The authors would like to thank all the staff at the Advanced Optical Microscopy Facility (AOMF) at University Health Network for their time and assistance in developing this protocol.

All procedures were approved by the University Health Network Animal Care Committee and performed in accordance with guidelines and regulations set by the Canadian Council on Animal Care.
1. Stereotactic injection
<ol>
	<li>Pair-house adult female Sprague-Dawley rats (250-280 g) in cages with wood bedding and ad lib access to food and water. Maintain the animal colony in a regular 12 h light/dark cycle (lights on 06:30) with constant temperature and humidity.</li>
	<li>Perform unilateral ste...

<ol>
	<li>Kalia, L. V., Lang, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parkinson's+disease.">Parkinson's disease.</a> Lancet. 386 (9996), 896-912 (2015).</li><li>West, M. J., Slomianka, L., Gundersen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+stereological+estimation+of+the+total+number+of+neurons+in+thesubdivisions+of+the+rat+hippocampus+using+the+optical+fractionator.">Unbiased stereological estimation of the total number of neurons in thesubdivisions of the rat hippocampus using the optical fractionator.</a> The Anatomical Record. 231 (4), 482-497 (1991).</li><li>Nair-Roberts, R. G., 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=Stereological+estimates+of+dopaminergic,+GABAergic+and+glutamatergic+neurons+in+the+ventral+tegmental+area,+substantia+nigra+and+retrorubral+field+in+the+rat.">Stereological estimates of dopaminergic, GABAergic and glutamatergic neurons in the ventral tegmental area, substantia nigra and retrorubral field in the rat.</a> Neuroscience. 152 (4), 1024-1031 (2008).</li><li>Golub, V. M., 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=Neurostereology+protocol+for+unbiased+quantification+of+neuronal+injury+and+neurodegeneration.">Neurostereology protocol for unbiased quantification of neuronal injury and neurodegeneration.</a> Frontiers in Aging Neuroscience. 7, 196 (2015).</li><li>Schmitz, C., Hof, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design-based+stereology+in+neuroscience.">Design-based stereology in neuroscience.</a> Neuroscience. 130 (4), 813-831 (2005).</li><li>Penttinen, A. M., 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=Implementation+of+deep+neural+networks+to+count+dopamine+neurons+in+substantia+nigra.">Implementation of deep neural networks to count dopamine neurons in substantia nigra.</a> European Journal of Neuroscience. 48 (6), 2354-2361 (2018).</li><li>Yousef, A., 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=Neuron+loss+and+degeneration+in+the+progression+of+TDP-43+in+frontotemporal+lobar+degeneration.">Neuron loss and degeneration in the progression of TDP-43 in frontotemporal lobar degeneration.</a> Acta Neuropathologica Communications. 5 (1), 68 (2017).</li><li>Koprich, J. B., 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=Expression+of+human+A53T+alpha-synuclein+in+the+rat+substantia+nigra+using+a+novel+AAV1/2+vector+produces+a+rapidly+evolving+pathology+with+protein+aggregation,+dystrophic+neurite+architecture+and+nigrostriatal+degeneration+with+potential+to+model+the+pathology+of+Parkinson's+disease.">Expression of human A53T alpha-synuclein in the rat substantia nigra using a novel AAV1/2 vector produces a rapidly evolving pathology with protein aggregation, dystrophic neurite architecture and nigrostriatal degeneration with potential to model the pathology of Parkinson's disease.</a> Molecular Neurodegeneration. 5, 43 (2010).</li><li>Koprich, J. B., 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=Progressive+neurodegeneration+or+endogenous+compensation+in+an+animal+model+of+Parkinson's+disease+produced+by+decreasing+doses+of+alpha-synuclein.">Progressive neurodegeneration or endogenous compensation in an animal model of Parkinson's disease produced by decreasing doses of alpha-synuclein.</a> PLoS One. 6 (3), 17698 (2011).</li><li>McKinnon, C., 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=Early-onset+impairment+of+the+ubiquitin-proteasome+system+in+dopaminergic+neurons+caused+by+alpha-synuclein.">Early-onset impairment of the ubiquitin-proteasome system in dopaminergic neurons caused by alpha-synuclein.</a> Acta Neuropathologica Communications. 8 (1), 17 (2020).</li><li>Henderson, M. X., 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=Spread+of+alpha-synuclein+pathology+through+the+brain+connectome+is+modulated+by+selective+vulnerability+and+predicted+by+network+analysis.">Spread of alpha-synuclein pathology through the brain connectome is modulated by selective vulnerability and predicted by network analysis.</a> Nature Neuroscience. 22 (8), 1248-1257 (2019).</li><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 Neuropathologica Communications. 5 (1), 11 (2017).</li><li>Webster, J. D., Dunstan, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-slide+imaging+and+automated+image+analysis:+considerations+and+opportunities+in+the+practice+of+pathology.">Whole-slide imaging and automated image analysis: considerations and opportunities in the practice of pathology.</a> Veterinary Pathology. 51 (1), 211-223 (2014).</li></ol>

The reliable assessment of the integrity of the dopaminergic system in pre-clinical models of PD is critical to determine the effectiveness of potential disease-modifying therapeutics. Therefore, it is important to control and minimize potential confounds that may reduce the reliability and reproducibility of histopathological data. Careful quantitative outcomes can provide more information than qualitative or semi-quantitative descriptions alone. At the same time, we must recognize that constraints in time and resources...

Parkinson&#39;s disease (PD) is a prevalent neurodegenerative movement disorder characterized by the presence of protein aggregates containing &#945;-synuclein (&#945;-syn) and the preferential loss of dopaminergic neurons in the substantia nigra pars compacta&#160;(SNpc)1. Quantification of dopaminergic neuron number is a vital part of PD research as it permits the evaluation of the integrity of the nigrostriatal system, thus, providing an important endpoint to assess the effectiveness of potential disease-modifying therapeutics. Currently, the standard for quantification of cell number is unbiased stereological counting, which utilizes two-dimensional (2D) cross-sections of tissue to estimate volumetric features in three-dimensional (3D) structures2,3,4. Modern design-based stereological methods employ comprehensive random sampling procedures and apply counting protocols (known as probes) to avoid potential artifacts and systematic errors, allowing for reliable detection of differences only slightly greater than inter-animal variation5. While stereology provides a powerful analytical tool for in vivo histological studies, it is time intensive, assumes uniform specimen preparation, and requires validation at several steps, which can impact the efficiency increasingly required for pre-clinical translational investigation.
Recent technological advances in digital science make it possible to adopt novel applications for more efficient assessments of pathology without a stereomicroscope, while filling a need as a surrogate of unbiased stereology. These methods increase speed, reduce human error, and improve the reproducibility of stereological techniques6,7. HALO is one such image analysis platform for quantitative tissue analysis in digital pathology. It comprises a variety of different modules and reports morphological and multiplexed expression data on a cell-by-cell basis across entire tissue sections using pattern recognition algorithms. The cytonuclear FL module measures the immunofluorescent positivity of fluorescent markers in the nucleus or cytoplasm. This allows for reporting of the number of cells positive for each marker, and the intensity score for each cell. The module can be adapted to provide individual cell sizes and intensity measurements, although this feature is not required for quantification of dopaminergic neurons.
The aim of this study is to verify this method with a previously validated viral vector-based &#945;-syn rat model of nigral neurodegeneration8,9,10. In this model, human mutant A53T &#945;-syn is expressed in the SNpc by stereotactic injection of adeno-associated virus hybrid serotype 1/2 (AAV1/2), resulting in significant neurodegeneration over a period of 6 weeks. The contralateral uninjected SNpc may, in some studies, serve as an internal control for the injected side. More commonly, injection of AAV-Empty Vector (AAV-EV) in a control cohort of animals is used as a negative control. We present a step-by-step guide to estimate the density of dopaminergic neurons remaining in the injected SNpc after 6 weeks using an automated image analysis software (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A-Syn Antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>32-8100</td>
			<td></td>
		</tr>
		<tr>
			<td>ABC Elite</td>
			<td>Vector Labs</td>
			<td>PK-6102</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 secondary antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>A-28180</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline phosphatase-conjugated anti-rabbit igG</td>
			<td>Jackson Immuno</td>
			<td>111-055-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated anti-mouse IgG</td>
			<td>Vector Labs</td>
			<td>BA-9200</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO fluorescent mouting medium</td>
			<td>Agilent</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>HALO&#8482;</td>
			<td>Indica Labs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Histo-Clear II</td>
			<td>Diamed</td>
			<td>HS202</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmPACT DAB Peroxidase substrate</td>
			<td>Vector Labs</td>
			<td>SK-4105</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM880 Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NeuN Antibody</td>
			<td>Millipore</td>
			<td>MAB377</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Vector Labs</td>
			<td>S-1000-20</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Tissue-Tek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>BioShop</td>
			<td>PAR070.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sliding microtome</td>
			<td>Leica</td>
			<td>SM2010 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Investigator</td>
			<td>MBF Bioscience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>BioShop</td>
			<td>SUC700</td>
			<td></td>
		</tr>
		<tr>
			<td>TH Antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>P21962</td>
			<td></td>
		</tr>
		<tr>
			<td>VectaMount mounting medium</td>
			<td>Vector Labs</td>
			<td>H-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vector Blue Alkaline Phosphatase substrate</td>
			<td>Vector Labs</td>
			<td>SK-5300</td>
			<td></td>
		</tr>
		<tr>
			<td>Zen Black Software</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zen Blue Software</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

semi-quantitative determination of dopaminergic neuron density in the substantia nigra of rodent models using automated image analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

By applying the above methods to brain tissue collected 6 weeks after AAV injections, we demonstrated that stereotactic injection of AAV expressing mutant A53T &#945;-syn (AAV-A53T) in the SNpc of rat brain results in a significant reduction in the density of dopaminergic neurons compared to injection of empty vector AAV (AAV-EV) as a control (Figure 5A,B). The mean number of TH-positive neurons/mm2 in the SNpc of rats injected with AAV-EV was 276.2 &#177; 34.7, a...

Watch this Scientific Journal Video about Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis at JoVE.com

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

Estimation of dopaminergic neuron number in the substantia nigra is a key outcome measure in Parkinson's disease research. This protocol significantly shortens the workload required to estimate neuron number. This method provides a time efficient and accurate process for detecting changes in dopaminergic neuron number and is suitable for determining the effect of interventions on cell survival.This method could be easily adapted to provide accurate counts of neurons in different brain regions while maintaining cost and time advantages over traditional stereological methods. To capture IHC images use the software coupled to a confocal microscope at 10X magnification. Open the pin hole to 1.5 area units to capture a wide plane totaling 1.5 micro meters and set the focus on the injected side of the brain.On the acquisition tab check the tile scan imaging option and set the dimensions to 10 by four. Under the acquisition mode panel set the zoom to 1.1 to avoid any stitching marks between the tile scan images. Set the frame size to 1024 by 1024 pixels and the averaging to two to ensure high quality image acquisition.In the channels panel set track one to Alexa 488 and track two to Alexa 555. Load the slide onto the stage and choose a section with a strong TH staining. Click on live on the acquisition panel.In the channels panel set the laser strength and gain to levels that maximize signal and limit the background noise. Use the range indicator to ensure that the signals are not overexposed. Repeat this with multiple slides.On the acquisition tab check the positions box. To begin imaging, use the eye piece and choose the first section showing positive TH staining. Then set the focus at the point of interest and move the stage to the midline of the section.This saves the position in the X, Y, and Z axes and will image a tile scan capturing the whole section. Repeat this for all other slides including an un-injected site. Separate image files using appropriate software and import them to automated image analysis software.Define a region of interest by selecting the pen annotation tool to draw an annotation around the substantia nigra pars compacta. Move to the analysis tab and select real time tuning from the dropdown analyze menu. This opens a separate window on the section image allowing for real time modification of analysis parameters.Under the analysis magnification section select the appropriate image zoom. Under the cell detection section select nuclear dye as the dye used for TH staining. Adjust the nuclear contrast threshold, minimum nuclear intensity, nuclear segmentation aggressiveness and nuclear size settings while carefully watching the time tuning window.Repeat this process with multiple samples. Once an appropriate number of images have been sampled and real-time tuning has been adjusted accordingly save the analysis settings in the settings actions drop-down menu. Select all images to be analyzed and click on analyze.Choose the analysis setting that was just saved and in the region of analysis window check the annotation layers box, then check layer one and click on analyze. Once complete export the summary analysis data for all sections by selecting the option to export object analysis data. This data set could be used to examine changes in cell size in response to a toxin or therapeutic.Six weeks after adeno associated virus or AAV injections, stereotactic injection of AAV expressing mutant A53T alpha synuclein into the substantia nigra in the rat brain resulted in a significant reduction in the density of dopaminergic neurons. The mean number of TH positive neurons per millimeter squared in the substantia nigra of rats injected with AAV-A35T was significantly decreased as compared to that of rats injected with the AAV empty vector. Similar observations were made using unbiased stereology.When attempting this protocol keep in mind the software is only as useful as it is trained to be. Therefore time and care must be taken to ensure a single cell is identified as a single cell. The software can be adapted to measure a fluorescence intensity of other proteins of interests such as alpha-synuclein to determine if treatments can modulate protein levels.This technique has allowed for greater efficiency when testing the effect of multiple potential therapeutics on dopaminergic neuron density.

semi-quantitative-determination-dopaminergic-neuron-density

Research

JoVE Journal

Neuroscience

4.3K vistas.  University Health Network. La estimación del número dopaminérgico de la neurona en el nigra del substantia es una medida dominante del resultado en la investigación de la enfermedad de Parkinson. Este protocolo acorta significativamente la carga de trabajo necesaria para estimar el número de neuronas. Este método proporciona un proceso eficiente y preciso para detectar cambios en el número de neuronas dopaminérgicas y es adecuado para determinar el efecto de las intervenciones sobre la supervivencia celular.