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

Summary

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.

Abstract

In pre-clinical Parkinson'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.

Introduction

The quantification of neuronal cell number is pivotal in pre-clinical Parkinson'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 standard^3,4,5.

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 sigh....

Protocol

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

1. Euthanize mice with CO₂ or any other approved method.
2. 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 infusion pump, followed by 70 mL of 4% paraformaldehyde (PFA) in 0.1 M PBS.
   CAUTION: PFA is toxic, allergenic, and carcinogenic.
....

Results

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 (± SEM) was 7,647 ± 83 cells for the right SN and 7,675 ± 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 commercially availa.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Paxinos mouse atlas			The Mouse Brain George Paxinos Keith B.J.FranklinCopyright @2001 by Academic Press CD Rom designet & created by Paul Halasz
brain matrix slicer mouse	Zivic Instruments	BSMAS 001-1
paraformaldehyde	Merck	1040051000
sucrose /D(+) Saccharose	Roth	4621.1
isopentane	Roth	3927.1
glycerol	Merck	1040931000
Ethanol	Sigma Aldrich	32205-1L
Name	Company	Catalog Number	Comments
phosphate buffered saline ingredients:
sodium chloride	Sigma Aldrich	31434-1KG-R
potassium dihydrogen phosphate	Merck	1048731000
di-sodium hydrogen phosphate dihydrate	Merck	1065801000
potassium chloride	Merck	1049360500
normal goat serum	Dako	X0907
bovine serum albumin	Sigma	A4503-100G
Triton X-100	Sigma Aldrich	X100-100ml	detergent
3,3-Diaminobenzidine-tetrahydrochlorid/DAB tablets 10mg pH 7.0	Kem En Tec	4170
H2O2/ Hydrogen peroxide 30%	Merck	1072090250
avidin/biotin reagent	Thermo Scientific	32050	Standard Ultra Sensitive ABC Staining Kit, 1:100
rabbit anti mouse tyrosine hydroxylase antibody	abcam	Ab112	1:1000
biotinylated goat-anti-rabbit IgG H+L	vector laboratories	BA-1000	1:100
StereoInvestigator version 11.07	MBF
BX53 microscope	Olympus
Visiview	Visitron Systems GmbH	3.3.0.2
Axiophot2	Zeiss
ImageJ software	NIH	Version 4.7
Tissue-TEK OCT	Sakura	4583
dry ice
grid overlay plugin	Wayne Rasband		https://imagej.nih.gov/ij/plugins/graphic-overlay.html
cell counter plugin	Kurt de Vos		https://imagej.nih.gov/ij/plugins/cell-counter.html).
optical disector macro	Christopher Ward
C57Bl/6N male mice	Charles River, Germany
SuperFrost Plus coated object slides	Langenbrinck, Germany
25G needle Microlance 3	BD	300400
REGLO Analog Infusion pump	Ismatec	ISM 829
StereoInvestigator system			StereoInvestigator version 11.07
BX53 microscope			BX53 microscope
self-assorted stereology			Visiview
Axiophot2			Axiophot2