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.

The authors are grateful to Keali R&#246;hm, Louisa Frie&#223;, and Heike Menzel for their expert technical assistance; to Helga Br&#252;nner for the animal care; and to Chistopher S. Ward for the generation and distribution of the optical disector grid plugin for the ImageJ software.

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The protocol was approved by local authorities at the Regierung von Unterfranken, Wuerzburg, Germany.
1. Tissue Processing and Immunohistochemistry
<ol>
	<li>Euthanize mice with CO2 or any other approved method.</li>
	<li>Perfuse six 12-week-old C56Bl/6N male mice transcardially with 10 mL of 0.1 M phosphate-buffered saline (PBS) using a 25-G needle and an infusio...

<ol>
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Stereology starts with tissue processing. The serial cutting of SN tissue must be performed carefully to prevent the loss of sections during stereological analysis. Additionally, one essential step is to mark one hemisphere in order to distinguish the right from the left SN when performing stereology. Placing a tiny hole at the upper part of the brainstem generated the best results in the presented study. Moreover, since working with the optical fractionator method demands that the tissue is cut in thick sections of abou...

The quantification of neuronal cell number is pivotal in pre-clinical Parkinson&#39;s disease research to determine the level of neurodegeneration within the substantia nigra (SN)1,2. The unbiased stereological estimation of cell number in a region of interest is considered the gold standard3,4,5.
Before the advent of unbiased stereology, the number of neurons in sections was assessed by manipulating counted cell profiles to correct for the variable probabilities that neurons come into sight in a section. One of the most commonly used methods was the correction of quantified cell counts described by Abercrombie6. This method attempted to take into account that cells can be quantified more than once if fragments of the same cell are found in adjacent thin sections. Therefore, Abercrombie and other authors generated equations that required assumptions about the shape, size, and orientation of the counted cells7,8. However, these assumptions were usually not realized and therefore led to systematic errors and divergence from the actual cell number (i.e., bias). Moreover, the bias could not be reduced by additional sampling3.
For the stereological estimation of cell numbers using the optical fractionator, mathematical principles are applied to directly estimate the cell numbers directly in a defined, 3-dimensional volume. The advantage of this method is that it does not involve assumptions about the shape, size, and orientation of the cells being counted. Thus, the estimated cell numbers are closer to the true values and get closer as the sample size increases (i.e., unbiased)3. Because many rules must be followed when using stereology to keep the method unbiased, ready-to-use commercial stereology systems have been developed (for review, see Schmitz and Hof, 20054). Specialized stereology systems implement design-based stereological methods with a priori defined probes and sampling schemes for stereological assessments that lead to independence from shape, size, spatial distribution, and orientation of the cells to be analyzed4,9. However, commercially available stereology systems are expensive; this may limit implementation in new research.
The aim of this study was to develop a usable technique for the design-based stereological estimation of dopaminergic cell counts in the mouse SN, employing the optical fractionator method and using standard microscopy equipment (i.e., light microscope, standard microscope software, and a motorized x, y, z stage). For this, a step-by-step guide on how to process mouse brain tissue and how to estimate SN cell numbers using design-based unbiased stereology is presented. Moreover, templates for the calculation of the estimated cell numbers and coefficients of error (CE) are provided.
The method described here is not limited to the analysis of the SN, but can be adapted for use in other anatomically defined regions of the mouse or rat brain. For instance, unbiased stereology has been used to estimate neuronal cell numbers in the hippocampus10 and the locus coeruleus11. Additionally, cell types other than neurons, such as astrocytes12 and microglia13, can be assessed as well. Therefore, this method can be useful to scientists who intend to implement unbiased stereology in their research but are not willing to spend a lot of money to purchase a 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>
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			<td height="21" style="height:21px;width:216px;">paraformaldehyde</td>
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			<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>
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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 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>
			<td></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>
			<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>
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			<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>
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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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		<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>
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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>
		</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>
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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>
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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>
			<td></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>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<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>
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			<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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			<td style="width:119px;"></td>
			<td style="width:467px;">Visiview</td>
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			<td height="21" style="height:21px;width:216px;">Axiophot2</td>
			<td style="width:217px;"></td>
			<td style="width:119px;"></td>
			<td style="width:467px;">Axiophot2</td>
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stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fractionator and standard microscopy equipment

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

Using the presented method, the estimated number of TH+ dopaminergic neurons in the right SN ranged between 7,363 and 7,987 cells and, in the left SN, between 7,446 and 7,904 cells. Thus, the mean number of dopaminergic neurons (&#177; SEM) was 7,647 &#177; 83 cells for the right SN and 7,675 &#177; 66 for the left SN. The calculated CE for each animal was lower than 0.08 (range: 0.073-0.079) (Figure 7). To ascertain the comparability of this method with comm...

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

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

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

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Neuroscience

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