Name: determining bile duct density in the mouse liver
Uploaded: 2019-04-30T14:45:00
Description: Watch this Scientific Journal Video about Determining Bile Duct Density in the Mouse Liver at JoVE.com

De auteurs erkennen de steun van de National Institutes of Health (NIH) (R01 GM084135 en R01 DK109982), een pilot/haalbaarheids Award van de Texas Medical Center spijsvertering ziekte centrum onder NIH P30 DK56338, en een Alagille syndroom Accelerator Award van De medische basis.

Alle dieren werden ondergebracht in een barrière dierenfaciliteit aan Baylor College of Medicine per institutionele richtlijnen voor dierenverzorging en-gebruik en onder erkende dier protocollen.
1. verzameling van muis leverweefsel
<ol><li>
Bereiding van de muis voor de oogst van de lever
	<ol>
<li>Euthanas de muis met behulp van isoflurane.</li>
		<li>Voer cervicale dislocatie van de muis om de dood te garanderen.</li>
		<li>Maak een dwarse incisie ongeveer één inch ond...

<ol>
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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=Jagged1+heterozygosity+in+mice+results+in+a+congenital+cholangiopathy+which+is+reversed+by+concomitant+deletion+of+one+copy+of+Poglut1+(Rumi).">Jagged1 heterozygosity in mice results in a congenital cholangiopathy which is reversed by concomitant deletion of one copy of Poglut1 (Rumi).</a> Hepatology. 63 (2), 550-565 (2016).</li><li>Hadchouel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paucity+of+interlobular+bile+ducts.">Paucity of interlobular bile ducts.</a> Seminars in Diagnostic Pathology. 9 (1), 24-30 (1992).</li><li>Emerick, K. 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=Features+of+Alagille+syndrome+in+92+patients:+frequency+and+relation+to+prognosis.">Features of Alagille syndrome in 92 patients: frequency and relation to prognosis.</a> Hepatology. 29 (3), 822-829 (1999).</li><li>Poncy, 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=Transcription+factors+SOX4+and+SOX9+cooperatively+control+development+of+bile+ducts.">Transcription factors SOX4 and SOX9 cooperatively control development of bile ducts.</a> Dev Biol. 404 (2), 136-148 (2015).</li><li>Hofmann, J. 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=Jagged1+in+the+portal+vein+mesenchyme+regulates+intrahepatic+bile+duct+development:+insights+into+Alagille+syndrome.">Jagged1 in the portal vein mesenchyme regulates intrahepatic bile duct development: insights into Alagille syndrome.</a> Development. 137 (23), 4061-4072 (2010).</li><li>McCright, B., Lozier, J., Gridley, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+Alagille+syndrome:+Notch2+as+a+genetic+modifier+of+Jag1+haploinsufficiency.">A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency.</a> Development. 129 (4), 1075-1082 (2002).</li><li>Andersson, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Model+of+Alagille+Syndrome+and+Mechanisms+of+Jagged1+Missense+Mutations.">Mouse Model of Alagille Syndrome and Mechanisms of Jagged1 Missense Mutations.</a> Gastroenterology. 154 (4), 1080-1095 (2018).</li><li>Loomes, K. 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M. <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+the+intrahepatic+biliary+tree.">Development of the intrahepatic biliary tree.</a> Semin Liver Dis. 22 (3), 213-226 (2002).</li><li>Kaneko, K., Kamimoto, K., Miyajima, A., Itoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+remodeling+of+the+biliary+architecture+underlies+liver+homeostasis.">Adaptive remodeling of the biliary architecture underlies liver homeostasis.</a> Hepatology. 61 (6), 2056-2066 (2015).</li><li>Schaub, J. 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Analyse van de ontwikkeling en reparatie van BD bij muizen is een belangrijk hulpmiddel bij het bestuderen van de pathogenese en het mechanisme van cholestatische aandoeningen. Daarnaast is de ontwikkeling van nieuwe therapieën deels afhankelijk van het tot stand brengen van een reproduceerbare en bij voorkeur kwantificeerbaar fenotype. Huidige fenotyping in muismodellen omvat meestal serum chemie, lever histologie en immunokleuring voor cel-type specifieke markers. Hoewel deze technieken waardevolle informatie generere...

De biliaire boom is een cruciaal onderdeel van de lever van zoogdieren, waardoor de passage van gal van hepatocyten in de darm. Intrahepatische galwegen (BDs) worden gevormd door cholangiocyten, die differentiëren van bipotentiaal hepatoblasten door Inkeping en tgfβ signalering1,2. De juiste specificatie en toewijding van cholangiocyten en hun assemblage in volwassen BDs zijn van cruciaal belang voor de ontwikkeling van de Intrahepatische biliaire boom. Naarmate de lever groeit tijdens de ontwikkeling of bij orgaan regeneratie, moet het biliaire systeem langs de lever worden ontwikkeld om een goede galdrainage te garanderen. Bovendien resulteren een aantal syndromische en niet-syndromische ziekten in de paustad van de Intrahepatische BDs3. Bovendien geven een aantal acute en chronische leverziekten aanleiding tot zogenaamde ductular reacties in de lever, die worden gedefinieerd als de aanwezigheid van een significant aantal cellen die biliaire markers uitdrukken, maar niet noodzakelijkerwijs voortvloeien uit biliaire cellen of vorm Patent BDs4. In de multi systeem stoornis Alagille syndroom (algs), haploinsufficiëntie van de inkeping ligand jagged1 (JAG1) resulteert in een slechte BD vorming en cholestase5,6. Ons lab heeft onlangs aangetoond dat een eerder gegenereerde Jag1 heterozygoot Mouse line7 een diermodel is van BD gebrek aan in algs8. In dit muismodel van ALGS zijn cholangiocyten nog steeds aanwezig. Ze zijn echter niet verplicht om zich te integreren in volwassen, patent BDs8. Daarom, analyse van de lever in een model van BD gebrek aan vereist meer dan de schijnbare aanwezigheid of afwezigheid van cholangiocyten. Het is belangrijk om de mate waarin volwassen BDs aanwezig zijn in de lever nauwkeurig te beoordelen.
In anatomische pathologie zijn er geaccepteerde kwantitatieve methoden om te beoordelen of BD gebrek aan9bestaat. Bijvoorbeeld, studies over algs bij menselijke patiënten kwantificeren vaak de BD naar portal ader (PV) ratio door het analyseren van ten minste 10 portaal vaten per leverbiopsie9,10. Analyse van de vorm en de algehele aanwezigheid of afwezigheid van patent BDs, gecombineerd met serum chemie, kan waardevolle informatie verschaffen over de ontwikkeling van BD in muizen11,12,13. Muizen kunnen echter een significant aantal Bdeen verliezen met slechts een bescheiden toename van het serum bilirubine niveau8. Dienovereenkomstig kan een kwantitatieve methode die het aantal aanwezige bd's per PV evalueert, een directere meting van de mate van BD gebrek aan bij muizen opleveren. In een recent rapport kwantificeren we het aantal Bd's per PV over alle lever lobben en rapporteerden we een significante afname in de BD-naar-PV-verhouding in Jag1 +/– dieren8. In de loop van onze analyse merkten we dat ondanks de significante variatie in de mate van inflammatoire respons en ductular reacties, de BD-naar-PV-verhouding niet veel variabiliteit toont8. Bovendien konden we met de kwantificering van de BD-naar-PV-ratio aantonen dat het verwijderen van één exemplaar van het glycosyltransferase gen Poglut1 in Jag1 +/– dieren hun BD gebrek aan8aanzienlijk kan verbeteren. In een Jag1 +/+ achtergrond resulteert voorwaardelijk verlies van Poglut1 in vasculaire gladde spiercellen in een progressieve toename van BD-getallen, wat bescheiden is (20-30%) bij P7 maar wordt prominent bij volwassenen8. Nogmaals, deze techniek liet ons toe om te laten zien dat zelfs bij P7, de toename van BD dichtheid bij deze dieren statistisch significant is. De verhoogde BD-dichtheid in dit genotype op vier maanden leeftijd werd ook gevalideerd door middel van hars giet analyse. 8 deze observaties en andere rapporten die de dichtheid van BD in verschillende algs-muizen modellen14,15 hebben gemeten, hebben ons ertoe aangezet deze methode in onze algemene strategie op te nemen om biliaire defecten in verschillende mutanten te analyseren en transgene muizen.
Hier, we detail een eenvoudige techniek die kan worden gebruikt om te onderzoeken de mate van BD gebrek aan in muismodellen van leverziekte (Figuur 1). Bij deze methode wordt co-kleuring met cholangiocyte markers cytokeratine (CK) 8 en CK19 (hierna Wide-spectrum CK, wsCK) gebruikt om Bd's en cholangiocyten in de muis te visualiseren. Een antilichaam tegen alpha-glad spier actine (αsma) wordt toegevoegd aan de kleuring om schepen te labelen. Systematische analyse van de BD-naar-PV-verhouding in een gedeelte dat alle lever lobben omvat, zorgt ervoor dat een groot aantal Pv's voor elk genotype wordt geanalyseerd. Omdat onze methode gebaseerd is op het kwantificeren van BDs en PVs in 2D-beelden, is het niet geschikt voor het bestuderen van de effecten van een bepaalde mutatie op de 3D-structuur van de galstructuur of de integriteit van de kleine galbuizen. Niettemin biedt het een eenvoudige en objectieve strategie voor onderzoekers om de biliaire ontwikkeling in de muis te beoordelen.

<table>
	<tbody>
		<tr>
			<td>Isothesia (Isoflurane)</td>
			<td>Henry Schein</td>
			<td>11695-6776-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator</td>
			<td>Bel-Art</td>
			<td>16-800-552</td>
			<td></td>
		</tr>
		<tr>
			<td>10% PFA</td>
			<td>Electron Microscopy Sciences</td>
			<td>15712</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL tube</td>
			<td>ThermoScientific</td>
			<td>339653</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2401</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2801</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Decon Laboratories</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoChoice</td>
			<td>VWR Life Sciences</td>
			<td>H103-4L</td>
			<td>clearing agent</td>
		</tr>
		<tr>
			<td>Omnisette Tissue Cassette</td>
			<td>Fisher HealthCare</td>
			<td>15-197-710E</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrosette</td>
			<td>Simport</td>
			<td>M512</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast X-TRA</td>
			<td>McCormick Scientific</td>
			<td>39503002</td>
			<td>Parrafin</td>
		</tr>
		<tr>
			<td>Tissue Mold</td>
			<td>Fisher Scientific</td>
			<td>62528-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Microm</td>
			<td>HM 325</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>C8H10</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Based Antigen Retrieval</td>
			<td>Vector Laboratories</td>
			<td>H-3301</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Cooker</td>
			<td>Instant Pot</td>
			<td>Lux Mini</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Pap Pen</td>
			<td>Life Technologies</td>
			<td>8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyoxyethylene 20 Sorbitan Monolaurate (Tween-20)</td>
			<td>J.T. Baker</td>
			<td>X251-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Octyl Phenol Ethoxylate (Triton-X-100)</td>
			<td>J.T. Baker</td>
			<td>X198-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Jackson Immunoresearch</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CK8</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-I</td>
			<td>Antibody Registry ID AB531826</td>
		</tr>
		<tr>
			<td>anti-CK19</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-III</td>
			<td>Antibody Registry ID AB2133570</td>
		</tr>
		<tr>
			<td>anti-&#945;SMA</td>
			<td>Sigma Aldrich</td>
			<td>A2547, Clone 1A4</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rat-Alexa488</td>
			<td>ThermoFisher</td>
			<td>A21208</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse-Cy5</td>
			<td>Jackson Immunoresearch</td>
			<td>715-175-151</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>22x50 micro cover glass</td>
			<td>VWR Life Sciences</td>
			<td>48393 059</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>Leica</td>
			<td>DMI6000 B</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimtech Science</td>
			<td>05511</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Antifade Mounting Medium</td>
		</tr>
	</tbody>
</table>

determining bile duct density in the mouse liver

Muis wordt in grote lijnen gebruikt als modelorganisme om biliaire ziekten te bestuderen. Om de ontwikkeling en functie van het biliaire systeem te evalueren, worden verschillende technieken gebruikt, waaronder serum chemie, histologische analyse en immunokleuring voor specifieke markers. Hoewel deze technieken belangrijke informatie over de galwegen kunnen bieden, presenteren ze vaak geen volledig beeld van de ontwikkelings defecten van galwegen (BD) over de hele lever. Dit is deels te wijten aan het robuuste vermogen van de muis lever om de gal af te voeren, zelfs bij dieren met een significante beperking in de biliaire ontwikkeling. Hier presenteren we een eenvoudige methode voor het berekenen van het gemiddelde aantal BDs dat is gekoppeld aan elke portaal ader (PV) in secties die alle lobben van Mutante/transgene muizen beslaan. Bij deze methode worden levers op type wijze gemonteerd en gesectioneerd om de vergelijking tussen verschillende genotypen en experimentele omstandigheden te vergemakkelijken. BDs worden geïdentificeerd door middel van lichte microscopie van cytokeratine-gekleurd cholangiocyten, en vervolgens geteld en gedeeld door het totale aantal Pv's aanwezig in de lever sectie. Als voorbeeld laten we zien hoe deze methode duidelijk onderscheid kan maken tussen wild-type muizen en een muismodel van Alagille syndroom. De hier gepresenteerde methode kan geen vervanging zijn voor technieken die de driedimensionale structuur van de biliaire boom visualiseren. Het biedt echter een eenvoudige en directe manier om de ontwikkeling van BD en de mate van ductulaire reactie vorming bij muizen kwantitatief te beoordelen.

We eerder gedocumenteerde biliaire gebreken in Jag1 +/– dieren, een muismodel van algs8. Om de BD-naar-PV-verhouding te bepalen, hebben we P30-muis levers verdeeld en ze samen met de vasculaire marker αSMA aan CK8 en CK19 (wsCK) gekoppeld. Vervolgens hebben we alle Pv's in elk van de lever lobben gefotografeerd. Zoals weergegeven in Fig. 2a, hebben we pv's gedefinieerd als αsma-bevlekte schepen met aangrenzende wsck-kleuri...

Watch this Scientific Journal Video about Determining Bile Duct Density in the Mouse Liver at JoVE.com

Determining Bile Duct Density in the Mouse Liver

We presenteren een vrij eenvoudige en gevoelige methode voor nauwkeurige kwantificering van de dichtheid van de gal duct in de lever van de muis. Deze methode kan helpen bij het bepalen van de effecten van genetische en omgevings modificatoren en de effectiviteit van mogelijke therapieën in muizen modellen van galziekten.

Bepaling van de Galkanaal dichtheid in de lever van de muis

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

Dit protocol stelt de onderzoeker in staat om galkanaalontwikkeling te kwantificeren in muismodellen van leverziekte. Het maakt het ook mogelijk voor de evaluatie van de effecten van genetische modifiers op galkanaal ontwikkeling. Het voordeel van deze techniek is dat het een eenvoudige, gevoelige en kwantitatieve methode is voor de directe beoordeling van de ontwikkeling van galwegen.Om te beginnen, met de huid strak gehouden door tangen, gebruik kleine schaar om een dwarse incisie ongeveer een centimeter onder de ribbenkast van een geëuthanaseerde muis te maken. Stel het hele ventrale oppervlak van de lever bloot. Gebruik kleine schaar om zorgvuldig te snijden door de ligamenten die de lever aan de andere organen in de buik.Vervolgens, snijd door de gemeenschappelijke galkanaal om de lever los te maken van de darm. Houd de galblaas vast en verwijder voorzichtig de lever. Plaats het onmiddellijk in een 50 milliliter buis gevuld driekwart met 4%paraformaldehyde.Om het leverweefsel te repareren, incubeer het in 4%PFA bij vier graden Celsius gedurende 48 uur. Na een reeks ethanol wast, was de lever met clearing agent drie keer gedurende 30 minuten elk bij kamertemperatuur. Om te beginnen met inbedden, was een weefsel cassette in een weefsel schimmel drie keer gedurende 30 minuten in paraffine wax voorverwarmd tot 60 graden Celsius.Vul vervolgens de weefselmal met paraffinewas tot driekwart hoogte en bewaar deze op een verwarmingsblok op 60 graden Celsius. Plaats de lever in de mal met de ventrale kant naar boven gericht. Na het zorgvuldig verwijderen van de mal uit het verwarmingsblok, plaats de bovenkant van de cassette op de mal.Top af met hete vloeibare paraffine en laat het afkoelen tot kamertemperatuur 's nachts. Na het verwijderen van het leverblok uit de mal, gebruik een microtome om te beginnen met het doorsneed door de oppervlakkige rugzijde van de lever, waardoor vijf micrometersecties. Controleer de oppervlakkige secties onder een dissectiemicroscoop om ervoor te zorgen de secties niet worden gevouwen of gevouwen.Als u dia's voor immunohistochemie wilt verwerken, selecteert u één dia per te analyseren genotype en 15 minuten wassen in xyleen, 100%ethanol, 95% ethanol en ten slotte 70%ethanol. Na het wassen van de glijbaan in het geïoniseerde water, dompel het onder in Tris-gebaseerde hoge pH antigeen retrieval oplossing. Dan, verwarm het onder druk in een snelkookpan gedurende drie minuten op 10 pond per vierkante inch.Nadat u de dia hebt laten afkoelen tot kamertemperatuur, gebruikt u een PAP-pen om de secties op de dia te schetsen. Na het wassen met PBS Tween, blokkeer de weefselsecties door 100 microliters van blokkerende oplossing per sectie toe te voegen en incbaat behandeld bij vier graden Van Celsius gedurende één uur toe te voegen. Breng 100 microliter van de verdunde antilichaamoplossing aan die alle drie de primaire antilichamen op elke sectie bevat en broed ze 's nachts bij vier graden Celsius uit.Voeg na het wassen van de glijbanen 100 microliter van de secundaire antilichaamoplossing toe die zowel secundaire antilichamen bevat als een uur lang bij kamertemperatuur incuvoeert. Tot slot, breng montagemedium en een coverslip aan op de dia's. Gebruik een fluorescerende microscoop om 20x beelden te maken bij 1x zoom van elke sectie.Zorg ervoor dat elke poortader over de lever wordt afgebeeld. Maak een spreadsheet met de volgende kolommen:Dier/Voorbeeldnummer, Afbeeldingsnummer, aantal portaladders en het aantal galwegen. Identificeer en noteert het aantal portaladders per afbeelding voor elke afbeelding.Identificeer vervolgens octrooigalewegen in elk beeld, door de aanwezigheid van cholangiocyten rond een definieerbaar lumen. Deze structuren moeten worden gescheiden door mesenchyme van andere breedspectrum cytokeratine-positieve cellen. Een belangrijke uitdaging van deze techniek is het identificeren van octrooigale kanalen.Het bepalen van wat een octrooikanaal is vereist een analyse van de vorm van het kanaal en de cellen rond het lumen. Tel elk octrooigalekanaal en plaats in dezelfde kolom als het afbeeldingsnummer en herhaal voor elke poortaderafbeelding. Bereken ten slotte de som van alle portaladders en alle galwegen in het levermonster en bereken de galkanaal-poort-poortaderverhouding voor het levermonster.P30 muis levers werden gesectied en mede-gekleurd voor CK8 en CK19 samen met de vasculaire marker, alpha-SMA, om de BD-PV-verhouding te bepalen. Wanneer alle portaaladeren in elke leverkwab worden afgebeeld, werden portaladders gedefinieerd als alfa-SMA bevlekte vaten met aangrenzende cytokeratininvlekken met een breed spectrum. De alfa-SMA vlekstructuren zonder breedspectrumcytokeratine waren centrale aderen die van de analyse werden uitgesloten.Octrooikanalen hebben een duidelijk definieerbaar lumen dat wordt omringd door breedspectrumcytokeratine-positieve cholangioctyes en zijn meestal gescheiden van nabijgelegen kanalen of cholangiocyten door mesenchyme. Breedspectrum cytokeratine-positieve cellen die geen definieerbaar lumen hebben, worden niet meegeteld voor het totale aantal galwegen. Lever sectie van een wild-type dier toont een portaal ader geassocieerd met een volledig octrooikanaal, samen met een aantal niet-opgenomen cellen.Representatieve lever sectie van een JAG1 heterozygoot dier toont geen octrooi galwegen aanwezig zijn rond de drie portaal aderen. Alle breedspectrumcytokeratine-positieve cellen hier zijn niet opgenomen en daarom niet mogen worden geteld. Analyse van de BD-pv-verhouding voor drie wilde en drie JAG1 heterozygoot dieren laat zien hoe de twee genotypen gemakkelijk kunnen worden onderscheiden op basis van galkanaaltellingen.Het is belangrijk om alle portaalschepen voor galwegen te beoordelen. Het is vooral van cruciaal belang voor het bepalen van galkanaaldichtheid als er fenotypische variabiliteit over de lever. Deze techniek werd gebruikt om een genetische suppressor van het galfenotype te identificeren in een muismodel van Helgason.Daarom kan het nuttig zijn bij het beoordelen van de behandelingsmodaliteiten van diermodellen van galziekte.

determining-bile-duct-density-in-the-mouse-liver

Research

JoVE Journal

Developmental Biology

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Stimulatie van Notch signalering in Mouse osteoclastvoorlopers

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

Histological Analyses of Acute Alcoholic Liver Injury 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 morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

Histologische analyses van acute alcoholische leverbeschadiging bij zebravis

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 ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

In Vitro Groei van de Preantral follikels muis onder gesimuleerde microzwaartekracht

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The effects these exposures have on mammary gland development are typically evaluated using either basic dimensional measurements or by scoring morphological characteristics. However, the broad range of methods for interpreting developmental changes could lead to inconsistent translations across laboratories. A common method of assessment is needed so that proper interpretations can be formed from data being compared across studies. The present study describes the application of the Sholl analysis method to quantify mammary gland branching characteristics. The Sholl method was originally developed for use in quantifying neuronal dendritic patterns. By using ImageJ, an open-source image analysis software package, and a plugin developed for this analysis, the mammary gland branching density and the complexity of a mammary gland from a peripubertal female rat were determined. The methods described here will enable the use of the Sholl analysis as an effective tool for quantifying an important characteristic of mammary gland development.

Mammary gland development in the rodent has typically been evaluated using descriptive assessments or by measuring basic physical attributes. Branching density is an indicator of mammary development that is difficult to quantify objectively. This protocol describes a reliable method for the quantitative assessment of mammary gland branching characteristics.

Kwantificerende vertakkingsdichtheid in Rat Mammary Gland Whole-mounts Met behulp van de Sholl Analyse Methode

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Dissectie en immunefluorescentie kleuring van Mushroom lichaam en fotoreceptor neuronen in de hersenen van de volwassen Drosophila melanogaster

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

Een roman gebruik van driedimensionale hoogfrequente Ultrasonografie voor vroege zwangerschap karakterisering in de muis

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

Een familiale hypercholesterolemie menselijke lever chimeer muismodel met behulp van geïnduceerde pluripotente stamcel afkomstige hepatocyten

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualiseren van het knooppunt en de Notochordal plaat In Gastrulating muis embryo's met behulp van Scanning elektronen microscopie en hele Mount immunofluorescentie

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

Generatie van Organoids van muis Extrahepatic galwegen

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...