Gli autori vorrebbero riconoscere il dottor Katy Murray al Centro Risorse Internazionali di Zebrafish; Il dottor Stacey Huppert e Kari Huppert al CCHMC, per i loro consigli utili sul protocollo; E il servizio veterinario CCHMC, per la cura dei pesci. Questo lavoro è stato sostenuto dalla sovvenzione NIH R00AA020514 e da una borsa di studio del Centro per la Genomica Pediatrica a CCHMC (a CY). È stato anche sostenuto in parte da NIH concedere P30 DK078392 (Integrative Morphology Core) del Centro di Ricerca Digestiva Disease Core a Cincinnati.

<ol>
	<li>Yoon, Y. H., Chen, C. M., Yi, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Surveillance report #100: Liver cirrhosis mortality in the United States: National, State, and regional trends. , 2000-2011 (2014).</li><li>Singal, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolving+frequency+and+outcomes+of+liver+transplantation+based+on+etiology+of+liver+disease.">Evolving frequency and outcomes of liver transplantation based on etiology of liver disease.</a> Transplantation. 95 (5), 755-760 (2013).</li><li>Louvet, A., Mathurin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcoholic+liver+disease:+mechanisms+of+injury+and+targeted+treatment.">Alcoholic liver disease: mechanisms of injury and targeted treatment.</a> Nat Rev Gastroenterol Hepatol. 12 (4), 231-242 (2015).</li><li>Ki, S. 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=Interleukin-22+treatment+ameliorates+alcoholic+liver+injury+in+a+murine+model+of+chronic-binge+ethanol+feeding:+role+of+signal+transducer+and+activator+of+transcription+3.">Interleukin-22 treatment ameliorates alcoholic liver injury in a murine model of chronic-binge ethanol feeding: role of signal transducer and activator of transcription 3.</a> Hepatology. 52 (4), 1291-1300 (2010).</li><li>Tsuchiya, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interstrain+differences+in+liver+injury+and+one-carbon+metabolism+in+alcohol-fed+mice.">Interstrain differences in liver injury and one-carbon metabolism in alcohol-fed mice.</a> Hepatology. 56 (1), 130-139 (2012).</li><li>Koteish, A., Yang, S., Lin, H., Huang, X., Diehl, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+ethanol+exposure+potentiates+lipopolysaccharide+liver+injury+despite+inhibiting+Jun+N-terminal+kinase+and+caspase+3+activation.">Chronic ethanol exposure potentiates lipopolysaccharide liver injury despite inhibiting Jun N-terminal kinase and caspase 3 activation.</a> J Biol Chem. 277 (15), 13037-13044 (2002).</li><li>Leo, M. A., Lieber, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+fibrosis+after+long-term+administration+of+ethanol+and+moderate+vitamin+A+supplementation+in+the+rat.">Hepatic fibrosis after long-term administration of ethanol and moderate vitamin A supplementation in the rat.</a> Hepatology. 3 (1), 1-11 (1983).</li><li>Tsukamoto, 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=Severe+and+progressive+steatosis+and+focal+necrosis+in+rat+liver+induced+by+continuous+intragastric+infusion+of+ethanol+and+low+fat+diet.">Severe and progressive steatosis and focal necrosis in rat liver induced by continuous intragastric infusion of ethanol and low fat diet.</a> Hepatology. 5 (2), 224-232 (1985).</li><li>Tsukamoto, H., Mkrtchyan, H., Dynnyk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intragastric+ethanol+infusion+model+in+rodents.">Intragastric ethanol infusion model in rodents.</a> Methods Mol Biol. 447, 33-48 (2008).</li><li>Howarth, D. L., Passeri, M., Sadler, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drinks+like+a+fish:+using+zebrafish+to+understand+alcoholic+liver+disease.">Drinks like a fish: using zebrafish to understand alcoholic liver disease.</a> Alcohol Clin Exp Res. 35 (5), 826-829 (2011).</li><li>Howarth, D. L., Yin, C., Yeh, K., Sadler, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+hepatic+dysfunction+parameters+in+two+models+of+fatty+liver+disease+in+zebrafish+larvae.">Defining hepatic dysfunction parameters in two models of fatty liver disease in zebrafish larvae.</a> Zebrafish. 10 (2), 199-210 (2013).</li><li>Lin, J. 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=Development+of+an+Animal+Model+for+Alcoholic+Liver+Disease+in+Zebrafish.">Development of an Animal Model for Alcoholic Liver Disease in Zebrafish.</a> Zebrafish. , (2015).</li><li>Passeri, M. J., Cinaroglu, A., Gao, C., Sadler, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+steatosis+in+response+to+acute+alcohol+exposure+in+zebrafish+requires+sterol+regulatory+element+binding+protein+activation.">Hepatic steatosis in response to acute alcohol exposure in zebrafish requires sterol regulatory element binding protein activation.</a> Hepatology. 49 (2), 443-452 (2009).</li><li>Tsedensodnom, O., Vacaru, A. M., Howarth, D. L., Yin, C., Sadler, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+metabolism+and+oxidative+stress+are+required+for+unfolded+protein+response+activation+and+steatosis+in+zebrafish+with+alcoholic+liver+disease.">Ethanol metabolism and oxidative stress are required for unfolded protein response activation and steatosis in zebrafish with alcoholic liver disease.</a> Dis Model Mech. 6 (5), 1213-1226 (2013).</li><li>Yin, C., Evason, K. J., Maher, J. J., Stainier, 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=The+bHLH+transcription+factor+Hand2+marks+hepatic+stellate+cells+in+zebrafish:+Analysis+of+stellate+cell+entry+into+the+developing+liver.">The bHLH transcription factor Hand2 marks hepatic stellate cells in zebrafish: Analysis of stellate cell entry into the developing liver.</a> Hepatology. , (2012).</li><li>Lassen, 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=Molecular+cloning,+baculovirus+expression,+and+tissue+distribution+of+the+zebrafish+aldehyde+dehydrogenase+2.">Molecular cloning, baculovirus expression, and tissue distribution of the zebrafish aldehyde dehydrogenase 2.</a> Drug Metab Dispos. 33 (5), 649-656 (2005).</li><li>Reimers, M. J., Hahn, M. E., Tanguay, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+zebrafish+alcohol+dehydrogenases+share+common+ancestry+with+mammalian+class+I,+II,+IV,+and+V+alcohol+dehydrogenase+genes+but+have+distinct+functional+characteristics.">Two zebrafish alcohol dehydrogenases share common ancestry with mammalian class I, II, IV, and V alcohol dehydrogenase genes but have distinct functional characteristics.</a> J Biol Chem. 279 (37), 38303-38312 (2004).</li><li>Zhang, C., Ellis, J. L., Yin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+vascular+endothelial+growth+factor+signaling+facilitates+liver+repair+from+acute+ethanol-induced+injury+in+zebrafish.">Inhibition of vascular endothelial growth factor signaling facilitates liver repair from acute ethanol-induced injury in zebrafish.</a> Dis Model Mech. , (2016).</li><li>Vonghia, 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=Acute+alcohol+intoxication.">Acute alcohol intoxication.</a> Eur J Intern Med. 19 (8), 561-567 (2008).</li><li>Wittekind, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traditional+staining+for+routine+diagnostic+pathology+including+the+role+of+tannic+acid.+1.+Value+and+limitations+of+the+hematoxylin-eosin+stain.">Traditional staining for routine diagnostic pathology including the role of tannic acid. 1. Value and limitations of the hematoxylin-eosin stain.</a> Biotech Histochem. 78 (5), 261-270 (2003).</li><li>Westerfield, 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> The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio Rerio). , (2007).</li><li>Theise, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+alcoholic+liver+disease.">Histopathology of alcoholic liver disease.</a> Clinical Liver Disease. 2 (2), (2013).</li><li>Lorent, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+Jagged-mediated+Notch+signaling+disrupts+zebrafish+biliary+development+and+generates+multi-organ+defects+compatible+with+an+Alagille+syndrome+phenocopy.">Inhibition of Jagged-mediated Notch signaling disrupts zebrafish biliary development and generates multi-organ defects compatible with an Alagille syndrome phenocopy.</a> Development. 131 (22), 5753-5766 (2004).</li><li>Huang, M., Xu, J., Shin, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+Ethanol-induced+Fibrotic+Liver+Model+in+Zebrafish+to+Study+Progenitor+Cell-mediated+Hepatocyte+Regeneration.">Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration.</a> J Vis Exp. (111), (2016).</li><li>Paredes, J. F., Lopez-Olmeda, J. F., Martinez, F. J., Sanchez-Vazquez, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Daily+rhythms+of+lipid+metabolic+gene+expression+in+zebra+fish+liver:+Response+to+light/dark+and+feeding+cycles.">Daily rhythms of lipid metabolic gene expression in zebra fish liver: Response to light/dark and feeding cycles.</a> Chronobiol Int. 32 (10), 1438-1448 (2015).</li><li>Meeker, N. D., Hutchinson, S. A., Ho, L., Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+PCR-ready+genomic+DNA+from+zebrafish+tissues.">Method for isolation of PCR-ready genomic DNA from zebrafish tissues.</a> Biotechniques. 43 (5), 610-614 (2007).</li><li>van der Velden, Y. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serine-threonine+kinase+LKB1+is+essential+for+survival+under+energetic+stress+in+zebrafish.">The serine-threonine kinase LKB1 is essential for survival under energetic stress in zebrafish.</a> Proc Natl Acad Sci U S A. 108 (11), 4358-4363 (2011).</li></ol>

Gli autori dichiarano di non avere interessi finanziari concorrenti.

Il protocollo attuale descrive una procedura dettagliata per il trattamento acuto di etanolo nelle larve di zebrafish e le successive analisi istopatologiche con la colorazione H &amp; E. Il trattamento acuto di etanolo dovrebbe essere effettuato non prima di 96 ore dopo la fecondazione, in quanto questo è il momento in cui il fegato di zebra inizia a esprimere enzimi che metabolizzano l&#39;alcool 13 . 2% di etanolo è la dose massima che le larve possono tollerare 13 <s...

La malattia alcolica del fegato (ALD), causata dal consumo di alcol, è una delle principali cause della morbilità e della mortalità correlate all&#39;alcol. Negli Stati Uniti, quasi la metà delle morti per malattie epatiche coinvolge l&#39;alcool 1 e l&#39;ALD è responsabile di quasi 1 su 3 trapianti di fegato 2 . ALD ha un ampio spettro. La steatoside, caratterizzata da un eccesso di accumulo lipidico negli epatociti, si verifica nella fase iniziale del consumo pesante ed è reversibile alla cessazione dell&#39;uso dell&#39;alcol. Sotto l&#39;influenza dei fattori genetici e ambientali e dell&#39;assunzione continua dell&#39;alcol, la steatosi epatica può progredire all&#39;epatite alcolica e, alla fine, alla cirrosi 3 . Gli studi che utilizzano i modelli di roditore ALD hanno fornito conoscenze approfondite sulla malattia, ma hanno limitazioni (esaminate nel riferimento 3 ). L&#39;alimentazione orale di una dieta dell&#39;alcool provoca solo steatosi nei roditori 4 , &lt;/ Sup&gt; 5 . Lo sviluppo di infiammazione e fibrosi richiede un secondo insulto 6 , 7 o infusione intragastrica cronica, che è invasiva e tecnicamente impegnativa 8 , 9 . Il teleost zebrafish sviluppa anche lesioni epatiche in risposta sia al trattamento alcolico cronico che acuto 10 , 11 , 12 , 13 , 14 , 15 . In particolare, il pesce zebra di larve rappresenta un attraente organismo modello complementare in cui studiare lesioni al fegato alcoliche acute , 10 , 11 , 13 , 15 . Il fegato di zebrafish è funzionale e produce enzimi chiave per il metabolismo dell&#39;etanolo da 4 giorni(Dpf) 13 , 16 , 17. L&#39; etanolo può essere aggiunto direttamente all&#39;acqua e l&#39;esposizione al 2% di etanolo per 24 ore è sufficiente a indurre steatosi epatica e risposte fibrogeniche nelle larve 13 e 15 dello zebrafish. È stato riportato che il trattamento con 2% di etanolo per 24 ore ha determinato una concentrazione di etanolo tessuto di 80 mM nelle larve 13 di zebrafish. Altri hanno dimostrato che le larve tollerano questa concentrazione ei fenotipi epatici osservati negli animali trattati sono specifici per l&#39;esposizione di etanolo 11 , 13 , 15 , 18 . Tuttavia, poiché 80 mM è quasi letale negli esseri umani 19 , è importante valutare l&#39;istologia epatica del pesce zebra trattato con etanolo e rilevareDistruggere la rilevanza fisiologica dell&#39;uomo. Il rapido sviluppo esterno e la traslucenza delle larve dello zebrafish permettono di caratterizzare l&#39;azione dell&#39;alcool nel fegato in tempo reale e in campioni fissi. La disponibilità di linee transgeniche fluorescenti specifiche di tipo cellulare e dei recenti progressi nella microscopia confocale facilitano lo studio di come diversi tipi di cellule epatiche cambino la loro morfologia e il loro comportamento in risposta al trattamento acuto di etanolo 11,15. Tuttavia, l&#39;imaging confocale dei pesci zebra transgenici fluorescenti non può completamente sostituire la colorazione di ematossilina e di Eosin (H &amp; E) durante lo studio dell&#39;istologia del fegato. Marcando tutti i tipi di cellule epatiche contemporaneamente utilizzando zebrafish transgenici richiede la generazione di singole linee transgeniche, ciascuna etichettatura di un tipo di cellule epatiche con un unico fluoroforo. Introdurre diversi sfondi transgenici nello stesso pesce richiede la razzaG generazioni multiple, che richiede tempo e sono costosi. È necessaria una colorazione immunofluorescenza aggiuntiva per rilevare componenti di matrice extracellulare. La colorazione H &amp; E, invece, identifica contemporaneamente tutti i tipi di cellule epatiche e le componenti della matrice extracellulare, fornendo così una panoramica del fegato 20 . Inoltre, rivela facilmente varie caratteristiche istopatologiche delle malattie epatiche, come la morte epatocitaria, la steatosi e la fibrosi. Anche se H &amp; E è una macchia di routine nell&#39;istologia del fegato mammifero, non è comunemente utilizzata nella ricerca epatica del zebrafish e il protocollo è meno ben definito. Questo lavoro descrive un protocollo per il trattamento acuto di etanolo nelle larve di zebrafish e per le analisi istologiche di follow-up con la colorazione H &amp; E. Il protocollo di colorazione H &amp; E può essere utilizzato in tutti gli studi di sviluppo e funzionalità del fegato. Inoltre, le sezioni di paraffina possono essere utilizzate per immunohistochemistry, come pure per altri sta sta specialiIns nella patologia epatica, tra cui la macchia tricromica, la macchia di reticulina, ecc .

<table border="0" cellpadding="0" cellspacing="0" width="1137">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">1.5 mL centrifuge tubes</td>
			<td style="width:207px;">E &#38; K Scientific</td>
			<td style="width:119px;">280150</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL conical tubes</td>
			<td>VWR International</td>
			<td>89039-664</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL conical tubes</td>
			<td>VWR International</td>
			<td>89039-658</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">95% ethanol</td>
			<td>Decon Labs, Inc.</td>
			<td>2801</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic acid</td>
			<td>Newcomer Supply</td>
			<td>10010A</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Agarose</td>
			<td style="width:207px;">Research Products International</td>
			<td style="width:119px;">9012-36-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Aluminum jar rack holder</td>
			<td style="width:207px;">Newcomer Supply</td>
			<td style="width:119px;">5300JRK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacteriological petri dishes with lid</td>
			<td>Corning</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Biopsy pads</td>
			<td style="width:207px;">Simport</td>
			<td style="width:119px;">M476.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Charged slides</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td>12-550-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Clear mounting media</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:119px;">8310-16</td>
			<td>Can be substituted with other clear mounting media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Commercial sea salts</td>
			<td>Instant Ocean</td>
			<td>SS15-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable microtome blades</td>
			<td>Fisher Scientific</td>
			<td>4280L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>Leica Biosystems</td>
			<td>Leica Mz 95</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Enclosed tissue processor</td>
			<td>Leica Biosystems</td>
			<td>ASP300 S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eosin-Phloxine stain set</td>
			<td>Newcomer Supply</td>
			<td>1082A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Ethyl alcohol</td>
			<td style="width:207px;">Sigma-Aldrich</td>
			<td style="width:119px;">E7023</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde solution, ACS reagent, 37 WT. % in H20, contains 10-15% methanol as stabilizer (to prevent polymerization)</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td>A suspected carcinogen; irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Formalin, Buffered, 10%</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:119px;">SF100-4</td>
			<td>A suspected carcinogen; irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Graduated media bottle</td>
			<td style="width:207px;">VWR International</td>
			<td style="width:119px;">16159-520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Harris hematoxylin</td>
			<td>Poly Scientific R&#38;D Corp.</td>
			<td>s212</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Histology molds</td>
			<td style="width:207px;">Sakura Finetek USA Inc</td>
			<td style="width:119px;">4557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Hot plate/Stirrer</td>
			<td style="width:207px;">VWR International</td>
			<td style="width:119px;">47751-148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>A144</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Incubator</td>
			<td style="width:207px;">VWR International</td>
			<td style="width:119px;">97058-220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Insulin syringes</td>
			<td style="width:207px;">BD Medical</td>
			<td style="width:119px;">BD-309301</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:645px;">Inverted compound microscope</td>
			<td style="width:207px;">Carl Zeiss Microscopy</td>
			<td style="width:119px;">491912-9850-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isopropanol</td>
			<td>Newcomer Supply</td>
			<td>12094E</td>
			<td>Flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtome</td>
			<td>Leica Biosystems</td>
			<td colspan="2">Leica Jung BioCut 2035&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nutating mixer</td>
			<td>VWR International</td>
			<td>82007-202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Paraformaldehyde</td>
			<td style="width:207px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148-1KG</td>
			<td>A suspected carcinogen; irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Pasteur pipet</td>
			<td style="width:207px;">VWR International</td>
			<td style="width:119px;">53283-916</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Pipette pump (10 mL)</td>
			<td style="width:207px;">VWR International</td>
			<td style="width:119px;">53502-233</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium phosphate, monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades</td>
			<td>Grainger</td>
			<td>4A807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Slide Staining Kit</td>
			<td style="width:207px;">Newcomer Supply</td>
			<td style="width:119px;">5300KIT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide (NaOH)</td>
			<td>Fisher BioReagents</td>
			<td>S318-500</td>
			<td>Very hazardous</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate, dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S3264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless steel strainer (5 inch diameter)</td>
			<td>Adaptive Science Tools</td>
			<td>L0906045in</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Tissue cassettes</td>
			<td style="width:207px;">Simport</td>
			<td style="width:119px;">M505.12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Tissue embedding center</td>
			<td style="width:207px;">Sakura Finetek USA Inc</td>
			<td style="width:119px;">#5100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue wipers, 1-Ply</td>
			<td>Fisher Scientific</td>
			<td>06666A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Transfer pipets</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:119px;">137117M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tricaine powder/Ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris base, primary standard and buffer</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Wash bottle, low-density polyethylene, wide mouth</td>
			<td style="width:207px;">Nalge Nunc International</td>
			<td style="width:119px;">2402-0750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:645px;">Xylenes</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:119px;">X3S-4</td>
			<td>Irritant</td>
		</tr>
	</tbody>
</table>

histological analyses of acute alcoholic liver injury in zebrafish

Malattia alcolica del fegato (ALD) si riferisce a danni al fegato a causa di abuso acuto o cronico di alcol. È tra le principali cause della morbilità e della mortalità correlate all&#39;alcol e colpisce più di 2 milioni di persone negli Stati Uniti. Una migliore comprensione dei meccanismi cellulari e molecolari alla base del danno epatico indotto da alcool è cruciale per lo sviluppo di un trattamento efficace per ALD. Le larve di Zebrafish mostrano steatosi epatica e fibrogenesis dopo solo 24 ore di esposizione al 2% di etanolo, rendendole utili per lo studio di lesioni al fegato alcoliche acute. Questo lavoro descrive la procedura per il trattamento acuto di etanolo nelle larve di zebrafish e mostra che provoca steatosi e gonfiori dei vasi sanguigni epatici. Viene descritto anche un protocollo dettagliato per la colorazione di ematossilina e di Eosin (H &amp; E) ottimizzato per l&#39;analisi istologica del fegato di larve zebrafish. La colorazione H &amp; E ha diversi vantaggi unici rispetto all&#39;immunofluorescenza, in quanto segna tutti i livelliEr cellule e componenti extracellulari contemporaneamente e può facilmente rilevare lesioni epatiche, come la steatosi e la fibrosi. Dato l&#39;aumento dell&#39;uso di zebrafish nella modellizzazione della tossina e della lesione epatica indotta dal virus, nonché delle patologie epatiche ereditarie, questo protocollo serve come riferimento per le analisi istologiche eseguite in tutti questi studi.

Formalin tampone 10% e 4% paraformaldeide (PFA) sono due dei fixatives più comuni utilizzati per le pratiche istologiche. Tuttavia, non forniscono risultati di fissazione ottimali per i tessuti epatici di zebrafish ( Figura 1 e Tabella 1 ). La fissazione con il 10% di formalina o il 4% di PFA provoca spesso dei restringimenti, creando grandi spazi tra il fegato e i tessuti circostanti ( Figura 1A , <strong clas...

Watch this Scientific Journal Video about Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish at JoVE.com

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Questo protocollo descrive le analisi istologiche dei fegati delle larve di zebrafish che sono state trattate con 2% di etanolo per 24 ore. Tale trattamento di etanolo acuto provoca steatosi epatica e gonfiore della vasculatura epatica.

Analisi istologiche di lesioni al fegato acuto alcolico in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

histological-analyses-of-acute-alcoholic-liver-injury-in-zebrafish

Research

JoVE Journal

Developmental Biology

14.2K Views.  Cincinnati Children's Hospital Medical Center.  Questo protocollo descrive le analisi istologiche dei fegati delle larve di zebrafish che sono state trattate con 2% di etanolo per 24 ore. Tale trattamento di etanolo acuto provoca steatosi epatica e gonfiore della vasculatura epatica. 

Video: Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

Rene Rigenerazione in Zebrafish adulti da Gentamicina Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Ricerca

Biologia dello sviluppo

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Mechanical Injury Vessel in embrioni di zebrafish

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

Automated quantificazione di cellule ematopoietiche - Interazioni cellule stromali in istologici Immagini di Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

Ablazione specifiche Hepatocyte in Zebrafish allo Studio biliare-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Analisi delle larve Zebrafish muscolo scheletrico Integrità con Blu di Evans Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Sviluppo di un modello di fegato Fibrotic etanolo-indotta in zebrafish per studiare progenitrici cellulo-mediata epatociti Rigenerazione

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Acute and Chronic Models of Hyperglycemia in Zebrafish: A Method to Assess the Impact of Hyperglycemia on Neurogenesis and the Biodistribution of Radiolabeled Molecules

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological problems after stroke and is shown to impair neurogenic processes. Interestingly, the adult zebrafish has recently emerged as a relevant and useful model to mimic hyperglycemia/diabetes and to investigate constitutive and regenerative neurogenesis. This work provides methods to develop zebrafish models of hyperglycemia to explore the impact of hyperglycemia on brain cell proliferation under homeostatic and brain repair conditions. Acute hyperglycemia is established using the intraperitoneal injection of D-glucose (2.5 g/kg bodyweight) into adult zebrafish. Chronic hyperglycemia is induced by immersing adult zebrafish in D-glucose (111 mM) containing water for 14 days. Blood-glucose-level measurements are described for these different approaches. Methods to investigate the impact of hyperglycemia on constitutive and regenerative neurogenesis, by describing the mechanical injury of the telencephalon, dissecting the brain, paraffin embedding and sectioning with a microtome, and performing immunohistochemistry procedures, are demonstrated. Finally, the method of using zebrafish as a relevant model for studying the biodistribution of radiolabeled molecules (here,[18F]-FDG) using PET/CT is also described.

This work describes methods to establish acute and chronic hyperglycemia models in zebrafish. The aim is to investigate the impact of hyperglycemia on physiological processes, such as constitutive and injury-induced neurogenesis. The work also highlights the use of zebrafish to follow radiolabeled molecules (here, [18F]-FDG) using PET/CT.

Modelli acuti e cronici di iperglicemia in Zebrafish: un metodo per valutare l&#39;impatto dell&#39;iperglicemia sulla neurogenesi e la biodistribuzione delle molecole radiomarcate

Hyperglycemia is a major health issue that leads to cardiovascular and cerebral dysfunction. For instance, it is associated with increased neurological ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Approccio preciso di ablazione cellulare per la modellazione di lesioni acute del rene nello sviluppo di zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Modelli di lesione di Zebrafish adulto per studiare gli effetti del Prednisolone nella rigenerazione di tessuto osseo

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Controlled Cortical Impact Model of Mouse Brain Injury with Therapeutic Transplantation of Human Induced Pluripotent Stem Cell-Derived Neural Cells

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical insult to secondary injury processes, including apoptosis and inflammation. Animal modeling has been valuable in the search to unravel injury mechanisms and evaluate potential neuroprotective therapies. This protocol describes the controlled cortical impact (CCI) model of focal, open-head TBI. Specifically, parameters for producing a mild unilateral cortical injury are described. Behavioral consequences of CCI are analyzed using the adhesive tape removal test of bilateral sensorimotor integration. Regarding experimental therapy for TBI pathology, this protocol also illustrates a process for transplanting cultured cells into the brain. Neural cell cultures derived from human induced pluripotent stem cells (hiPSCs) were chosen for their potential to show superior functional restoration in human TBI patients. Chronic survival of hiPSCs in the host mouse brain tissue is detected using a modified DAB immunohistochemical process.

This protocol demonstrates methodologies for a mouse model of open-skull traumatic brain injury and transplantation of cultured human induced pluripotent stem cell-derived cells into the injury site. Behavioral and histologic tests of outcomes from these procedures are also described in brief.

Modello di impatto corticale controllato di lesioni cerebrali del topo con trapianto terapeutico di cellule neurali derivate da cellule staminali indotte dall'uomo

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Disease pathology due to TBI progresses from the primary mechanical ...