Name: stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fractionator and standard microscopy equipment
Uploaded: 2017-09-01T14:00:00
Description: Watch this Scientific Journal Video about Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment at JoVE.

Författarna är tacksamma mot Keali Röhm, Louisa Frieß och Heike Menzel för deras teknisk experthjälp; att Helga Brünner för djurens vård; och att Chistopher S. Ward för generering och distribution av den optiska disector rutnät plugin för programmet ImageJ.

alla tillämpliga internationella, nationella och/eller institutionella riktlinjer för skötsel och användning av djur följdes. Protokollet godkändes av lokala myndigheter på den Regierung von Unterfranken, Würzburg, Tyskland.
 1. vävnad behandling och immunohistokemi <ol><li>Euthanize möss med CO 2 eller någon annan godkänd metod.</li>
	<li>Perfuse sex 12 veckor gamla C56Bl/6N hanmöss transcardially med 10 mL 0,1 M fosfatbuffrad koksaltlösning (PBS) med en 25-G nål ...

<ol>
	<li>Ip, C. W., 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=AAV1/2-induced+overexpression+of+A53T-alpha-synuclein+in+the+substantia+nigra+results+in+degeneration+of+the+nigrostriatal+system+with+Lewy-like+pathology+and+motor+impairment:+a+new+mouse+model+for+Parkinson's+disease.">AAV1/2-induced overexpression of A53T-alpha-synuclein in the substantia nigra results in degeneration of the nigrostriatal system with Lewy-like pathology and motor impairment: a new mouse model for Parkinson's disease.</a> Acta Neuropathol Commun. 5 (1), 11 (2017).</li><li>Ip, C. W., Beck, S. 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Grid_Overlay.java Available from: <a target="_blank" href="https://imagej.nih.gov/ij/plugins/graphic-overlay.html">https://imagej.nih.gov/ij/plugins/graphic-overlay.html</a> (2010)</li><li>. cell_counter.jar Available from: <a target="_blank" href="https://imagej.nih.gov/ij/plugins/cell-counter.html">https://imagej.nih.gov/ij/plugins/cell-counter.html</a> (2010)</li><li>Baquet, Z. C., Williams, D., Brody, J., Smeyne, R. 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Stereology börjar med vävnad behandling. Seriell styckning av SN vävnad måste utföras noga för att förhindra förlust av sektioner under beteendetester analys. Dessutom är ett viktigt steg att markera en halvklotet för att skilja höger från vänster SN när du utför stereology. Att placera ett litet hål på den övre delen av hjärnstammen genereras de bästa resultaten i studien presenteras. Dessutom sedan arbetar med optisk naftafraktioneringskolonn metod krav att vävnaden är skuren i tjocka delar av ca ...

Kvantifiering av antalet neuronala celler är avgörande i prekliniska Parkinsons Sjukdomforskning för att avgöra nivån på neurodegeneration inom det substantia nigra (SN)1,2. Opartisk beteendetester uppskattning av antalet celler i ett område av intresse anses vara den gyllene standard3,4,5.
Före tillkomsten av opartisk stereology uppskattades antalet neuroner i sektioner genom att manipulera räknade cell profiler att korrigera för variabel sannolikheterna att nervceller kommer i sikte i ett avsnitt. En av de vanligaste metoderna var korrigering av kvantifierade blodkroppar som beskrivs av Abercrombie6. Denna metod försökte ta hänsyn till att celler kan kvantifieras mer än en gång om fragment av samma cell finns i intilliggande tunnslip. Därför, Abercrombie och andra författare genereras ekvationer som krävs antaganden om form, storlek och orientering av räknade celler7,8. Dessa antaganden var dock oftast inte insett och därför ledde till systematiska fel och avvikelser från den faktiska mobilnummer (dvs bias). Bias kunde dessutom inte minskas med ytterligare provtagning3.
För beteendetester uppskattning av cell nummer med hjälp av den optiska naftafraktioneringskolonn, tillämpas matematiska principer för att direkt beräkna den cell nummer direkt i en definierad, 3-dimensionell volym. Fördelen med denna metod är att det inte innebär antaganden om form, storlek och orientering av cellerna räknas. Således, de uppskattade cell nummer är närmare sanna värden och komma närmare stickprovsstorleken ökar (dvs opartisk)3. Eftersom många regler måste följas när du använder stereology för att hålla metoden opartisk, klar att använda kommersiella stereology system har utvecklats (för granskning, se Schmitz och Hof, 20054). Specialiserade stereology system implementera design-baserade beteendetester metoder med en priori definierats sonder och provtagning system för beteendetester bedömningar som leder till självständighet från form, storlek, rumslig fördelning och orientering av den celler att vara analyserade4,9. Kommersiellt tillgängliga stereology system är dock dyra; Detta kan begränsa genomförandet i ny forskning.
Syftet med denna studie var att utveckla en användbar teknik för design-baserade beteendetester uppskattning av dopaminerga blodkroppar i musen SN, anställa den optiska naftafraktioneringskolonn metoden och använder standard mikroskopi utrustning (dvs ljus Mikroskop, standard Mikroskop programvara och en motoriserad x, y, z skede). För detta presenteras en steg för steg guide om hur att bearbeta mus hjärnvävnad och uppskatta SN cell nummer med hjälp av design-baserade opartisk stereology. Dessutom finns mallar för beräkningen av det uppskattade cellantal och koefficienter av fel (CE).
Den metod som beskrivs här är inte begränsat till analysen av SN, men kan anpassas för användning i andra anatomiskt definierade regioner av hjärnan som mus eller råtta. Opartisk stereology har exempelvis använts för att uppskatta neuronala cell nummer i hippocampus10 och locus coeruleus11. Dessutom kan celltyper än neuroner, såsom astrocyter12 och mikroglia13, bedömas liksom. Denna metod kan därför användbar till forskare som avser att genomföra opartisk stereology i deras forskning men är inte villig att spendera en massa pengar att köpa ett stereology system.

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			<td height="25" style="height:25px;width:216px;">Paxinos mouse atlas</td>
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			<td style="width:467px;">The Mouse Brain George Paxinos Keith B.J.FranklinCopyright @2001 by Academic Press CD Rom designet &#38; created by Paul Halasz</td>
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			<td height="21" style="height:21px;width:216px;">brain matrix slicer mouse</td>
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			<td height="21" style="height:21px;width:216px;">isopentane</td>
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			<td style="width:119px;">3927.1</td>
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			<td height="21" style="height:21px;width:216px;">glycerol</td>
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			<td style="width:119px;">1040931000</td>
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			<td height="21" style="height:21px;width:216px;">Ethanol</td>
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			<td style="width:119px;">32205-1L</td>
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			<td height="21" style="height:21px;width:216px;">Name</td>
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			<td style="width:217px;">Sigma Aldrich</td>
			<td style="width:119px;">31434-1KG-R</td>
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			<td height="42" style="height:42px;width:216px;">potassium dihydrogen phosphate</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1048731000</td>
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			<td height="42" style="height:42px;width:216px;">di-sodium hydrogen phosphate dihydrate</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1065801000</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">potassium chloride</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1049360500</td>
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			<td height="21" style="height:21px;width:216px;">normal goat serum</td>
			<td style="width:217px;">Dako</td>
			<td style="width:119px;">X0907</td>
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			<td height="21" style="height:21px;width:216px;">bovine serum albumin</td>
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			<td style="width:119px;">A4503-100G</td>
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			<td height="21" style="height:21px;width:216px;">Triton X-100</td>
			<td style="width:217px;">Sigma Aldrich</td>
			<td style="width:119px;">X100-100ml</td>
			<td>detergent</td>
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			<td height="63" style="height:63px;width:216px;">3,3-Diaminobenzidine-tetrahydrochlorid/DAB tablets 10mg pH 7.0</td>
			<td style="width:217px;">Kem En Tec</td>
			<td style="width:119px;">4170</td>
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			<td height="26" style="height:26px;width:216px;">H2O2/ Hydrogen peroxide 30%</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1072090250</td>
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			<td height="21" style="height:21px;width:216px;">avidin/biotin reagent</td>
			<td style="width:217px;">Thermo Scientific</td>
			<td style="width:119px;">32050</td>
			<td>Standard Ultra Sensitive ABC Staining Kit, 1:100</td>
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			<td height="42" style="height:42px;width:216px;">rabbit anti mouse tyrosine hydroxylase antibody</td>
			<td style="width:217px;">abcam</td>
			<td style="width:119px;">Ab112</td>
			<td>1:1000</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">biotinylated goat-anti-rabbit IgG H+L</td>
			<td style="width:217px;">vector laboratories</td>
			<td style="width:119px;">BA-1000</td>
			<td>1:100</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">StereoInvestigator version 11.07</td>
			<td style="width:217px;">MBF</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BX53 microscope</td>
			<td style="width:217px;">Olympus</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Visiview</td>
			<td style="width:217px;">Visitron Systems GmbH</td>
			<td style="width:119px;">3.3.0.2</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Axiophot2</td>
			<td style="width:217px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">ImageJ software</td>
			<td style="width:217px;">NIH</td>
			<td style="width:119px;">Version 4.7</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue-TEK OCT</td>
			<td style="width:217px;">Sakura</td>
			<td style="width:119px;">4583</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">dry ice</td>
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			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">grid overlay plugin</td>
			<td style="width:217px;">Wayne Rasband</td>
			<td style="width:119px;"></td>
			<td>https://imagej.nih.gov/ij/plugins/graphic-overlay.html</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cell counter plugin</td>
			<td style="width:217px;">Kurt de Vos</td>
			<td style="width:119px;"></td>
			<td>https://imagej.nih.gov/ij/plugins/cell-counter.html).&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">optical disector macro</td>
			<td style="width:217px;">Christopher Ward</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C57Bl/6N male mice</td>
			<td style="width:217px;">Charles River, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SuperFrost Plus coated object slides</td>
			<td style="width:217px;">Langenbrinck, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25G needle Microlance 3</td>
			<td style="width:217px;">BD&#160;</td>
			<td align="right" style="width:119px;">300400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">REGLO Analog Infusion pump</td>
			<td style="width:217px;">Ismatec</td>
			<td style="width:119px;">ISM 829</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">StereoInvestigator system</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">StereoInvestigator version 11.07</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BX53 microscope</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">BX53 microscope</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">self-assorted stereology</td>
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stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fractionator and standard microscopy equipment

I prekliniska Parkinsons Sjukdomforskning är analys av nigrostriatala tarmkanalen, inklusive kvantifiering av dopaminerga neuron i substantia nigra, viktigt. För att uppskatta antalet totala dopaminerga neuron, anses opartisk stereology med metoden optisk naftafraktioneringskolonn för närvarande guldmyntfoten. Eftersom teorin bakom metoden optiska naftafraktioneringskolonn är komplex och stereology är svårt att uppnå utan specialutrustning, finns flera kommersiellt tillgängliga komplett stereology system som inkluderar den nödvändiga programvaran, rent för cell räknar skäl. Sedan köpa en specialiserad stereology setup inte är alltid möjligt, av många skäl, denna rapport beskriver en metod för beteendetester uppskattning av dopaminerga neuronala cell räknar med standard mikroskopi utrustning, inklusive ett ljusmikroskop, en motoriserade objekttabellen (x, y, z plan) med programvara för bildåtergivning och en dator för analys. En steg för steg förklaring ges om hur man utför beteendetester kvantifiering med metoden optisk naftafraktioneringskolonn och förprogrammerade filer för beräkning av uppskattade blodkroppar finns. För att bedöma riktigheten av denna metod, utfördes en jämförelse till uppgifter som erhållits från en kommersiellt tillgängliga stereology apparatur. Jämförbara cell nummer hittades med hjälp av detta protokoll och stereology enheten, vilket visar att detta protokoll för opartisk stereology precision.

Använda denna metod, det uppskattade antalet TH + dopaminerga nervceller i rätt SN varierade mellan 7,363 och 7,987 celler och i vänster SN, mellan 7,446 och 7,904 celler. Således var det genomsnittliga antalet dopaminerga neuron (± SEM) 7,647 ± 83 celler för rätt SN och 7,675 ± 66 för vänster SN. Den beräknade CE för varje djur var lägre än 0,08 (intervall: 0,073-0.079) (figur 7). För att säkerställa jämförbarheten av denna metod med komm...

Watch this Scientific Journal Video about Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment at JoVE.

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

Detta arbete presenterar ett stegvisa protokoll för opartisk beteendetester uppskattning av dopaminerga neuronala cell nummer i mus substantia nigra med standard mikroskopi utrustning (dvs. ett ljusmikroskop, en motoriserad objekttabellen (x, y, z planet), och offentlig domän programvara för digital bildanalys.

Beteendetester uppskattning av dopaminerga Neuron antal mus Substantia Nigra med hjälp av optisk naftafraktioneringskolonn och Standard mikroskopi utrustning

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

The overall goal of this protocol is to describe the design-based stereological estimation of dopaminergic cell number in the mouse SN using standard microscopy equipment. This method is useful to estimate the number of dopaminergic neurons in the mouse substantia nigra for preclinical Parkinson's disease research using design-based unbiased stereology. The main advantage of this technique is that it can be accomplished with standard microscopy equipment without need for a commercially available complete stereology setup.Demonstrating the procedure will be Louisa Friez, a technician from my laboratory. Begin by placing a paraformaldehyde fixed mouse brain into a brain matrix and making a cut plus 0.74 millimeters from bregma. Transfer the caudal part of the brain including the SN into 4%PFA in 0.1 molar and fix overnight at four degrees Celsius.The next day, exchange the PFA for 30%sucrose in 0.1 molar PBS and incubate for another two days at four degrees Celsius to cryo protect the sample. After two days when the brain has sunk in the sucrose, place the tissue into a Cryomold filled with optimal cutting temperature compound and slowly freeze the tissue in liquid dry ice cooled isopentane. Use a cryostat to serially cut 30 micron cryo sections in the coronal plane.Mark the right hemisphere by puncturing a small hole into the upper region of the brain stem. Collect four series of sections starting at minus 2.46 millimeters from bregma and ending at 4.04 millimeters from bregma. Transfer each series of sections into a tube filled with cryoprotectant and store at minus 20 degrees Celsius.Capture digital images of tyrosine hydroxylase immunohistochemically stained SN sections using imaging software coupled to a microscope. Start by determining the actual mount and thickness of the sections at three randomly selected counting sites per section. First click on view top offset to define the top of the section.Then click on stop view top. Thereafter, click on view bottom offset and then click on stop view bottom. Write down the thickness of the section.Calculate the guard zone by subtracting three microns from the top offset and type the result into the top offset box. Then subtract the 13 micron height of the optical dissector from the top offset number and type the result into the bottom offset box. Select the following parameters, steps 14, size 1.00.Select the directory to save the file. Click on sequence to start the acquisition. After the images have been acquired, click on processing and select the following parameters, all planes from sequence, montage and the fast and stitching boxes with an image overlap of 10%Start to stitch the images by clicking on start.This will generate stack images for analysis. To begin, open the image by clicking on file and open. Set the image scale by using the analyze and set scale command.From this step, change the defined size from pixels to microns. Select the plugins then grid then grid type lines command to randomly insert a grid. Check the random offset box.Define the size of one square within the grid as 130 microns by 130 microns to equal 16, 900 square microns. Change image type from 16-bit to RGB color by clicking on image then type then RGB color. Locate the substantia nigra pars compacta.Encircle the region with the paintbrush tool using a brush width of 11. Undo the set scale command by clicking on analyze then set scale then click to remove scale to calculate in pixels rather than microns. Take a screenshot, save the image file and open the new file with ImageJ.Select brush and a brush width of 25. Mark every grid square that contacts the SN for placement of an optical dissector. Select one grid square that includes parts of the SN.Measure the upper left corner of the square with the cursor in ImageJ to define the starting x and y coordinates.Select the spreadsheet template entitled optical dissector position and calculate the optical dissector position. Then estimate the cell number using the calculation of cell count spreadsheet as described in the written portion of the protocol. This low power image shows a section of mouse brain after TH staining for dopaminergic neurons.Higher magnification depicts TH positive dopaminergic neurons. The inset shows the boxed region at increased magnification. This image shows one series of TH stained SN sections covering the whole mouse SN from rostral to caudal.Each section is separated from the consecutive section by 120 microns. The number of TH positive neurons in the right and left mouse SN were estimated using the method shown in this video and using a commercially available stereology system. As seen here, both methods resulted in comparable cell counts and there were no significant differences between data sets.Don't forget that working with paraformaldehyde can be extremely hazardous and precautions such as wearing of personal protection equipment like protective gloves should always be taken while performing this procedure. After watching this video, you should have a good understanding of how to perform unbiased stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fraction method and standard microscopy equipment.

Research

JoVE Journal

Neuroscience

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