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.

Gli autori sono grati a Keali Röhm, Louisa Frieß e Heike Menzel per loro assistenza tecnica; a Helga Brünner per la cura degli animali; e a Chistopher S. Ward per la generazione e la distribuzione del disector ottico griglia plugin per il software ImageJ.

tutte le linee guida internazionali, nazionali e/o istituzionali applicabili per la cura e l'uso di animali sono state seguite. Il protocollo è stato approvato dalle autorità locali presso il Regierung von Unterfranken, Wuerzburg, Germania.
 1. trasformazione del tessuto e Immunohistochemistry <ol><li>Euthanize topi con CO 2 o qualsiasi altro metodo approvato.</li>
	<li>Profumi sei via di topo maschio C56Bl/6N 12-week-old con 10 mL di 0.1 M tampone fosfato salino (PBS) utilizza...

<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 inizia con lavorazione del tessuto. Il taglio seriale del tessuto SN deve essere eseguito con attenzione per evitare la perdita delle sezioni durante l'analisi STEREOLOGICHE. Inoltre, un passo essenziale consiste nel contrassegnare un emisfero al fine di distinguere la destra dalla sinistra SN durante l'esecuzione di ansia. Immissione di un piccolo foro nella parte superiore del tronco cerebrale generato i risultati migliori nello studio presentato. Inoltre, dal lavorare con le esigenze di metodo frazionatore ...

La quantificazione del numero di cellulare di un neurone è fondamentale nella ricerca pre-clinica la malattia del Parkinson per determinare il livello della neurodegenerazione nel nigra di substantia (SN)1,2. La valutazione imparziale stereologiche del numero delle cellule in una regione di interesse è considerata il gold standard3,4,5.
Prima dell'avvento di stereology imparziale, il numero dei neuroni nelle sezioni è stato valutato mediante la manipolazione delle cellule contate profili per correggere le probabilità variabile che neuroni entrano in vista in una sezione. Uno dei metodi più comunemente usati era la correzione dell'emocromo quantificati descritto da Abercrombie6. Questo metodo ha tentato di prendere in considerazione che le cellule possono essere quantificate in più di una volta se frammenti della stessa cella si trovano nelle sezioni sottili adiacenti. Di conseguenza, Abercrombie e altri autori generato equazioni che ha richiesto le ipotesi circa la forma, dimensione e orientamento delle cellule contate7,8. Tuttavia, questi presupposti erano solitamente non realizzati e quindi portati a errori sistematici e divergenza dal numero effettivo della cella (cioè, polarizzazione). Inoltre, il bias non potrebbe essere ridotto di campionamento supplementare3.
Per la stima stereologiche di numeri di cellulare usando il frazionatore ottico, principi matematici sono applicati per valutare direttamente i numeri di cellulare direttamente in un volume definito, 3-dimensionale. Il vantaggio di questo metodo è che non comporta ipotesi circa la forma, dimensione e orientamento delle cellule contate. Così, i numeri di cellulare stimato sono più vicini ai valori true e avvicinarsi man mano che aumenta la dimensione del campione (cioè, imparziali)3. Perché molte regole devono essere seguite quando usando stereology per mantenere il metodo imparziale, sono stati sviluppati sistemi di ready-to-use commerciale stereology (per la revisione, Vedi Schmitz e Hof, 2005,4). Sistemi specializzati stereology implementano metodi stereologiche basati su progettazione con aprioristicamente definite sonde e sistemi di campionamento per le valutazioni stereologiche che portano all'indipendenza dalla forma, dimensione, distribuzione spaziale e orientamento della cellule per essere analizzati4,9. Tuttavia, i sistemi di stereology commercialmente disponibili sono costosi; Questo potrebbe limitare implementazione nella ricerca di nuove.
Lo scopo di questo studio era quello di sviluppare una tecnica utilizzabile per la stima stereologiche basati su progettazione della conta delle cellule dopaminergiche nel topo SN, impiegando il metodo ottico frazionatore e utilizzando attrezzature di microscopia standard (cioè, luce microscopio, microscopio standard software e un motorizzato x, y, z fase). Per questo, è presentata una guida passo passo su come elaborare del tessuto di cervello di topo e come stimare i numeri di cell SN utilizzando stereology imparziali basati su progettazione. Inoltre, sono disponibili modelli per il calcolo dei numeri di cellulare stimato e coefficienti di errore (CE).
Il metodo qui descritto non è limitato all'analisi dello SN, ma può essere adattato per l'uso in altre regioni anatomicamente definite del cervello di topo o ratto. Per esempio, stereology imparziale è stato utilizzato per stimare i numeri di cellulare di un neurone l' ippocampo10 e il locus coeruleus11. Inoltre, tipi cellulari diversi da neuroni, astrociti12 e microglia13, possono essere valutati come bene. Pertanto, questo metodo può essere utile agli scienziati che intendono attuare stereology imparziale nella loro ricerca, ma non sono disposti a spendere un sacco di soldi per l'acquisto di un sistema di ansia.

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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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		<tr height="21">
			<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>
			<td></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>
			<td></td>
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		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glycerol</td>
			<td style="width:217px;">Merck</td>
			<td style="width:119px;">1040931000</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:217px;">Sigma Aldrich</td>
			<td style="width:119px;">32205-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:217px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
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			<td height="42" style="height:42px;width:216px;">phosphate buffered saline ingredients:</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="63">
			<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>
			<td></td>
		</tr>
		<tr height="26">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
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stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fractionator and standard microscopy equipment

Nella ricerca pre-clinica la malattia del Parkinson, analisi del tratto nigrostriatal, compresa la quantificazione di perdita di neuroni dopaminergici nel nigra di substantia, è essenziale. Per stimare il numero di neuroni dopaminergici totale, imparziale stereology utilizzando il metodo ottico frazionatore è attualmente considerato il gold standard. Poiché la teoria dietro il metodo ottico frazionatore è complessa e stereology è difficile da raggiungere senza attrezzature specializzate, esistono vari sistemi commercialmente disponibili stereology completi che includono il software necessario, puramente per cella contando i motivi. Dal momento che l'acquisto di un setup stereology specializzato non è sempre fattibile, per molte ragioni, questo rapporto descrive un metodo per la stima stereologiche del cellulare di un neurone dopaminergico conteggi mediante microscopia standard attrezzature, compreso un microscopio ottico, un tavola di oggetto (x, y, z aereo) motorizzata con software di imaging e un computer per l'analisi. È dato una spiegazione dettagliata su come eseguire stereologiche quantificazione mediante il metodo ottico frazionatore, e file di pre-programmati per il calcolo dei conteggi delle cellule stimato vengono forniti. Per valutare la precisione di questo metodo, è stato effettuato un confronto con i dati ottenuti da un apparato di stereology commercialmente disponibili. Numeri paragonabili delle cellule sono stati trovati usando questo protocollo e il dispositivo stereology, dimostrando così la precisione di questo protocollo per stereology imparziali.

Usando il metodo presentato, il numero stimato di TH + neuroni dopaminergici in SN ha variato fra cellule 7.363 e 7.987 a destra e, nella parte sinistra SN, tra le celle di 7.446 e 7.904. Così, il numero medio dei neuroni dopaminergici (± SEM) era 7.647 ± 83 cellule per il diritto SN e 7.675 ± 66 per la sinistra SN. La CE calcolata per ciascun animale è stato inferiore a 0,08 (gamma: 0,073-0,079) (Figura 7). Per accertare la comparabilità di questo meto...

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

Questo lavoro presenta un protocollo passo-passo per la valutazione imparziale stereologiche dei numeri di un neurone delle cellule dopaminergiche nel nigra di substantia del mouse utilizzando attrezzature di microscopia standard (cioè, un microscopio ottico, una tabella di oggetto motorizzato (x, y, z aereo) e software di pubblico dominio per analisi digitale delle immagini.

Stereologiche stima del numero di neuroni dopaminergici nel Nigra di Substantia del Mouse utilizzando il frazionatore ottica e microscopia Standard attrezzature

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.

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