Name: quantifying branching density in rat mammary gland whole-mounts using the sholl analysis method
Uploaded: 2017-07-12T13:00:00
Description: Watch this Scientific Journal Video about Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method at JoVE.com

Forfatterne vil gerne anerkende Dr. Michael Easterling (Social and Scientific Systems, Inc., Durham, NC) for hans hjælp til validering af denne metode og Dr. Tiago Ferreira (McGill University, Montreal, Quebec, Canada) for hans løbende Hjælp til Sholl ansøgningen.

Alle dyreanvendelser og procedurer til denne undersøgelse blev godkendt af NIEHS Laboratory Animal Care and Use Committee og udført i en Association for Assessment og Akkreditering af Laboratory Animal Care-akkrediterede facilitet. 1. punktafgift mammakirtler <ol><li> Mærk alle lysbilleder med en xylen-korrekt metode (blyant fungerer bedst). Dæk dem med monteringsløsning i enden for at bevare etiketten. </li><li> Euthanize dyret ved en godkendt metode til institutionel dyresundhed og ...

<ol>
	<li>Sholl, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+organization+of+the+neurons+in+the+visual+and+motor+cortices+of+the+cat.">Dendritic organization of the neurons in the visual and motor cortices of the cat.</a> J Anat. 87 (4), 387-406 (1953).</li><li>Ferreira, T. A., Iacono, L. L., Gross, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+receptor+1A+modulates+actin+dynamics+and+restricts+dendritic+growth+in+hippocampal+neurons.">Serotonin receptor 1A modulates actin dynamics and restricts dendritic growth in hippocampal neurons.</a> Eur J Neurosci. 32 (1), 18-26 (2010).</li><li>Stanko, J. P., Easterling, M. R., Fenton, S. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Mammary+Gland+Development+and+Function+in+Mouse+Models.">Evaluation of Mammary Gland Development and Function in Mouse Models.</a> J. Vis. Exp. (53), e2828 (2011).</li><li>ImageJ User Guide. IJ 1.46r. ImageJ Available from: <a target="_blank" href="https://imagej.nih.gov/ij/docs/guide/146.html">https://imagej.nih.gov/ij/docs/guide/146.html</a> (2012)</li><li>Tucker, D. K., Foley, J. F., Hayes-Bouknight, S. A., Fenton, S. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+abnormalities+in+the+rat+mammary+gland+following+gestational+and+lactational+exposure+to+2,3,7,8-tetrachlorodibenzo-p-dioxin+(TCDD).">Persistent abnormalities in the rat mammary gland following gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).</a> Toxicol Sci. 67 (1), 63-74 (2002).</li><li>Murray, T. J., Maffini, M. V., Ucci, A. A., Sonnenschein, C., Soto, 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=Induction+of+mammary+gland+ductal+hyperplasias+and+carcinoma+in+situ+following+fetal+bisphenol+A+exposure.">Induction of mammary gland ductal hyperplasias and carcinoma in situ following fetal bisphenol A exposure.</a> Reprod Toxicol. 23 (3), 383-390 (2007).</li><li>Moral, R., Santucci-Pereira, J., Wang, R., Russo, I. H., Lamartiniere, C. A., Russo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+exposure+to+butyl+benzyl+phthalate+induces+modifications+in+the+morphology+and+the+gene+expression+profile+of+the+mammary+gland:+an+experimental+study+in+rats.">In utero exposure to butyl benzyl phthalate induces modifications in the morphology and the gene expression profile of the mammary gland: an experimental study in rats.</a> Environ Health. 10 (1), 5 (2011).</li><li>Johnson, M. D., Mueller, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+dimensional+multiphoton+imaging+of+fresh+and+whole+mount+developing+mouse+mammary+glands.">Three dimensional multiphoton imaging of fresh and whole mount developing mouse mammary glands.</a> BMC Cancer. 13, 373 (2013).</li><li>Enoch, R. R., Stanko, J. P., Greiner, S. N., Youngblood, G. L., Rayner, J. L., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+gland+development+as+a+sensitive+end+point+after+acute+prenatal+exposure+to+an+atrazine+metabolite+mixture+in+female+Long-Evans+rats.">Mammary gland development as a sensitive end point after acute prenatal exposure to an atrazine metabolite mixture in female Long-Evans rats.</a> Environ Health Persp. 115 (4), 541-547 (2007).</li><li>Hovey, R. C., Coder, P. S., Wolf, J. C., Sielken, R. L., Tisdel, M. O., Breckenridge, C. 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Fra fødslen til puberteten er brystkirtlen vækst allometrisk. Efter pubertet udvikler brystkirtlen gennem omfattende duktal forgrening og forlængelse, som fortsætter indtil brystepitelet optager hele fedtpuden. Forgreningsegenskaber er et vigtigt aspekt ved udviklingen af ​​brystkirtlen, og evnen til objektivt at kvantificere disse egenskaber kan være yderst nyttig til vurdering af normal brystudvikling og til at identificere unormal udvikling efter tidlig livseksponering for brystgiftige stoffer. <p class=...

Mammekirtlen forgrening er en egenskab, der almindeligvis vurderes som en indikator for kirteludvikling, men det er svært at objektivt kvantificere. I 1953 beskrev Sholl 1 en metode til måling af neuronal dendritisk arborisering i katte visuelle og motoriske cortices, og et plugin til denne teknik blev udviklet af Ferriera et al. 2 . Da både neuroner og brystkirtler udviser en lignende trælignende struktur, blev plugin brugt til at kvantificere brystepithelialforgreningsdensiteter i 2D-billeder af peripubertal-rottebrystkirtlen. Peripubertaletrinnet blev udvalgt til analyse, fordi fravænning er et livsstadium, der ofte bedømmes i akademiske laboratorier og testretningslinjer. Sholl analysen er et plugin distribueret med FIJI, som er open source billedbehandling pakke ImageJ, med yderligere plugins inkluderet. Pluggen skaber en række koncentriske ringe, der omgiver en predefCenter (typisk soma af en neuron eller oprindelsen af ​​den primære kanal i en brystkirtle) og strækker sig ud til den fjerneste del af objektet (den omgivende radius). Det tæller derefter antallet af kryds (N), der forekommer på hver af ringene. Plugin&#39;en returnerer også en Sholl-regressionskoefficient ( k ), som er en måling af hastigheden af ​​henfald af epithelialforgrening. Ved hjælp af ImageJ er der skabt et skelettet billede af en brystkirtel helmontering, og brystepithelområdet (MEA) måles. Billedet analyseres ved hjælp af Sholl analyse plugin, og værdierne for N og k , blandt andre værdier, der ikke benyttes her, returneres. Mammary epithelial branching density bestemmes ved at beregne N / MEA. I hvilket omfang forgrening fortsætter i de yderste områder af kirtlenepitelet er forgrenings-kompleksiteten og er en indikator for ensartet distal epithelvækst. Da k er et mål for det distale fald i epithElial branching, er det et effektivt mål for forgreningskompleksiteten og en pålidelig indikator for brystudvikling. Denne protokol beskriver en computerassisteret metode til at skabe skeletoniserede billeder af brystkirtler i hele mund og kvantitativt evaluerende brystforgreningsegenskaber hos peripubertal han- og hunrotter. Denne metode er forholdsvis hurtig og kræver ikke brug af specialiseret mikroskopi udstyr. Udvikling og validering af denne fremgangsmåde er beskrevet i Stanko et al. (2015) 3 . Denne rapport beskriver også forberedelse af rotte brystkirtler hele = mounts. Lignende helbredsfremgangsmåder for bryst er blevet beskrevet i de Assis et al. (2010) 4 og Plante et al. (2011) 5 .

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			<td height="21" style="height:21px;width:245px;">Dissecting board</td>
			<td style="width:161px;">NA</td>
			<td style="width:224px;">NA</td>
			<td style="width:439px;">A piece of styrofoam roughly 10&#34;x12&#34; is suitable.</td>
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			<td height="21" style="height:21px;width:245px;">Dissecting T-Pins</td>
			<td>Daigger</td>
			<td style="width:224px;">EF7419A</td>
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			<td height="21" style="height:21px;width:245px;">Spray bottle with ethanol</td>
			<td>NA</td>
			<td style="width:224px;">NA</td>
			<td>70% ethanol is suitable.</td>
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			<td height="21" style="height:21px;width:245px;">Curved dissecting scissors</td>
			<td>Fine Science Tools</td>
			<td style="width:224px;">14569-09</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Straight dissecing scissors</td>
			<td>Fine Science Tools</td>
			<td style="width:224px;">14568-09</td>
			<td></td>
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			<td height="21" style="height:21px;width:245px;">Curved forceps</td>
			<td>Fine Science Tools</td>
			<td style="width:224px;">11003-12</td>
			<td></td>
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			<td height="160" style="height:160px;width:245px;">Superfrost Plus 24x75x1 mm microscope slides</td>
			<td>ThermoFisher Scientific</td>
			<td>4951PLUS-001</td>
			<td style="width:439px;">Thermo Scientific Superfrost Plus &#38; Colorfrost Plus slides hold tissue sections on permanently without the need for expensive coatings in IHC and Anatomical Pathology applications. This treatment reduces tissue loss during staining as well as hours of slide preparation. Slides electro-statically attract frozen tissue sections and cytology preparations and feature a chemistry similar to silane, although optimized to improve application performance. 
			https://www.thermofisher.com/order/catalog/product/4951PLUS4.&#160;</td>
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			<td height="42" style="height:42px;width:245px;">Fisherfinest Premium Cover Glass 24x60x1 mm</td>
			<td>Fisher scientific</td>
			<td style="width:224px;">12-548-5P</td>
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			<td height="42" style="height:42px;width:245px;">Bemis Parafilm M Laboratory Wrapping Film</td>
			<td>Fisher scientific</td>
			<td style="width:224px;">13-374-12</td>
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			<td height="21" style="height:21px;">Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>C2432</td>
			<td></td>
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			<td height="21" style="height:21px;">Glacial acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A9967</td>
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			<td height="21" style="height:21px;width:245px;">Ethanol absolute, &#8805;99.8% (GC)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:224px;">24102</td>
			<td></td>
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			<td height="21" style="height:21px;">Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>214736</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Carmine alum&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C1022</td>
			<td></td>
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			<td height="21" style="height:21px;">Aluminum potassium sulfate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A6435</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Permount mounting media</td>
			<td>Fisher Scientific</td>
			<td style="width:224px;">SP15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Macroscope</td>
			<td>Leica</td>
			<td>Z16 APO&#160;</td>
			<td rowspan="3" style="width:439px;">This is the image capturing hardware and software used in this laboratory.&#160; As there are many different options, the methods and applications may vary between laboratories.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Digital camera</td>
			<td>Leica</td>
			<td style="width:224px;">DFC295</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Camera software</td>
			<td>Leica</td>
			<td style="width:224px;">Leica Application Suite v3.1&#160;</td>
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			<td height="21" style="height:21px;width:245px;">ImageJ software</td>
			<td>Open source</td>
			<td>http://imagej.net/Welcome</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sholl analysis&#160;</td>
			<td>Open source</td>
			<td>http://imagej.net/Sholl_Analysis</td>
			<td></td>
		</tr>
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quantifying branching density in rat mammary gland whole-mounts using the sholl analysis method

Et stigende antal undersøgelser udnytter gnaverbrystkirtlen som et slutpunkt til vurdering af den udviklingstoksicitet af en kemisk eksponering. Virkningerne disse eksponeringer har på udvikling af brystkirtlen evalueres typisk ved hjælp af enten basisdimensionelle målinger eller ved at score morfologiske egenskaber. Den brede vifte af metoder til tolkning af udviklingsmæssige ændringer kan dog føre til inkonsekvente oversættelser på tværs af laboratorier. En fælles vurderingsmetode er nødvendig, så der kan dannes ordentlige fortolkninger ud fra data sammenlignet på tværs af undersøgelser. Den foreliggende undersøgelse beskriver anvendelsen af ​​Sholl analysemetoden til at kvantificere brystkirtlen forgreningsegenskaber. Sholl-metoden blev oprindeligt udviklet til brug ved kvantificering af neuronale dendritiske mønstre. Ved hjælp af ImageJ, en softwarepakke til open source-billedanalyse og et plugin udviklet til denne analyse, forgreningsdensiteten i brystkirtlen og kompleksiteten af ​​amAmmerkirtlen fra en peripubertal hunrot blev bestemt. De metoder, der beskrives her, muliggør brugen af ​​Sholl-analysen som et effektivt redskab til kvantificering af et vigtigt kendetegn ved udvikling af brystkirtler.

Værdierne for den målte lukkede radius, MEA, N, k og beregnet forgreningsdensitet for brystkirtlen analyseret i denne protokol er angivet i tabel 1 . Sholl analysen genererer lineære og semi-log plots af antallet af krydsninger i hver radius ( Figur 9 ) og, hvis valgt, et varmekort af krydsene ( Figur 10 ). Mindre udviklede kirtler udviser færre krydsninger inden for samme MEA og har derfor en laver...

Watch this Scientific Journal Video about Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method at JoVE.com

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

Udvikling af mammakirtler i gnaveret er typisk blevet vurderet ved hjælp af beskrivende vurderinger eller ved at måle grundlæggende fysiske egenskaber. Forgreningsdensitet er en indikator for brystudvikling, der er vanskeligt at kvantificere objektivt. Denne protokol beskriver en pålidelig metode til kvantitativ vurdering af brystkirtelsforgreningsegenskaber.

Kvantificering af forgreningsdensitet i rotte mammary Gland Whole-mounts ved hjælp af Sholl analysemetoden

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