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.

Forfatterne er takknemlig Keali Röhm, Louisa Frieß og Heike Menzel for deres ekspert teknisk assistanse; til Helga Brünner for dyr omsorg; og Chistopher S. Ward for generasjon og distribusjon av optisk disector rutenett plugin for ImageJ programvaren.

alle gjeldende internasjonale, nasjonale eller institusjonelle retningslinjer og bruk av dyr ble fulgt. Protokollen ble godkjent av lokale myndigheter på Regierung von Unterfranken, Würzburg, Tyskland.
 1. vev behandling og Immunohistochemistry <ol><li>Euthanize mus med CO 2 eller andre godkjent metode.</li>
	<li>Perfuse seks 12-uke-gamle C56Bl/6N mannlige mus transcardially med 10 mL 0.1 M fosfat-bufret saltoppløsning (PBS) bruker en 25-G nål og en infusjon pumpe, etterfulgt...

<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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Stereology starter med vev behandling. Seriell skjæring av SN vev må utføres med forsiktighet for å hindre tap av delene under stereological analyse. Dessuten er en viktig skritt å merke en halvkule for å skille rett fra venstre SN når du utfører stereology. Plassere et lite hull i den øvre delen av hjernestammen generert beste resultatene presentert studien. Videre siden jobbet med optisk fractionator metoden krav som vevet skjær i tykke snitt ca 30-40 µm, i stedet for 5-10 µm mer vanlig i immunohistochemist...

Kvantifisering av neuronal celle nummer er sentral i pre-klinisk Parkinsons sykdom forskning for å bestemme nivået av neurodegeneration innen substantia nigra (SN)1,2. Upartiske stereological estimering av celle nummer i et område av interesse anses gullstandarden3,4,5.
Før objektiv stereology, ble antall nevroner i delene vurdert ved å manipulere telt celle profiler korrigere variabel sannsynligheten at nervecellene kommer til syne i en inndeling. En av de mest brukte metodene var korreksjon av kvantifisert celletall beskrevet av Abercrombie6. Denne metoden forsøkte å ta hensyn til at cellene kan kvantifiseres mer enn én gang hvis fragmenter av samme celle finnes i tilstøtende tynne snitt. Derfor generert Abercrombie og andre forfattere ligninger som nødvendige forutsetninger om form, størrelse og retning av telles cellene7,8. Men var dette vanligvis ikke realisert og førte til systematisk feil og avvik fra hvor selve cellen (dvs. bias). Videre kunne bias ikke bli redusert med ytterligere prøvetaking3.
For stereological estimering celle tall ved hjelp av den optiske fractionator, brukes matematiske prinsipper for å anslå direkte celle tall direkte i et definert, 3-dimensjonale volum. Fordelen med denne metoden er at det ikke involverer antagelser om form, størrelse og retning av cellene blir regnet. Dermed anslagsvis cellen tallene er nærmere den sanne verdiene og komme nærmere som utvalgsstørrelsen (dvs. objektiv)3. Fordi mange regler må følges ved stereology for å holde metoden upartisk, klar til bruk kommersielle stereology systemer er utviklet (for vurdering, se Schmitz og Hof, 20054). Spesialisert stereology systemer implementere design-baserte stereological metoder med en priori definert sonder og prøvetaking ordninger for stereological vurderinger som fører til uavhengighet fra form, størrelse, romlige fordelingen og retningen på de cellene skal analysert4,9. Tilsvarede stereology systemer er imidlertid dyrt; Dette kan begrense implementering i ny forskning.
Målet med denne studien var å utvikle en brukervennlig teknikk for design-baserte stereological estimering dopaminergic celletall musen SN, sysselsetter metoden optiske fractionator og bruker standard mikroskopi utstyr (dvs. lys mikroskopet, standard mikroskop programvare og motoriserte x, y, z scenen). For dette vises en trinnvis veiledning på hvordan å behandle musen hjernevevet og beregne SN celle tall ved hjelp av design-baserte upartisk stereology. Dessuten tilbys maler for beregning av beregnet celle tall og koeffisientene til feil (CE).
Metoden beskrevet her er ikke begrenset til analyse av SN, men kan tilpasses for bruk i andre anatomisk definerte områder av mus eller rotte hjernen. For eksempel har upartisk stereology blitt brukt til å beregne neuronal cellen tallene i hippocampus10 og locus coeruleus11. I tillegg kan celletyper enn neurons, som astrocyttene12 og microglia13, vurderes også. Derfor kan denne metoden være nyttig for forskere som ønsker å implementere upartisk stereology i sin forskning, men er ikke villig til å bruke mye penger å kjøpe en stereology system.

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			<td height="25" style="height:25px;width:216px;">Paxinos mouse atlas</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<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>
			<td style="width:217px;">Zivic Instruments&#160;</td>
			<td style="width:119px;">BSMAS 001-1</td>
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			<td height="21" style="height:21px;width:216px;">paraformaldehyde</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1040051000</td>
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			<td height="21" style="height:21px;width:216px;">sucrose /D(+) Saccharose&#160;</td>
			<td style="width:217px;">Roth</td>
			<td style="width:119px;">4621.1</td>
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			<td height="21" style="height:21px;width:216px;">isopentane</td>
			<td style="width:217px;">Roth</td>
			<td style="width:119px;">3927.1</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">glycerol</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1040931000</td>
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			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:217px;">Sigma Aldrich</td>
			<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>Comments</td>
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			<td height="42" style="height:42px;width:216px;">phosphate buffered saline ingredients:</td>
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			<td height="21" style="height:21px;width:216px;">sodium chloride</td>
			<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>
			<td></td>
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		<tr height="42">
			<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>
			<td></td>
		</tr>
		<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>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">normal goat serum</td>
			<td style="width:217px;">Dako</td>
			<td style="width:119px;">X0907</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin</td>
			<td style="width:217px;">Sigma&#160;</td>
			<td style="width:119px;">A4503-100G</td>
			<td></td>
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		<tr height="21">
			<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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		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<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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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dry ice</td>
			<td style="width:217px;"></td>
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">self-assorted stereology</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">Visiview</td>
		</tr>
		<tr height="21">
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stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fractionator and standard microscopy equipment

I pre-klinisk Parkinsons sykdom forskning er analyse av nigrostriatal-område, inkludert kvantifisering av dopaminergic Nevron tap i substantia nigra, avgjørende. For å beregne totalt dopaminergic Nevron nummeret, anses for tiden upartisk stereology med metoden optisk fractionator gull standard. Fordi teorien bak optisk fractionator metoden er komplekse og fordi stereology er vanskelig å oppnå uten spesialisert utstyr, eksisterer flere kommersielt tilgjengelig komplett stereology systemer som inkluderer nødvendig programvare, rent teller grunner for cellen. Siden kjøpe en spesialisert stereology oppsett ikke er alltid mulig, for mange grunner, denne rapporten beskriver en metode for dopaminergic neuronal celle stereological estimering teller bruker standard mikroskopi utstyr, inkludert en lett mikroskop, en motorisert objekttabellen (x, y, z-fly) med bildebehandlingsprogramvare og en datamaskin for analyse. Gis en trinnvis forklaring på hvordan du utfører stereological kvantifisering med metoden optiske fractionator og pre-programmerte filene for beregning av beregnet celletall tilbys. For å vurdere nøyaktigheten av denne metoden, ble en sammenligning til data fra et kommersielt tilgjengelig stereology apparatet utført. Sammenlignbare cellen tallene ble funnet ved hjelp av denne protokollen og stereology enheten, dermed demonstrere presisjonen for denne protokollen for upartisk stereology.

Med metoden presentert, beregnet antall TH + dopaminergic neurons SN varierte mellom 7,363 og 7,987 celler til høyre og venstre SN, mellom 7,446 og 7,904 celler. Dermed var gjennomsnittlig antall dopaminergic nevroner (± SEM) 7,647 ± 83 celler for høyre SN og 7,675 ± 66 for venstre SN. Beregnet CE for hvert dyr var lavere enn 0,08 (område: 0.073-0.079) (figur 7). For å konstatere sammenlignbarheten metoden med kommersielt tilgjengelig og spesialisert s...

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

Dette arbeidet presenterer en trinnvis protokoll for upartisk stereological estimering dopaminergic neuronal cellen tallene i musen substantia nigra bruker standard mikroskopi utstyr (dvs. en lys mikroskop, en motorisert objekttabellen (x, y, z fly), og offentlige programvare for digital bildeanalyser.

Stereological estimering av Dopaminergic Neuron nummer i musen Substantia Nigra optiske Fractionator og Standard mikroskopi utstyr

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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