Name: sodium taurocholate induced severe acute pancreatitis in c57bl/6 mice
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De auteurs dankzij het Post Graduation Program in de medische kliniek van de Universiteit van São Paulo; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) en University of São Paulo Medical School (FMUSP).

Dit protocol werd goedgekeurd door de Ethische Commissie voor het gebruik van dieren van de USP Medicine School, Num. Project: 1343/2019-CEUA: FMUSP. Voor dit protocol werden C57BL/6 muizen van 6 weken oud met een gewicht van 20 ± 2 g gebruikt (n = 9/groep).
1. Laparotomie
<ol><li>Verdoof dieren met xylazine (10 mg/kg) en ketamine-oplossing (80 mg/kg), subcutaan (0,1 ml/10 g lichaamsgewicht) met behulp van een spuit van 1 ml en een naald van 13x0,45 mm 26G 1/2. Controleer op voldoende anest...

<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. 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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>

De methode voor het induceren van acute pancreatitis door retrograde natrium taurocholaatinfusie is al aangetoond bij ratten22,23,24. Drie soortgelijke werken, gepubliceerd in 2008, 2010 en 2015, dienden als referentie voor het protocol3,14,15. In dit werk vermelden we alle kritieke stappen voor het reproduceren van deze methode in C57B...

Bij mensen is de aanwezigheid van galstenen de meest voorkomende oorzaak van pancreatitis als gevolg van de obstructie van het terminale deel van de choledochal, waardoor de stroom van pancreassecreties wordt onderbroken en een intens ontstekingsproces in de pancreas wordt veroorzaakt, met een toename van de concentratie van spijsverteringsenzymen in het serum en ontstekingsmediatoren1,2.
Er zijn twee verschillende theorieën voorgesteld om de ontwikkeling van acute pancreatitis (AP) te verklaren. De "common channel" -theorie suggereert dat de stenen die in de galblaas aanwezig zijn, het distale gemeenschappelijke galkanaalsysteem belemmeren, waardoor galsecretie retrograde in de pancreaskanalen kan stromen. De tweede theorie (de "kanaalobstructie" -theorie) suggereert dat de obstructie van het pancreaskanaal door overtollige galstenen een blokkade veroorzaakt in de stroom van pancreassecretie naar de twaalfvingerige darm, waardoor ductale hypertensie3 ontstaat. Hoewel de mechanismen die leiden tot acute gal pancreatitis niet volledig worden begrepen, is het resultaat een intens ontstekingsproces. Spijsverteringsenzymuitbarsting en zelfvertering van de alvleesklier leiden tot histopathologische veranderingen, een toename van inflammatoire cytokines (IL-1β, IL-6, TNF-α) in ascitisch vocht en serum, en een toename van acute fase-eiwitten4,5,6.
Ernstige acute pancreatitis is een aandoening die klinische aandacht verdient vanwege de betrokkenheid van meerdere organen en een hoog sterfterisico. Diermodellen voor de reproductie van acute pancreatitis (AP) zijn belangrijk omdat deze de pathofysiologische mechanismen van de ziekte verklaren en helpen bij het volgen van de evolutie van ontstekingsgebeurtenissen, te beginnen met de beginfasen van de ziekte. Dit is meestal niet mogelijk in de klinieken2,7. Bovendien is de toegang tot pancreasweefsels gemakkelijk in preklinische studies, wat de opheldering van veranderingen in verband met klinische omstandigheden8 bevordert, samen met de mogelijkheid om met isogene soorten te werken, ongewenste variabelen te elimineren en klinische gelijkenis te weerspiegelen met de uitkomsten waargenomen in de menselijke conditie9.
Gal- en niet-galmodellen voor de inductie van acute pancreatitis bij ratten en muizensoorten zijn vaak bestudeerd in de wetenschappelijke literatuur. Niet-galwegmethoden voor inductie omvatten toediening van supramaximale stimulerende doses van de cholecystokinine secretagogue of zijn analoge cerulein10; toediening van bijna dodelijke doses L-arginine; of toediening van een choline-deficiënt dieet aangevuld met ethionine11. Hoewel deze methoden gemakkelijk te reproduceren zijn en resulteren in pancreasontsteking, repliceren ze niet de mechanismen die in theorie AP veroorzaken (d.w.z. de reflux van galsecretie in de pancreaskanalen). De techniek die het galmodel aanpakt, is gebaseerd op de retrograde infusie van galzuren in de pancreaskanalen en vereist goed opgeleide onderzoekers om dit protocol uit te voeren. Er zijn verschillende studies gepubliceerd met behulp van deze methode bij ratten (blijkbaar om technische redenen, aangezien deze experimenten chirurgische ingrepen omvatten)12,13. De aanpak bij muizen kan echter interessantere uitkomsten bieden in de studie van ontsteking3,14,15. In deze studie zullen we een checklist laten zien van de stappen die moeten worden gevolgd voor de reproductie van ernstige acute pancreatitis door infusie van natrium taurocholaat bij C57BL/6 verdoofde muizen.
Voor werken die de noodzaak van experimenten met antilichamen en analyse van de gen- en eiwitexpressie met zich meebrengen, heeft het gebruik van muizen de voorkeur vanwege het grotere arsenaal aan materialen voor deze dieren en de mogelijkheid om te werken met isogene en knock-out soorten, onder andere die relevant kunnen worden gebruikt voor studies16. Muizen C57BL/6 is een inteelt muizenstam die oorspronkelijk is ontwikkeld voor de studie van antitumoractiviteit en immunologie. Deze stam krijgt steeds meer de voorkeur van onderzoekers omdat het isogeen is, waardoor een grotere reproduceerbaarheid van de resultaten mogelijk is, wat het gebruik van een kleiner aantal dieren in een experiment en minder variabiliteit van de resultaten tussen dezelfde groep kan impliceren17,18.
Perides et al. (2010)14 publiceerden een protocol voor AP-inductie bij muizen door natrium taurocholaatinfusie. Hier werken we dit model bij met behulp van een hogere natrium taurocholaatconcentratie (2,5%) in C57BL/6 muizen, met een gedefinieerd volume en snelheid van infusie (figuur 1). Het maximale niveau van ernst wordt bereikt binnen 12 uur na inductie bij muizen. De verhoging van de concentratie van IL-6, zowel in het serum als in de peritoneale holte, is gecorreleerd met de progressie van AP. Met de praktijk is de totale geschatte tijd vanaf de inductie van anesthesie tot de voltooiing van de infusie 25 minuten per dier. Het is essentieel dat een getrainde onderzoeker dit experiment uitvoert. Om ervoor te zorgen dat de oplossing op de juiste manier in het gemeenschappelijke galkanaal wordt geïnjecteerd, voert u verschillende pilotentrainingen uit met methyleenblauw in plaats van natrium taurocholaat.

<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

Biliaire acute pancreatitis inductie door natrium taurocholaat infusie is op grote schaal gebruikt door de wetenschappelijke gemeenschap als gevolg van de representatie van de menselijke klinische conditie en reproductie van inflammatoire gebeurtenissen die overeenkomen met het begin van klinische gal pancreatitis. De ernst van pancreasschade kan worden beoordeeld door de concentratie, snelheid en het volume van het geïnfundeerde galzuur te meten. Deze studie biedt een bijgewerkte checklist van de materialen en methoden die worden gebruikt in de protocolreproductie en toont de belangrijkste resultaten van dit acute pancreatitis (AP) -model. De meeste eerdere publicaties hebben zich beperkt tot het reproduceren van dit model bij ratten. We hebben deze methode toegepast bij muizen, wat extra voordelen biedt (d.w.z. de beschikbaarheid van een arsenaal aan reagentia en antilichamen voor deze dieren, samen met de mogelijkheid om te werken met genetisch gemodificeerde muizenstammen) die relevant kunnen zijn voor de studie. Voor acute pancreatitis-inductie bij muizen presenteren we een systematisch protocol, met een gedefinieerde dosis van 2,5% natrium taurocholaat bij een infusiesnelheid van 10 μL / min gedurende 3 minuten bij C57BL / 6-muizen die zijn maximale ernstniveau bereikt binnen 12 uur na inductie en de resultaten markeren met resultaten die de methode valideren. Met oefening en techniek is de totale geschatte tijd, van de inductie van anesthesie tot de voltooiing van de infusie, 25 minuten per dier.

De ernst van pancreatitis werd gescoord tussen 0-3 volgens de schaal21 van Schmidt, waarbij nul overeenkomt met de afwezigheid, 1 overeenkomt met een milde aanwezigheid (<25%), 2 overeenkomt met een matige aanwezigheid (tussen 25 en 50%) en 3 overeenkomt met een intense aanwezigheid (> 50%) (tabel 1). De uitgevoerde metingen waren plasma amylase activiteit, pancreasoedeem, acinar cel, letsel/necrose, pancreasontsteking (door histologische analyse v...

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

Diermodellen voor ernstige acute pancreatitis maken de studie van pathofysiologische veranderingen in de beginfase mogelijk, waardoor de observatie van de evolutie van ontstekingsgebeurtenissen wordt vergemakkelijkt. Hier bieden we een protocol voor de inductie van ernstige acute gal pancreatitis door retrograde infusie van natrium taurocholaat in het pancreaskanaal van verdoofde C57BL / 6-muizen.

Natrium taurocholaat geïnduceerde ernstige acute pancreatitis bij C57BL/6 muizen

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

Deze methode kan worden gebruikt voor het reproduceren van de ontstekingsgebeurtenissen die optreden bij acute gal pancreatitis. Het is mogelijk om veranderingen in pancreasweefsel in de vroege stadia te bestuderen, wat over het algemeen niet mogelijk is in de kliniek. De belangrijkste voordelen van deze techniek zijn dat het snel en gemakkelijk te reproduceren is voor een getrainde onderzoeker, en het biedt vergelijkbare resultaten als acute pancreatitis bij mensen.De methode kan worden gebruikt om farmacologische immuunresponsen van geneesmiddelmoleculen en andere therapeutische interventies te bestuderen, omdat het de studie van ontstekingsbemiddelende cellen in de pancreas en aangrenzende organen mogelijk maakt. Bij het proberen van dit protocol is het lokaliseren van de organen en het vaststellen van het cannulatiegebied cruciaal. Onthoud de locatie van de organen en oefen de injectietoepassing eerst uit met behulp van een gekleurde oplossing zoals methyleenblauw.Na het controleren van de diepte van de anesthesie met een teen knijpen, reinigt u de buikstreek van een verdoofde muis met een 5% povidon jodiumoplossing. Verwijder met behulp van een trimmer het haar tussen de borst en de onderbuik en reinig een chirurgisch gebied van ongeveer twee vierkante centimeter met 70% alcohol. Immobiliseer het dier op de chirurgische plaat en gebruik een schaar om vijf millimeter van de huid horizontaal op het bovenste deel van de buik en één centimeter onder het xiphoid-proces te knippen.Herhaal de snede op het peritoneum om de laparotomie uit te voeren met minimale blootstelling van de holte. Trek met behulp van een retractor de lever naar het hoofd op ongeveer een centimeter van de darm en lokaliseer het gebied van de pancreas en de twaalfvingerige darm. Til met behulp van een tang de lever op naar de dierenkop.Door voorzichtig aan het deel van de dunne darm te trekken, bevestigt u de twee laterale uiteinden met een 6-0 polypropyleen hechtdraad voor een beter zicht op het distale deel van de gemeenschappelijke galweg. Sluit met behulp van een microvatclip de proximale gemeenschappelijke galweg tijdelijk af. Stel het orgaan bloot uit de buikholte.Maak vervolgens een tijdelijke occlusie van de distale gemeenschappelijke galwegen met een 8-0 hechting. Om toegang te krijgen tot het gemeenschappelijke galkanaal, doorprik het periampullaire gebied, dat verschijnt als een witachtig deel van de wand van de dunne darm. Met een naald van 0,4 millimeter aangesloten op een polyethyleenbuis van 0,54 millimeter, start u de infusiepomp voor infusie met 2,5% natrium taurocholaatoplossing met een constante snelheid van 10 microliter per 10 gram lichaamsgewicht gedurende drie minuten.Verwijder na de infusie de microvatclip, de tijdelijke 8-0 hechting en de injectienaald uit de galwegen van de pancreas. Hecht de buik met een 6-0 niet-absorberende monofilament polypropyleen hechting. De tijd tussen de laparotomie en de eindnaad mag niet meer dan 30 minuten zijn.Na de operatie huisvest je de dieren in polyethyleen dozen bekleed met houtkrullen en water en voedsel ad libitum. Houd het dier voorzichtig vast met de naar achteren getrokken huid, bevordert een licht uitsteeksel van de oogbol en plaats het dier met het oog naar boven gericht. Breng een druppel oogzalf met lokale verdoving in het oog van het dier.Plaats het uiteinde van een capillaire buis in de mediale ooghoek en breng deze voorzichtig onder de oogbol in een hoek van ongeveer 30 tot 45 graden. Draai de capillaire buis totdat de bloedstroom begint. Zodra de verzameling voorbij is, zorg dan voor homeostase door de oogleden gesloten te houden met lichte compressie.Injecteer later met behulp van een naald van 27 gauge vier milliliter ijskoude PBS in de peritoneale holte van de muis. Masseer na de injectie het peritoneum gedurende 10 seconden voorzichtig om aangehechte cellen te verwijderen. Maak met een schaar en pincet een kleine snede van 0,5 centimeter op de binnenhuid en spieren om de buikholte bloot te leggen.Plaats een bolpipet in het peritoneum en verzamel zoveel mogelijk vocht, zorg ervoor dat vetweefsel niet wordt opgespiratiseerd. Verzamel een deel van de alvleesklier uit het hoofdgebied voor verdere verwerking. Deponeer de verzamelde vloeistof in buizen die op ijs worden bewaard.Centrifugeer vervolgens de peritoneale vloeistof gedurende vijf minuten op 250 maal G en bewaar het supernatant voor interleukine-6 dosering. Om serum te verkrijgen, centrifugeert u de verzamelde bloedmonsters gedurende 15 minuten op 700 maal G en slaat u het supernatant op voor amylase en interleukine-6 dosering. Analyse van pancreasweefsel 12 uur na inductie van acute pancreatitis toonde intense ontsteking in het aangetaste orgaan in vergelijking met weefselmonsters uit de schijngroep.Vergelijkbare resultaten werden weergegeven door histologisch onderzoek na hematoxyline eosinekleuring van weefselmonsters van sham en APTA. Analyse van bloedserum toonde een significante toename van de serum amylase en interleukine-6 cytokineconcentratie in de AP-groep in vergelijking met de shamgroep. Tegelijkertijd was de interleukine-6-concentratie significant hoger in peritoneale holtevloeistof in de AP-groep.De ernst van pancreatitis in dit model is proportioneel afhankelijk van de concentratie, het volume en de druk van de infusie en het tijdstip van acute pancreatitisinductie. Met behulp van deze techniek kunnen de effecten van continue peritoneale lavage op ernstige acute pancreatitis worden bestudeerd.

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.

Verlopen CO 2 Meting in geïntubeerde of spontaan ademende patiënten van de Spoedeisende Hulp

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 ...

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.

Druk gecontroleerde ventilatie te Acute Lung Injury in Muizen Braken

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.

Ischemie-reperfusie model van Acute Kidney Injury en Post Letsel Fibrosis in Muizen

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.

Acute Brain Trauma in Muizen gevolgd door Longitudinale Twee-foton Imaging

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.

De bm12 Induceerbare model van systemische lupus erythematosus (SLE) in C57BL / 6 muizen

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.

Lymfkliertest distale colostoma, een nieuw Model van afleiding Colitis in C57BL/6 muizen

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.

Real-time druk-volume analyse van acuut myocardinfarct in muizen

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.

Inducerende acute Long blessure bij muizen door directe Intratracheal lipopolysaccharide instillatie

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.

Acuut nierletselmodel geïnduceerd door Cisplatine bij volwassen zebravissen

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.

Vruchtbaarheidsbehoud bij patiënten met ernstige ovariële disfunctie

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