Published: February 2nd, 2021
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 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 α-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.
Parkinson's disease (PD) is a prevalent neurodegenerative movement disorder characterized by the presence of protein aggregates containing α-synuclein (α-syn) and the preferential loss of dopaminergic neurons in the substantia nigra pars compacta (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-di....
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
By applying the above methods to brain tissue collected 6 weeks after AAV injections, we demonstrated that stereotactic injection of AAV expressing mutant A53T α-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 ± 34.7, a.......
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.......
|Alexa Fluor 488 secondary antibody
|Alexa Fluor 555 secondary antibody
|Alkaline phosphatase-conjugated anti-rabbit igG
|Biotinylated anti-mouse IgG
|Bovine Serum Albumin
|DAKO fluorescent mouting medium
|ImmPACT DAB Peroxidase substrate
|LSM880 Confocal Microscope
|Normal Goat Serum
|VectaMount mounting medium
|Vector Blue Alkaline Phosphatase substrate
|Zen Black Software
|Zen Blue Software
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