Analysis d software

Vessel length density (VLD) and diameters were measured in whole mounts of ears stained with L. esculentum lectin as previously described10 (link). Briefly, vessel diameters were measured by overlaying a captured microscopic image with a square grid. Squares were chosen at random, and the diameter of each vessel (if any) in the center of selected squares was measured. Two hundred total vessel diameter measurements were obtained from 6 10 ears per each group (n = 6–10). Vessel length density was measured on 1 to 3 fields per ear in 6–10 ears per each group (n = 6–10) by tracing the total length of vessels in the fields and dividing it by the area of the fields. All image measurements were performed with AnalySIS D software (Soft Imaging System, Gmbh, Münster, Germany). The number of arterioles was quantified in fluorescently immunostained cryosections. Briefly, arterioles were defined as vessels of regular shape and moderately larger than capillaries (15–30 μm) associated with a thick and homogeneous smooth muscle layer (positive for α-smooth muscle actin, α-SMA) coating the endothelial layer (positive for CD31), as previously described17 (link), and were counted on 14 microscopic fields/group, localized in proximity to the areas of induced angiogenesis (n = 2 muscles/group; field size = 180′710 μm²).

Deparaffinized 6-μm sections of the putamen were incubated with 3% H₂O₂ in methanol for 20 min to block endogenous peroxidase activity. Sections were microwaved in trisodium citrate solution (pH 6.5) for antigen retrieval and blocked with 1.5% normal sera for 30 min before incubation with the primary antibody for 1h at room temperature [glial fibrillary acidic protein: GFAP (1:500, Dako, Ely, UK); CD68 (1:100, Dako); CD163 (1:100, Serotec, Kidlington, UK); fibrinogen (1:400, Alere Ltd, Stockport, UK); ferritin (1:1000, Sigma, Poole, UK)]. The avidin-biotin horseradish peroxidase (ABC-HRP) complex method was used (Vectastain Elite kit, Vector Laboratories, Peterborough, UK), with diaminobenzidine (DAB) as the substrate. Five random regions within the area of interest were selected (×20 magnification; Cell∧R, Olympus, Southend-on-Sea, UK), and the percentage area immunoreactivity of the image analysed using analysis^∧D software (Olympus Biosystems, Planegg, Germany) following delineation and exclusion of vascular profiles and voids in the sections.

Images were captured in a belt‐transect pattern using the 20X objective, excluding layer I, as described in the previous section. Quantification of the total number of γH2AX⁺ nuclei was performed in the MCx and FACx, using Analysis^˄D software (Olympus Biosystems, Watford, UK). The number of γH2AX⁺ neuronal nuclei was determined using a size exclusion of >10 μm (500 pixels), and the number of positive small nuclei (glia) was determined by subtracting the number of pyramidal neuronal nuclei from the total number of positive nuclei. To assess the total number of cells (neurones and glia), the same detection protocol was applied to haematoxylin‐only stained sections, which allowed the determination of the percentage of immunopositive cells (total number of γH2AX⁺ neurones*100/total number of neurones; total number of γH2AX⁺ glia*100/total number of glia per subject).

Image analysis was performed blind to the clinical information. Selected regions of interest for the spinal cord (anterior horn, lateral corticospinal tract and the dorsal column), pons and the thalamus were marked on haematoxylin and eosin sections and mapped onto consecutive immunostained slides. Assessment of antigen-specific immunoreactivity was performed by capturing 20× bright-field microscopic images (Olympus Cell R, Olympus Biosystems, Watford, UK) in five random fields selected within the areas of interest. For the parietal cortex, images were taken across the entire cortical thickness in three adjacent transects. To quantitatively assess the immunoreactive profile of the candidate markers across the region of interest, the colour threshold was set, and the percentage area immunoreactivity exceeding the threshold was determined using analySIS D software (Olympus Biosystems, Watford, UK). The number of ionized calcium-binding adaptor molecule 1 (Iba-1)- or MHC-II-positive microglia was calculated based on size exclusion (250 pixels for MHC-II; 150 pixels for Iba-1).^{26 (link)}

Image capture of three adjacent 350 μm‐wide cortical ribbons through the full thickness of the FCx was performed using Cell R software (Olympus Biosystems, Watford, UK), using a × 20 objective. The image was automatically thresholded and the total number of positive nuclei was quantitated using Analysis^˄D software (Olympus Biosystems, Watford, UK). The number of DNA‐PKcs⁺ or γH2AX⁺ positive pyramidal neuronal nuclei was determined using a size exclusion of >250 pixels, and the number of positive small nuclei (glia and small neurones) determined by subtracting the number of pyramidal neuronal nuclei from the total number of positive nuclei. To assess the total number of pyramidal neurones and small cells in each case, an identical quantitation protocol was performed on haematoxylin‐only stained serial sections. Therefore, in addition to total counts of immunopositive neurones, the proportion of immunopositive neurones was also determined by dividing the number of positive neurones by the total number of neurones in the FCx.

Assessment of NDRG2-specific immunoreactivity was performed by capturing bright-field microscopic images in 3 adjacent 350 μm-wide cortical ribbons, consisting of contiguous fields to cover the total cortical thickness through the apex of the gyrus, using a ×20 objective (Nikon Eclipse Ni-U microscope, Nikon Instruments Europe BV, Amsterdam, Netherlands) and analysed using the Analysis ^D software (Olympus Biosystems, Watford, UK). The image was thresholded and the immunoreactive area (%) of the field determined per total area examined.