Fig 2: Multiple label immunofluorescence of mGlu3 labeling in microglia in layer V of rat PL mPFC. Tissue was labeled with antibodies recognizing the microglial protein Iba1 (cyan), mGlu3 (red), the dendritic marker Map2 (green), as well as the nuclear Hoechst stain (blue). (A) Similarly, to layer II/III, there are diffuse Iba1 positive microglial processes throughout the neuropil in layer V. (Ai–v) Magnified area of the yellow box in (A), showing an Iba1 positive microglial soma surrounding the Hoechst positive nucleus (blue) and emanating processes (cyan). The process contains mGlu3 (red) indicated by white arrows, which are Map2 negative. (B) In another region of layer V, there is an Iba1 positive microglial soma (cyan) with two visible processes in this plane. (Bi–v) Magnified area of the yellow box in (B), demonstrates mGlu3 localization within the processes extending from the soma, as demarcated by the white arrows. Scale bars, 10 µm (A,B); 5 µm (Ai–v,Bi–v).

Fig 3: Number of ?H2AX + cells was significantly increased in temporal pole and visual cortex of demented but not in resilient when compared to control brains (a). Representative photomicrographs of ?H2AX + cells on sections from control, resilient and demented cases containing visual cortex (b). Representative immunofluorescence images of colocalization of ?H2AX + cells (brown) with neurons (red) (7c 1–3, showing a progressively zoomed-in brain region), GFAP + astrocytes (green) (c 4–6, showing GFAP alone, 7c4, ?H2AX alone, 7c5, both GFAP-?H2AX, 7c6), and IBA1 + microglia (green) (7c 7–9, showing IBA1 alone, 7c7, ?H2AX alone, 7c8, both IBA1-?H2AX, 7c9). C Control (Braak 0-II); R Resilient (Braak III/IV); D Demented (Braak III/IV); **p < 0.01; ***p < 0.001. Scale bars 1 mm, 50 µm, and 20 µm. White arrows indicate colocalization between ?H2AX+ cells and neurons, astrocytes, and microglia, respectively

Fig 4: OATP4A1 expression correlate with hypoxia-related genes in PPN and PNZ regions in human GBM tissue. Representative immunohistochemical labelling of OATP4A1 labelled with pan myeloid marker IBA1, tumour cell/astrocyte marker, GFAP, endothelial cell marker, UAE-1 lectin, stromal cell marker, PDGFRß and Hoechst nuclear counterstain revealed increased expression of OATP4A1 within the PPN microenvironment (A1); scale bar = 200 µm. The yellow dotted line delineates the core tumour (left) from the PPN region (right). Immunofluorescent staining of OATP4A1 in PPN regions in three GBM tumour tissue cases (A2–A4). The white dotted line delineates PPN from PNZ regions. Images taken with a 20x objective; scale bar = 100 µm. Differential gene expression of PNZ (B) and PPN (C) compared to core tumour cells from the Ivy Atlas RNAseq dataset revealed a group of hypoxia-related genes, which were similar between PPN and PNZ (E). Spearman correlation matrix of OATPs and the top 40 DEGs in the PPN and PNZ regions reveal a positive correlation with only the OATP4A1 isoform with hypoxia-related genes (D) .

Fig 5: Example of the artificial intelligence based semiautomated quantification method used. Selection of cortical and subcortical regions of interest (a, b), tissue layer detection in green (b, c, f), P2RY12 counter in purple (c, d), IBA1 area detector in blue (f, g) and unannotated images stained with P2RY12 (e) and IBA1 (h)