+necrotizing+pancreatitis+in+mice+by+enhancing+SIRT1-mediated+deacetylation+of+p53+and+heat+shock+factor+1.">Resveratrol protects against L-arginine-induced acute necrotizing pancreatitis in mice by enhancing SIRT1-mediated deacetylation of p53 and heat shock factor 1.</a> International Journal of Molecular Medicine. 40 (2), 427-437 (2017).</li><li>Ma, Z. H., 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=Effect+of+resveratrol+on+peritoneal+macrophages+in+rats+with+severe+acute+pancreatitis.">Effect of resveratrol on peritoneal macrophages in rats with severe acute pancreatitis.</a> Inflammation Research. 54 (12), 522-527 (2005).</li><li>Souza, L. 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=Anti-inflammatory+effects+of+peritoneal+lavage+in+acute+pancreatitis.">Anti-inflammatory effects of peritoneal lavage in acute pancreatitis.</a> Pancreas. 39 (8), 1180-1184 (2010).</li><li>Perides, G., Acker, G. J. v., Laukkarinen, J. M., Steer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+acute+biliary+pancreatitis+induced+by+retrograde+infusion+of+bile+acids+into+the+mouse+pancreatic+duct.">Experimental acute biliary pancreatitis induced by retrograde infusion of bile acids into the mouse pancreatic duct.</a> Nature Protocols. 5 (2), 335-341 (2010).</li><li>Wittel, U. 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=Taurocholate-induced+pancreatitis:+a+model+of+severe+necrotizing+pancreatitis+in+mice.">Taurocholate-induced pancreatitis: a model of severe necrotizing pancreatitis in mice.</a> Pancreas. 36 (2), 9-21 (2008).</li><li>Tao, L., Reese, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+mouse+models+that+reflect+human+immune+responses.">Making mouse models that reflect human immune responses.</a> Trends Immunology. 38 (3), 181-193 (2017).</li><li>Vandamme, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+rodents+as+models+of+human+diseases.">Use of rodents as models of human diseases.</a> Journal of Pharmacy and Bioallied Science. 6 (1), 2-9 (2014).</li><li>Song, H. K., Hwang, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+C57BL/6N+mice+on+the+variety+of+immunological+researches.">Use of C57BL/6N mice on the variety of immunological researches.</a> Laboratory Animal Research. 33 (2), 119-123 (2017).</li><li>Bogdanske, J. J., Stelle, S. H. -. V., Riley, M. V., Schiffman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Suturing Principles and Techniques in Laboratory Animal Surgery. 1st edition. (1), (2010).</li><li>Ray, A., Dittel, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+peritoneal+cavity+cells.">Isolation of mouse peritoneal cavity cells.</a> Journal of Visualized Experiments. (35), e1488 (2010).</li><li>Schmidt, 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=A+better+model+of+acute+pancreatitis+for+evaluating+therapy.">A better model of acute pancreatitis for evaluating therapy.</a> Annals of Surgery. 215 (1), 44-56 (1992).</li><li>Liu, D. 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=Resveratrol+improves+the+therapeutic+efficacy+of+bone+marrow-derived+mesenchymal+stem+cells+in+rats+with+severe+acute+pancreatitis.">Resveratrol improves the therapeutic efficacy of bone marrow-derived mesenchymal stem cells in rats with severe acute pancreatitis.</a> International Immunopharmacology. 80, 106128 (2020).</li><li>Yang, X. F., 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=Chaiqin+chengqi+decoction+alleviates+severe+acute+pancreatitis+associated+acute+kidney+injury+by+inhibiting+endoplasmic+reticulum+stress+and+subsequent+apoptosis.">Chaiqin chengqi decoction alleviates severe acute pancreatitis associated acute kidney injury by inhibiting endoplasmic reticulum stress and subsequent apoptosis.</a> Biomedicine &amp; Pharmacotherapy. 125 (12), 110024 (2020).</li><li>Yang, X. F., 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=Chaiqin+chengqi+decoction+alleviates+severe+acute+pancreatitis+associated+acute+kidney+injury+by+inhibiting+endoplasmic+reticulum+stress+and+subsequent+apoptosis.">Chaiqin chengqi decoction alleviates severe acute pancreatitis associated acute kidney injury by inhibiting endoplasmic reticulum stress and subsequent apoptosis.</a> Biomedicine &amp; Pharmacotherapy. 125, 110024 (2020).</li><li>Venglovecz, V., Z, R., Hegyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+bile+acids+on+pancreatic+ductal+cells.">The effects of bile acids on pancreatic ductal cells.</a> Pancreapedia: The Exocrine Pancreas Knowledge Base. (1), (2019).</li><li>Roberts, S. E., Akbari, A., Thorne, K., Atkinson, M., Evans, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+incidence+of+acute+pancreatitis:+impact+of+social+deprivation,+alcohol+consumption,+seasonal+and+demographic+factors.">The incidence of acute pancreatitis: impact of social deprivation, alcohol consumption, seasonal and demographic factors.</a> Alimentary Pharmacology and Therapeutics. 38 (5), 539-548 (2013).</li><li>Lerch, M. M., Gorelick, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+acute+and+chronic+pancreatitis.">Models of acute and chronic pancreatitis.</a> Gastroenterology. 144 (6), 1180-1193 (2013).</li><li>Nakamura, K., Fukatsu, K., Sasayama, A., Yamaji, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+immune-modulating+formula+comprising+whey+peptides+and+fermented+milk+improves+inflammation-related+remote+organ+injuries+in+diet-induced+acute+pancreatitis+in+mice.">An immune-modulating formula comprising whey peptides and fermented milk improves inflammation-related remote organ injuries in diet-induced acute pancreatitis in mice.</a> Biosci Microbiota Food Health. 37 (1), 1-8 (2018).</li><li>Kui, B., 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=New+insights+into+the+methodolgy+of+L-Arginine-induced+acute+pancreatitis.">New insights into the methodolgy of L-Arginine-induced acute pancreatitis.</a> PLoS One. 10 (2), 011758 (2015).</li><li>Xue, 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=Alternatively+activated+macrophages+promote+pancreatic+fibrosis+in+chronic+pancreatitis.">Alternatively activated macrophages promote pancreatic fibrosis in chronic pancreatitis.</a> Nature Communication. 6, 7158 (2015).</li><li>Lesina, M., Wormann, S. M., Neuhofer, P., Song, L., Algul, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-6+in+inflammatory+and+malignant+diseases+of+the+pancreas.">Interleukin-6 in inflammatory and malignant diseases of the pancreas.</a> Seminars in Immunology. 26 (1), 80-87 (2014).</li><li>Rao, S. A., Kunte, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-6:+An+early+predictive+marker+for+severity+of+acute+pancreatitis.">Interleukin-6: An early predictive marker for severity of acute pancreatitis.</a> Indian Journal of Critical Care Medicine. 21 (7), 424-428 (2017).</li></ol>

タウロコレートナトリウム注入による急性膵炎を誘導する方法は、ラット22,23,24に既に示されている。2008年、2010年、2015年に出版された3つの同様の作品は、プロトコル3,14,15の参考文献として機能しました。本研究では、C57BL/6マウスでこの方法を再現す...

ヒトでは、胆石の存在は、胆汁の末端部の閉塞による膵炎の最も一般的な原因であり、膵臓分泌物の流れを中断し、膵臓で激しい炎症プロセスを引き起こし、血清および炎症性メディエーター中の消化酵素の濃度が増加する1,2。
急性膵炎(AP)の発症を説明するために、2つの異なる理論が提案されている。「共通チャネル」理論は、胆嚢に存在する石が遠位共通胆管系を妨げ、胆汁分泌が膵管に逆行して流れることを示唆している。第2の理論(「ダクト閉塞」理論)は、過剰胆石による膵管閉塞が十二指腸への膵分泌の流れの閉塞を引き起こし、管性高血圧症を引き起こすことを示唆している。急性胆道膵炎につながるメカニズムは完全には理解されていませんが、結果は激しい炎症プロセスです。消化酵素の噴火と膵臓の自己消化は、組織病理学的変化、アセンチズル液および血清中の炎症性サイトカイン(IL-1β、IL-6、TNF-α)の増加、および急性相タンパク質の増加を招く4,5,6.
重症急性膵炎は、複数の臓器の関与と高い死亡リスクのために臨床的な注意を払うに値する状態です。急性膵炎(AP)の再生のための動物モデルは、疾患の病態生理学的メカニズムを説明し、疾患の初期段階から始まる炎症性事象の進化を監視するのに役立つ重要である。これは通常、診療所2,7では不可能です。さらに、膵臓組織へのアクセスは前臨床試験で容易であり、臨床状態に関連する変化の解明を好む8イソジェニック種と共に、望ましくない変数を排除し、人間の状態で観察された結果と臨床的類似性をミラーリングする9。
ラットおよびマウス種における急性膵炎の誘導のための胆道モデルおよび非胆道モデルは、科学文献において頻繁に研究されてきた。誘導の非胆道方法には、コレシストキニン分泌酵素またはそのアナログセリフィン10の上格刺激用量の投与が含まれる。L-アルギニンのほぼ致死量の投与;またはエチオニン11を補充コリン欠損の食事の投与.これらの方法は、再現しやすく、膵炎をもたらすが、理論的にはAPを引き起こすメカニズム(すなわち、膵管内の胆汁分泌の還流)を複製しない。胆道モデルに対処する技術は、膵管への胆汁酸の逆行注入に基づいており、このプロトコルを実行するために十分な訓練を受けた研究者を必要とします。いくつかの研究は、ラット(これらの実験は外科的処置を伴うため、技術的な理由から)12,13でこの方法を使用して公開されています。しかし、マウスのアプローチは、炎症の研究でより興味深い結果を提供するかもしれない3,14,15。本研究では、C57BL/6麻酔マウスにタウロコレートナトリウムを注入して重症急性膵炎を再生するためのステップのチェックリストを示す。
抗体の実験や遺伝子やタンパク質発現の分析を必要とする研究では、これらの動物のための材料の武器が大きくなり、等原性およびノックアウト種で作業する可能性があるため、マウスの使用が好ましい。マウスC57BL/6は、もともと抗腫瘍活性および免疫学の研究のために開発されたマウスの近親交配株である。この株は、等性であることのために研究者によってますます好まされており、実験でより少ない数の動物の使用を意味し、同じグループ17,18間の結果の変動が少ないことを意味する結果のより大きな再現性を可能にする。
Peridesら (2010)14 は、タウロコール酸ナトリウム注入によるマウスにおけるAP誘導のためのプロトコルを発表した。ここでは、C57BL/6マウスのタウロコレートナトリウム濃度(2.5%)を使用して、定義された体積と注入速度を使用してこのモデルを更新します(図1)。重症度の最大レベルは、マウスの誘導の12時間以内に達する。血清中および腹腔におけるIL-6濃度の上昇は、APの進行と相関している。練習では、麻酔の誘導から注入の完了までの合計推定時間は、動物1匹につき25分である。訓練を受けた研究者がこの実験を行うことは不可欠です。溶液が一般的な胆管に適切に注入されるように、タウロコレートナトリウムの代わりにメチレンブルーを使用していくつかのパイロットトレーニングセッションを行います。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4 mm needle</td>
			<td>INTRAG MEDICAL TECH</td>
			<td>90183210</td>
			<td>30G</td>
		</tr>
		<tr>
			<td>&#160;0.54 mm polyethylene tube</td>
			<td>Tygon</td>
			<td>730010</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Styrofoam block</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>masking tape for mounting the mouse</td>
			<td>Missner</td>
			<td>1236</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Infusion pump scheduled to 10&#181;L / min.</td>
			<td>Havard aparatus-Peristaltic Pump Series</td>
			<td>MA1 55-7766</td>
			<td>&#160;Model 66 Small Peristaltic</td>
		</tr>
		<tr>
			<td>Scissors and forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic providine iodine</td>
			<td>Pfizer</td>
			<td>12086OR</td>
			<td>antisepsis</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>SIGMA</td>
			<td>459836</td>
			<td>Mix 700 mL 100% ethanol with 300 mL dH2O</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Lord</td>
			<td>bdk9a1ghk6</td>
			<td>For trichotomy</td>
		</tr>
		<tr>
			<td>Sodium taurocholate</td>
			<td>Sigma-Aldrich</td>
			<td>86339- 1G</td>
			<td>CAS NUMBER- 345909-26-4</td>
		</tr>
		<tr>
			<td>microvessel clip</td>
			<td>Medicon Surgical</td>
			<td>56.87.35</td>
			<td>Approximator, opening 4.0 mm, closing pressure 30 -&#160;40 g</td>
		</tr>
		<tr>
			<td>6-0 prolene</td>
			<td>Bioline</td>
			<td>5162</td>
			<td>Suture line</td>
		</tr>
		<tr>
			<td>Ketamin NP (cloridrato de dextrocetamina) 50mg/mL</td>
			<td>Crist&#225;lia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xilazine 2%</td>
			<td>Syntec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile saline solution (0.9% (wt/vol) saline)</td>
			<td>Farmace</td>
			<td>105851</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl Blue</td>
			<td>Sigma-Aldrich Chemicals</td>
			<td>M5528</td>
			<td></td>
		</tr>
		<tr>
			<td>MILLIPLEX&#160;MAP&#160;Mouse Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay</td>
			<td>MERCK</td>
			<td>MCYTOMAG-70K</td>
			<td>Simultaneously analyze multiple cytokine and chemokine biomarkers with Bead-Based Multiplex Assays using the Luminex technology, in mouse serum, plasma and cell culture samples.</td>
		</tr>
		<tr>
			<td>Amylase Assay</td>
			<td>Labtest</td>
			<td>11</td>
			<td></td>
		</tr>
		<tr>
			<td>Desmarres retractor 13-mm 
			width</td>
			<td>ROBOZ</td>
			<td>RS-6672</td>
			<td></td>
		</tr>
	</tbody>
</table>

sodium taurocholate induced severe acute pancreatitis in c57bl/6 mice

タウロキ酸ナトリウム注入による胆道急性膵炎誘導は、臨床胆汁性膵炎の発症に対応するヒト臨床状態および炎症性イベントの再現の表現により、科学界によって広く使用されている。膵臓の損傷の重症度は、注入胆汁酸の濃度、速度、および体積を測定することによって評価することができる。この研究は、プロトコルの再生に使用される材料と方法の最新のチェックリストを提供し、この急性膵炎(AP)モデルの主な結果を示しています。これまでの出版物のほとんどは、ラットでこのモデルを再現することに限定されています。我々は、この方法をマウスに適用し、研究に関連する可能性のある追加の利点(すなわち、これらの動物のための試薬および抗体の兵器の入手可能性とマウスの遺伝子組み換え株で作業する可能性)を提供する。マウスの急性膵炎誘導に関しては、C57BL/6マウスの3分間の注入速度10μL/minで2.5%タウロコレートナトリウムを定義した体系的なプロトコルを提示し、誘導の12時間以内に重症度の最大レベルに達し、その結果を検証する結果を強調する。練習と技術により、麻酔の誘導から注入の完了までの合計推定時間は、動物1匹につき25分である。

膵炎重症度は、0が不在に対応するシュミットのscale21 に従って0-3の間で採点され、1は軽度の存在(<25%)に対応し、2は中程度の存在(25〜50%)に対応し、3は強烈な存在(>50%)に対応する(表1)。測定は、血漿アミラーゼ活性、膵浮腫、アシナル細胞、傷害/壊死、膵炎(H&E染色切片の組織学的分析による)およびIL-6細胞カイン濃度を血清およびPerC液?...

Watch this Scientific Journal Video about Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice at JoVE.com

Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice

重度の急性膵炎の動物モデルは、初期段階での病態生理学的変化の研究を可能にし、炎症事象の進化の観察を促進する。ここでは、麻酔をしたC57BL/6マウスの膵管内へのタウロコレートナトリウムの逆行注入による重症急性胆道膵炎の誘導プロトコルを提供する。

C57BL/6マウスでタウロコレートナトリウム誘発重篤急性膵炎

Biliary acute pancreatitis induction by sodium taurocholate infusion has been widely used by the scientific community due to the representation of the ...

この方法は、急性胆道膵炎で起こる炎症事象を再生するために使用することができる。膵臓組織の変化を初期段階で研究することは可能であり、これは一般的に診療所では不可能である。この技術の主な利点は、訓練を受けた研究者のために迅速かつ容易に再現でき、ヒトの急性膵炎と同様の結果を提供することです。この方法は、膵臓および隣接する器官における炎症性媒介細胞の研究を可能にするため、薬物分子および他の治療介入の薬理学的免疫応答を研究するために使用することができる。このプロトコルを試みる際には、臓器を見つけ、カヌリケーション領域を確立することが重要です。臓器の位置を暗記し、メチレンブルーなどの着色溶液を使用して最初に注射用途を行使します。つま先ピンチで麻酔の深さを確認した後、5%ポビドネヨウ素溶液で麻酔マウスの腹部を洗浄します。トリマーを使用して、胸部と下腹部の間の毛を取り除き、70%のアルコールで約2平方センチメートルの手術領域をきれいにします。外科用基板上の動物を固定し、腹部の上部に水平に皮膚の5ミリメートルを切断し、xiphoidプロセスの下に1センチメートルを切断するためにはさみを使用しています。腹膜の切り傷を繰り返し、腔の暴露を最小限に抑え、腹腔切り出しを行います。レトラクタを使用して、腸から約1センチメートルで頭に向かって肝臓を引っ張り、膵臓と十二指腸の領域を見つける。鉗子を使用して、動物の頭に向かって肝臓を持ち上げます。小腸の一部を穏やかに引っ張ることによって、2つの側面端を6-0ポリプロピレン縫合糸で固定し、共通の胆管の遠位部分をよりよく見ることができる。マイクロ容器クリップを使用して、近位共通胆管を一時的に閉塞する。腹腔から臓器を露出する。その後、8-0で遠位共通胆管の一時的な閉塞を行う縫合。一般的な胆管にアクセスするには、小腸の壁の白っぽい部分として現れる百壁領域を穿刺する。0.54ミリメートルのポリエチレンチューブに接続された0.4ミリメートルの針で、3分間体重10グラムあたり10マイクロリットルの一定速度で2.5%ナトリウムタウロキロ酸溶液注入のための注入ポンプを開始します。注入後、マイクロ容器クリップを取り外し、一時的な8-0縫合、膵胆管からの注射針を用いた。6-0非吸収性モノフィラメントポリプロピレン縫合糸で腹部を縫合する。開腹術と縫合の終わりまでの時間は30分以下であるべきである。手術後、木の削りくずと水と食品のアドリビタムが並ぶポリエチレンボックスに動物を収容します。皮膚を引き戻して動物をそっと持ち、眼球のわずかな突起を促進し、目を上向きにして動物を配置します。動物の目に局所麻酔薬を含む目の軟膏の滴を植え付ける。眼の内側の隅に毛細管の端部を配置し、約30〜45度の角度で眼球の下にそっと挿入します。血流が始まるまで毛細管を回転させます。コレクションが終わったら、まぶたを軽い圧縮で閉じておくことでホメオスタシスを確実にします。その後、27ゲージ針を使用して、4ミリリットルの氷冷PBSをマウスの腹腔に注入する。注射後、腹骨を10秒間静かにマッサージし、接着した細胞を除去します。はさみとピンセットを使用して、内皮と筋肉に0.5センチメートルの小さな切り傷を作り、腹腔を露出させます。腹部に球根ピペットを挿入し、脂肪組織を吸引しないように、できるだけ多くの流体を収集します。さらに処理するためにヘッド領域から膵臓の一部を収集します。採取した流体を氷の上に保管されたチューブに堆積させます。その後、腹膜液をGの250倍5分間遠心分離し、インターロイキン6の加量に上清をストックする。血清を得るために、採取した血液サンプルを700回Gで15分間遠心分離し、アミラーゼおよびインターロイキン6投薬のために上清をストックする。急性膵炎の誘導から12時間後に膵臓組織を分析した結果、恥群の組織サンプルと比較して、患部の臓器に激しい炎症が見られた。同様の結果は、shamおよびAPTAからの組織サンプルのヘマトキシリン・エオシン染色後の組織学的検査によって表された。血清の分析は、シャム群と比較してAP群における血清アミラーゼおよびインターロイキン-6サイトカイン濃度の有意な増加を示した。同時に、インターロイキン−6濃度は、AP群において腹腔内流体において有意に高かった。このモデルにおける膵炎の重症度は、濃度、注入の体積および圧力、および急性膵炎誘導の時間に比例して依存する。この技術の助けを借りて、重度の急性膵炎に対する連続腹膜洗浄の効果を研究することができる。

sodium-taurocholate-induced-severe-acute-pancreatitis-in-c57bl6-mice

Research

JoVE Journal

Medicine

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth has solicited growing clinical interest among physicians in the emergency department for various indications: (1) surveillance et monitoring of the intubated patient; (2) verification of the correct positioning of an endotracheal tube; (3) monitoring of a patient in cardiac arrest; (4) achieving normocapnia in intubated head trauma patients; (5) monitoring ventilation during procedural sedation. The video allows physicians to familiarize themselves with the use of capnography and the text offers a review of the theory and principals involved. In particular, the importance of CO2 for the organism, the relevance of measuring expired CO2, the differences between arterial and expired CO2, the material used in capnography with their artifacts and traps, will be reviewed. Since the main reluctance in the use of expired CO2 measurement is due to lack of correct knowledge concerning the physiopathology of CO2 by the physician, we hope that this explanation and the video sequences accompanying will help resolve this limitation.

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

This video describes how to perform CO2 measurement in intubated as well as spontaneously breathing patients. The main clinical indications refer to emergency situations: (1) verifying adequate positioning of an endotracheal tube; (2) achieving normocapnia in trauma patients; (3) monitoring ventilation in the case of procedural sedation.

期限切れのCO 2</sub救急部から気管挿管または自発呼吸患者の>の測定

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth ...

医学

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

マウスにおける急性肺障害を誘発するために圧力制御された換気

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

マウスにおける急性腎障害とポスト傷害線維の虚血再灌流モデル

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

縦二光子イメージングに続いてマウスの急性脳外傷

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

C57BL / 6マウスにおける全身性エリテマトーデス（SLE）のBM12誘導モデル

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

マウスの遠位人工肛門、転用 c57bl/6 マウス大腸炎の新たなモデル

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

マウスにおける急性心筋梗塞のリアルタイム圧解析

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

直接気管内リポ多糖刺激によるマウスの急性肺損傷の誘導

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

成体ゼブラフィッシュにおけるシスプラチンによる急性腎損傷モデル

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Fertility Preservation in Patients with Severe Ovarian Dysfunction

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, as well as ovarian surgeries. In unmarried women with severe ovarian dysfunction, fertility preservation is important for future pregnancies. Although oocyte cryopreservation is an established method for fertility preservation, these patients could only preserve a limited number of oocytes even after ovarian hyperstimulation, leading to repeated stimulations to ensure sufficient oocytes to guarantee future pregnancy. To solve this issue, we have recently developed a drug-free in vitro activation (IVA) procedure, which enable us to stimulate early stages of ovarian follicles to develop to the preantral follicle stage. These preantral follicles can respond to the unique protocol of gonadotropin stimulation, resulting in increased number of retrieved oocytes per ovarian stimulation for cryopreservation. The drug-free IVA comprised from the surgical approach and ovarian stimulation. We removed a part of cortex from one or both ovaries from patients under laparoscopic surgery. The ovarian cortical tissues were cut into small cubes to disrupt the Hippo signaling pathway and stimulate the development of early stage follicles. These cubes were grafted orthotropically into remaining ovaries as well as beneath the serosa of both Fallopian tubes. We have already published the surgical procedure of the drug-free IVA and the protocol of subsequent ovarian stimulation, but herein we present the details of laboratory methods required for drug-free IVA.

We present details of laboratory procedures for drug-free in vitro activation (IVA) of ovarian follicles for patients with severe ovarian dysfunction. This method could increase the number of retrievable oocytes per ovarian hyperstimulation and benefit fertility preservation for those patients.

重度卵巣機能障害患者における不妊治療の保存

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, ...