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.
