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.