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Aβ deposits in grey and white matter. a 6E10 immunostaining for diffuse Aβ deposits was performed for the parietal cortex, fear and spatial memory-related areas, and the corpus callosum. The optic tract was immunonegative for 6E10 staining and thus not shown. b Quantification of (a) showing the percentage area stained by 6E10. Scale bar represents 100 μm. Data are plotted as mean ± SE. (PDF 1133 kb)

Study design. a Schematic diagram of the study design. Two mild TBIs at 0.5 J were induced at 5 to 6 months of age in APP/PS1 mice and WT littermates. Sham procedures were performed as controls. Mice were followed for 8 M post-injury and longitudinally assessed with various behavioural tasks. b LRR duration after TBI or sham injuries is shown. c The NSS of the animals at pre-injury and 1 h post-injury, as well as up to 7 days post-injury, is reported. A higher score indicates greater neurological deficits. d The percentage of mice that survived the entire 8 M post-injury period is reported. e EPM performance is plotted by reporting the time spent in open arms. A higher value suggests stronger risk-taking behaviour. f EPM plotted by reporting the time spent in closed arms. A higher value suggests greater anxiety-like behaviour. g RR performance of animals. A longer fall latency indicates better motor coordination. In (d), data are plotted as percentage of the whole. In all others, data are plotted as mean ± SE. (PDF 176 kb)

Summary of p values. This table summarizes the p values of the experiments and assays reported in this study. (DOCX 23 kb)

Brain cytokines and tau and plasma neurofilament-light. The carbonate-soluble fraction of brain homogenates were assayed for (a) inflammatory cytokines including IL-1β, IL-6, and TNF-α and (b) total tau, p-tau at Thr231, and the ratio of p-tau to total tau. c Plasma levels of neurofilament-light at 8 M post-injury is also reported. In (a), data are plotted as median ± interquartile range. In (b) and (c), data are plotted as mean ± SE. (PDF 50 kb)

White matter pathology in the corpus callosum. a At 8 M post-TBI, histopathological analyses were performed on the corpus callosum at the level of the dorsal hippocampus, using Iba1 (microglia), NeuroSilver (axonal injury), SMI312A (neurofilament) and GFAP (astrocytes). Representative images for each stain are shown. (b) Quantification of (a) is plotted by reporting the density and size of microglia, the stain area of NeuroSilver, the density of neurofilament-positive axonal bulbs, and the GFAP immunofluorescence intensity of astrocytes. Scale bar represents 200 μm. Data are plotted as mean ± SE. (PDF 1515 kb)

Cortical ICAM-1 expression is increased in AD. Cryopreserved cortex and cerebellum of AD or NCI patients were cut at 20 μm. After PFA fixation sections were washed with PBS and stained against (a) ICAM-I or (b) VCAM-1 and CD31 as a vascular marker and imaged using an inverted fluorescent microscope. Arrow demonstrates colocalization of ICAM-1 and CD31. Bar represents 50 μm (PDF 62676 kb)

HDL treatment does not alter LRP1 or RAGE protein levels in hCMEC/D3. (a-c) hCMEC/D3 were treated with HDL (0–400 μg/mL) for 5 h before lysing in RIPA. (a) LRP1 and (b) RAGE protein levels were quantified by immunoblotting (c). Graphs represent means ± SD relative to vehicle treated cells in 3 trials (PDF 1114 kb)

Aβ induced dose dependent PBMC adhesion and HDL attenuate dose dependent Aβ-induced PBMC adhesion to hCMEC/D3. In all conditions, hCMEC/D3 were stimulated with 0–1 μM Aβ40 (light grey) or Aβ42 (dark grey) monomers for 3 h. Fluorescently labelled PBMC were allowed to adhere to (a) Aβ40- or (b) Aβ42- stimulated cells for 3 additional hours. Cells were washed, fixed, and imaged to count adhered PBMC. hCMEC/D3 were primed with increasing doses (25–400 μg/mL) of HDL for 2 h and stimulated with 0.1 μM (c) Aβ40 (light grey) or (d) Aβ42 (dark grey) for 3 h. Fluorescently labelled PBMC were allowed to adhere to stimulated cells for 3 h followed by washing, fixation, imaging, and counting. Graphs represent mean ± SD of adhered PBMC relative to vehicle control from at least 3 independent trials where * p

Aβ does not activate phosphorylation of multifunctional serine/threonine protein kinases. hCMEC/D3 were stimulated with 0.1 mM of monomeric Aβ or 1 ng/mL of TNF-αfor 15 min before lysing cells in RIPA containing phosphostop. Phosphorylation of (a-b) p65, (c) STAT3, (d) Akt, (e) SAPK/JNK and (f) p42/44 MAPK were analysed by immunoblotting and compared to respective total p65, STAT3, Akt, SAPK/JNK and p42/44 MAPK respectively. (b) Nuclear translocation of p65 was analysed by immunofluorescence 15 min after Aβ stimulation. Graphs represent means ± SD relative to vehicle treated cells in 4 trials. *p

Adhesion molecules are enhanced by TNF-α but not Aβ.(a-c) hCMEC/D3 were primed with 100 μg/mL HDL for 2 h followed by stimulation with 1 ng/mL TNF-α for 3 h. Cell lysates were prepared in RIPA and protein levels of (a) ICAM-1 and (b) VCAM-1 were measured by denaturing immunoblotting (c). (d-g) hCMEC/D3 were stimulated with monomeric (d,f) Aβ40 or (e,g) Aβ42 at the indicated concentrations and (d,e) ICAM-1 and (f,g) VCAM-1 mRNA levels were measured by real-time PCR. (h-j) Following Aβ stimulation, cell surface proteins were biotinylated and isolated by immunoprecipitation. Protein levels of cell surface and total (h) ICAM-1 and (i) VCAM-1 were measured by denaturing immunoblotting (j). (k-m) hCMEC/D3 were pre-treated with 100 μg/mL of HDL for 2 h followed by stimulation with Aβ. After 3 h total and cell surface ICAM-1 expression were measured as above. Graphs represent means ± SD from at least 3 independent trials. ***p

HDL suppression of Aβ-induced inflammation is independent of eNOS and S1P in HUVEC. (a) L-NAME and (e) VPC23019 potency was tested by measuring intracellular NO production in HUVEC after incubating with 100 μg/mL HDL and 1 μM DAF-2 for 6 h. Fluorescence was measured at 485 nm. (b-d, f-h) In all conditions, HUVEC or hCMEC/D3 were stimulated with 0.1 μM monomeric Aβ40 or Aβ42 or 1 ng/mL of TNF-α for 3 h prior to measuring PBMC adherence. HUVEC were pre-treated for 1 h with (b-d) the eNOS inhibitor L-NAME or (f-h) the S1P1 and S1P3 inhibitor VPC23019 followed by 100 μg/mL HDL for 2 h. (i) hCMEC/D3 were pre-treated with 100 μg/mL of HDL for 2 h before simulating with Aβ40 or Aβ42. Total cellular expression of Annexin-1 (Anx1) was analysed by immunoblotting and compared to GAPDH. Graphs represent means ± SD from at least 3 independent trials. * p