Image Analysis
Immunostained sections images were taken using a VS120 Virtual Slide Microscope (Olympus, Southend-on-Sea, UK). Manual counts and density image analyses were performed using ImageJ software (ImageJ v1.53k, National Institute of Health, Bethesda, MD, USA). Automatic counting image analysis was achieved using Qupath softwear (QuPath v0.4.4, Cancer Research & Cell Biology, Queen’s University, Belfast, Northern Ireland). In an unbiased manner three images were selected along the rostrocaudal axis of the striatum or nigra based on their distance from bregma (striatal AP coordinates: +0.7, +1.0, +1.2 mm; nigral AP coordinates -5.6, -5.8, -6.04 mm). The optical density of the staining of α-synuclein in the substantia nigra and the striatum, and of tyrosine hydroxylase in the striatum was measured using ImageJ software. To do so, both the intact side and the lesioned side the mean grey value was measured. By applying the conversion formula in ImageJ (optical density = log10 (255/mean grey value)), these were then converted to optical densities, this was then expressed as a percentage of the intact side. To quantify the number of tyrosine hydroxylase immunopositive cell bodies in the substantia nigra, manual counts were conducted on both the ipsilateral and contralateral sides according to distinct boundaries. Specifically, immune-positive cells in the substantia nigra pars reticulata, pars lateralis and pars compacta were counted and immune-positive cells in the ventral tegmental area (VTA) were excluded. Cell counts data were expressed as a percentage of the intact side. The number of pS129-α-synuclein-positive accumulations in the substantia nigra were also counted on the side of the brain ipsilateral to the lesion surgery. Data were expressed as the average number of pS129-α-synuclein-positive accumulations in the three sections analysed. To ascertain the number of pS129-α-synuclein-positive accumulations in the striatum, QuPaths pixel classifier feature was used to create a threshold. Once the threshold was set, the annotation was saved pS129-α-synuclein and split objects was selected to give the number of annotations found within the striatum. Staining vectors were set on each image prior to running the programme to create the annotations on the lesioned side of the brain. Data were expressed as the average number of pS129-α-synuclein-positive accumulations in the three sections analysed. To assess the number of α-synuclein aggregates in the substantia nigra, analysis was conducted using QuPath’s built-in “cell detection”. Staining vectors were first set on each image for consistency and the same cell detection parameters used throughout the project. This give the number of aggregates detected in the ipsilateral side. Data were expressed as the average number of aggregated α-synuclein-positive accumulations in the three sections analysed.