Name: sodium taurocholate induced severe acute pancreatitis in c57bl/6 mice
Uploaded: 2021-06-28T13:00:00
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Yazarlar, São Paulo Üniversitesi Tıp Kliniği'ndeki Mezuniyet Sonrası Programı sayesinde; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) ve São Paulo Üniversitesi Tıp Fakültesi (FMUSP).

Bu protokol USP Tıp Fakültesi, Num. Projesi: 1343/2019-CEUA: FMUSP hayvanlarının kullanımı için Etik Kurulu tarafından onaylanmıştır. Bu protokol için 6 haftalık, 20 ± 2 g ağırlığında C57BL/6 fareler kullanılmıştır (n = 9/grup).
1. Laparotomi
<ol><li>Hayvanları 1 mL şırınga ve 13x0.45mm iğne 26G 1/2 kullanarak deri altından (0.1 mL/10g vücut ağırlığı) olmak için ksilazin (10 mg/kg) ve ketamin çözeltisi (80 mg/kg) ile uyuşturun. Parmağı kıstırarak ye...

<ol>
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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>

Retrograd sodyum taurokolat infüzyonu ile akut pankreatit indükleme yöntemi sıçanlarda zaten gösterilmiştir22,23,24. 2008, 2010 ve 2015'te yayınlanan üç benzer eser, protokol için referans olarak görev yaptı3,14,15. Bu çalışmada, bu yöntemi C57BL /6 farelerde yeniden üretmek için tüm kritik adımları ve doğrulamak ...

İnsanlarda, safra kesesi taşlarının varlığı, kolesochal'ın terminal kısmının tıkanması, pankreas salgılarının akışını kesmesi ve pankreasta yoğun bir enflamatuar sürece neden olması nedeniyle pankreatitin en yaygın nedenidir, serum ve enflamatuar mediatörlerde sindirim enzimlerinin konsantrasyonunda bir artış ile1,2.
Akut pankreatit (AP) gelişimini açıklamak için iki farklı teori önerilmiştir. "Ortak kanal" teorisi, safra kesesinde bulunan taşların distal ortak safra kanalı sistemini tıkadığını ve safra salgısının pankreas kanalına retrograd akmasını sağladığını öne sürmemektedir. İkinci teori ("kanal tıkanıklığı" teorisi), pankreas kanalının fazla safra kesesi taşları tarafından tıkanmasının duodenuma pankreas salgısının akışında tıkanmaya neden olduğunu ve düktal hipertansiyona neden olduğunu göstermektedir3. Akut safra pankreatitine yol açan mekanizmalar tam olarak anlaşılamasa da, sonuç yoğun bir enflamatuar süreçtir. Sindirim enzimi patlaması ve pankreas kendi kendine sindirim histopatolojik değişikliklere, assitik sıvı ve serumda enflamatuar sitokinlerde (IL-1β, IL-6, TNF-α) artışa ve akut faz proteinlerinde artışa yol açar4,5,6.
Şiddetli akut pankreatit, birden fazla organın tutulumu ve yüksek mortalite riski nedeniyle klinik dikkati hak eden bir durumdur. Akut pankreatit (AP) üremesi için hayvan modelleri önemlidir, çünkü bunlar hastalığın patofizyolojik mekanizmalarını açıklar ve hastalığın ilk aşamalarından başlayarak enflamatuar olayların evrimini izlemeye yardımcı olur. Bu genellikle kliniklerde mümkün değildir2,7. Ek olarak, preklinik çalışmalarda pankreas dokularına erişim kolaydır, klinik koşullara bağlı değişikliklerin aydınlatılmasını kolaylaştırır8 izojenik türlerle çalışma, istenmeyen değişkenleri ortadan kaldırma ve klinik benzerliği insan durumunda gözlenen sonuçlarla yansıtma olasılığı ile birlikte9.
Sıçanlarda ve fare türlerinde akut pankreatit indüksiyonu için safralı ve safrasız modeller bilimsel literatürde sıkça incelenmiştir. Safrasız indüksiyon yöntemleri arasında kolesistokinin secretagogunun veya analog cerulein10'un supramaximal uyarıcı dozlarının verilmesi; neredeyse ölümcül dozlarda L-arginin verilmesi; veya etionin11 ile desteklenmiş kolin eksikliği olan bir diyetin yönetimi. Bu yöntemlerin çoğaltılması ve pankreas iltihabı ile sonuçlanması kolay olsa da, teoride AP'yi tetikleyen mekanizmaları (yani safra salgısının pankreas kanalına reflü) çoğaltır. Safra modeline hitap eden teknik, safra asitlerinin pankreas kanalına retrograd infüzyonuna dayanır ve bu protokolü gerçekleştirmek için iyi eğitimli araştırmacılar gerektirir. Sıçanlarda bu yöntem kullanılarak çeşitli çalışmalar yayınlanmıştır (görünüşe göre bu deneyler cerrahi prosedürler içerdiğinden teknik nedenlerle)12,13. Bununla birlikte, farelerdeki yaklaşım iltihaplanma çalışmasında daha ilginç sonuçlar sunabilir3,14,15. Bu çalışmada, C57BL/6 anestezik farelerde sodyum taurokolat infüzyonu ile şiddetli akut pankreatit üremesi için izlenecek adımların bir kontrol listesini göstereceğiz.
Antikorlarla deneylere ve gen ve protein ekspresyonunun analiz edilmesine ihtiyaç duyan çalışmalar için, bu hayvanlar için daha fazla malzeme cephaneliği ve çalışmalarla ilgili kullanılabilecek diğerlerinin yanı sıra izojenik ve nakavt türleriyle çalışma olasılığı nedeniyle farelerin kullanımı tercih edilir16. Fareler C57BL/6, başlangıçta antitümör aktivite ve immünoloji çalışması için geliştirilen doğuştan gelen bir fare türüdür. Bu suş, araştırmacılar tarafından izojenik olduğu için giderek daha fazla tercih edilmekte ve bu da bir deneyde daha az sayıda hayvanın kullanıldığı ve aynı grup17,18 arasındaki sonuçların daha az değişkenliği anlamına gelebilecek sonuçların daha fazla tekrarlanabilirliğine izinmektedir.
Perides ve ark. (2010)14 , sodyum taurokolat infüzyonu ile farelerde AP indüksiyonu için bir protokol yayınladı. Burada bu modeli, tanımlanmış bir hacim ve infüzyon hızı ile C57BL / 6 farelerde daha yüksek sodyum taurokolat konsantrasyonu (% 2.5) kullanarak güncelleriz (Şekil 1). Maksimum şiddet seviyesine farelerde indüksiyondan 12 saat içinde ulaşılır. IL-6 konsantrasyonunun hem serumda hem de periton boşluğunda yükselmesi AP'nin ilerlemesi ile ilişkilidir. Pratikte, anestezinin indüksiyonundan infüzyonun tamamlanmasına kadar geçen toplam tahmini süre hayvan başına 25 dk'dır. Bu deneyi eğitimli bir araştırmacının yapması şarttır. Çözeltinin ortak safra kanalına düzgün bir şekilde enjekte edildiğinden emin olmak için sodyum taurocholate yerine metilen mavisi kullanarak birkaç pilot eğitim seansı gerçekleştirin.

<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

Sodyum taurokolat infüzyonu ile safra akut pankreatit indüksiyonu, insan klinik durumunun temsili ve klinik safra pankreatitinin başlangıcına karşılık gelen enflamatuar olayların üremesi nedeniyle bilim topluluğu tarafından yaygın olarak kullanılmaktadır. Pankreas hasarının şiddeti, aşılanan safra asidinin konsantrasyonu, hızı ve hacmi ölçülerek değerlendirilebilir. Bu çalışma, protokol üremesinde kullanılan malzeme ve yöntemlerin güncellenmiş bir kontrol listesini sağlar ve bu akut pankreatit (AP) modelinin ana sonuçlarını gösterir. Önceki yayınların çoğu, bu modeli sıçanlarda yeniden üretmekle sınırlıdır. Bu yöntemi, çalışmayla ilgili olabilecek ek avantajlar (yani, bu hayvanlar için reaktifler ve antikorlardan oluşan bir cephaneliğin mevcudiyeti ve genetiği değiştirilmiş fare suşlarıyla çalışma imkanı) sağlayan farelerde uyguladık. Farelerde akut pankreatit indüksiyonu için, C57BL/6 farelerde 3 dakika boyunca 10 μL/dk infüzyon hızında% 2.5 sodyum taurocholate tanımlanmış bir doz ile sistematik bir protokol sunuyoruz ve indüksiyonun 12 saat içinde maksimum şiddet seviyesine ulaşıyor ve yöntemi doğrulayan sonuçlarla sonuçları vurguluyoruz. Uygulama ve teknikle, anestezinin indüksiyonundan infüzyonun tamamlanmasına kadar toplam tahmini süre hayvan başına 25 dk'dır.

Pankreatit şiddeti Schmidt'in ölçeğine göre 0-3 arasında puanlanmıştır21 burada sıfır yokluğa karşılık gelir, 1 hafif bir varlığa karşılık gelir (%<25), 2 ılımlı bir varlığa karşılık gelir (%25 ila 50) ve 3 yoğun bir varlığa karşılık gelir (%> 50) (Tablo 1). Yapılan ölçümler plazma amilaz aktivitesi, pankreas ödemi, acınar hücre, yaralanma/nekroz, pankreas iltihabı (H&E-lekeli bölümlerin histoloji analizi...

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

Şiddetli akut pankreatit için hayvan modelleri, ilk aşamada patofizyolojik değişikliklerin incelenmesini sağlayarak enflamatuar olayların evriminin gözlemini kolaylaştırır. Burada, anestezi edilmiş C57BL/6 farelerin pankreas kanalına sodyum taurokolatın retrograd infüzyonu ile şiddetli akut safra pankreatit indüksiyonu için bir protokol sunuyoruz.

C57BL/6 Farelerde Sodyum Taurokolat İndüklenen Şiddetli Akut Pankreatit

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

Bu yöntem akut safra pankreatitinde meydana gelen enflamatuar olayların yeniden üretilmesi için kullanılabilir. Pankreas dokusundaki değişiklikleri erken evrelerinde incelemek mümkündür, bu da klinikte genellikle mümkün değildir. Bu tekniğin temel avantajları, eğitimli bir araştırmacı için hızlı ve kolay üremesi ve insanlarda akut pankreatit ile benzer sonuçlar sunmasıdır.Yöntem, pankreas ve bitişik organlardaki enflamatuar aracılık eden hücrelerin incelenmesine izin verdiğinden, ilaç moleküllerinin farmakolojik immün yanıtlarını ve diğer terapötik müdahaleleri incelemek için kullanılabilir. Bu protokolü denerken, organların yerini belirlemek ve cannülasyon bölgesini kurmak çok önemlidir. Organların yerini ezberle ve önce metilen mavisi gibi renkli bir çözelti kullanarak enjeksiyon uygulamasını yapın.Anestezinin derinliğini bir ayak parmağı sıkışması ile kontrol ettikten sonra, uyuşturulmış bir farenin karın bölgesini% 5 povidon iyot çözeltisi ile temizleyin. Bir düzeltici kullanarak, göğüs ve alt karın arasındaki saçları çıkarın ve yaklaşık iki santimetrekarelik bir cerrahi bölgeyi% 70 alkolle temizleyin. Hayvanı cerrahi tahtada hareketsiz hale getirmek ve cildin beş milimetresini karnın üst kısmında ve xiphoid işleminin bir santimetre altında yatay olarak kesmek için makas kullanın.Laparotomiyi boşluğun minimum maruziyetiyle gerçekleştirmek için periton üzerindeki kesimi tekrarlayın. Bir retraktör kullanarak, karaciğeri bağırsaktan yaklaşık bir santimetre uzaklıktaki başa doğru çekin ve pankreasın ve duodenumun bölgesini bulun. Kümesleri kullanarak karaciğeri hayvan kafasına doğru kaldırın.İnce bağırsağın kısmını hafifçe çekerek, ortak safra kanalının distal kısmının daha iyi görülmesi için iki yanal ucu 6-0 polipropilen dikişle sabitlayın. Bir mikro damar klipsi kullanarak, proksimal ortak safra kanalını geçici olarak tıkayın. Organı karın boşluğundan dışarı maruz bırak.Sonra distal ortak safra kanalının geçici bir tıkanıklığını 8-0 ile yapın Dikiş. Ortak safra kanalına erişmek için, ince bağırsağın duvarının beyazımsı bir parçası olarak görünen periampullary bölgesini delin. 0,54 milimetre polietilen tüpe bağlı 0,4 milimetrelik bir iğne ile, infüzyon pompasını üç dakika boyunca 10 gram vücut ağırlığı başına 10 mikrolitre sabit bir hızda% 2,5 sodyum taurokolat çözelti infüzyonu için başlatın.İnfüzyondan sonra, mikro kap klipsini çıkarın, geçici 8-0 dikiş ve pankreas safra kanalından enjeksiyon iğnesi. Karnı 6-0 nonabsorbent monofilament polipropilen dikiş ile dikin. Laparotomi ile son dikiş arasındaki süre 30 dakikadan fazla olmamalıdır.Ameliyattan sonra, hayvanları odun talaşları ve su ve yiyecek reklam libitum ile kaplı polietilen kutularda barındırın. Hayvanı deri geri çekilmiş olarak hafifçe tutun, göz küresinin hafif bir çıkıntısını teşvik edin ve hayvanı gözü yukarı bakacak şekilde konumlandırın. Hayvanın gözüne lokal anestezi içeren bir damla göz merhemi aşıla.Kılcal bir tüpün ucunu gözün medial köşesine yerleştirin ve yaklaşık 30 ila 45 derecelik bir açıyla göz küresinin altına hafifçe yerleştirin. Kan akışı başlayana kadar kılcal boruyu döndürün. Koleksiyon bittikten sonra göz kapaklarını hafif sıkıştırma ile kapalı tutarak homeostazdan emin olun.Daha sonra, 27 kalibrelik bir iğne kullanarak, farenin periton boşluğuna dört mililitre buz gibi PBS enjekte edin. Enjeksiyondan sonra, yapışık hücreleri çıkarmak için peritona 10 saniye boyunca hafifçe masaj yapın. Makas ve cımbız kullanarak, iç deride 0,5 santimetrelik küçük bir kesim yapın ve karın boşluğunu ortaya çıkarmak için kas yapın.Peritona bir ampul pipet yerleştirin ve yağ dokusunu aspire etmemeye dikkat ederek mümkün olduğunca fazla sıvı toplayın. Daha fazla işlem için pankreasın bir kısmını baş bölgeden toplayın. Toplanan sıvıyı buzda tutulan tüplere biriktirin.Daha sonra periton sıvısını beş dakika boyunca 250 kez G'de santrifüjleyin ve interlökin-6 dosing için süpernatantı stoklayın. Serum elde etmek için, toplanan kan örneklerini 15 dakika boyunca 700 kez G'de santrifüjleyin ve amilaz ve interlökin-6 dosing için süpernatantı stoklayın. Akut pankreatit indüksiyonundan 12 saat sonra pankreas dokusunun analizi, sham grubundan alınan doku örneklerine kıyasla etkilenen organda yoğun iltihaplanma olduğunu gösterdi.Sham ve APTA'dan alınan doku örneklerinin hematoksilin eozin lekelenimi sonrasında histolojik inceleme ile benzer sonuçlar temsil edildi. Kan serumunun analizi, AP grubunda serum amilaz ve interlökin-6 sitokin konsantrasyonunda sham grubuna göre anlamlı bir artış olduğunu gösterdi. Aynı zamanda AP grubunda periton kavite sıvısında interleukin-6 konsantrasyonu anlamlı olarak daha yüksekti.Bu modeldeki pankreatitin şiddeti orantılı olarak konsantrasyona, infüzyonun hacmine ve basıncına ve akut pankreatit indüksiyon süresine bağlıdır. Bu tekniğin yardımıyla sürekli periton lavajının şiddetli akut pankreatit üzerindeki etkileri incelenebilir.

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.

Süresi doldu CO 2</subAcil Servisten Entübe veya kendiliğinden nefes alamayan hastalarda> Ölçüm

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 ...

Tıp

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.

Farelerde Akut Akciğer Hasarı tetikleyebilecek Basınç Kontrollü Havalandırma

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.

Farelerde Akut Böbrek Hasarı ve Post Yaralanma Fibrozis iskemi-reperfüzyon Model

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.

Boyuna İki foton görüntüleme ile takip Farelerde Akut Beyin Travma

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 FARELERDE SLE bm12 İndüklenebilir Modeli (SLE)

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.

Fare Distal kolostomi, C57BL/6 farelerde saptırma kolit roman modeli

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.

Akut miyokard infarktüsünde farelerde gerçek zamanlı basınç-hacim Analizi

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.

Doğrudan Intratracheal Lipopolysaccharide Instilasyon ile farelerde akut akciğer hasarının İndükleme

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.

Yetişkin Zebra Balıklarında Cisplatin tarafından İndüklenen Akut Böbrek Yaralanması Modeli

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.

Şiddetli Yumurtalık Disfonksiyonu Olan Hastalarda Doğurganlığın Korunması

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