Fig 1.
PBM therapy restores neural stem cell pool to WT levels in 3xTg mice.
A. 20X multi-panel images were stitched together to quantify sox2+ cells in the dentate gyrus subgranular zone (outlined in yellow, scale bars = 100µm). B. NSC quantification was done using sox2+ cell counts normalized to SGZ length (mm). (One-way ANOVA *p<0.05).
Fig 2.
PBM does not affect the rate of neurogenesis in aged 3xTg mice.
A. Representative images of DCX (red) and calretinin (CR, green) stained dentate gyrus sections showing DCX+ cells (arrowheads) and CR+ cells in the subgranular zone and granule cell layer (Scale bars = 100µm) B. Quantification of DCX and CR staining showing significant reductions in the number of newborn neurons in both 3xTg groups compared to WT with no effect of PBM. (One-way ANOVA. *p<0.05, **p<0.01, ***p<0.001). C. Analysis of newborn granule neurons at different stages of development shows a dramatic decrease in newly generated DCX+ neural progenitors and an overrepresentation of DCX-/CR+ immature neurons. D. Schematic representation of neurogenesis and timeline of marker expression from NSC (left) to mature granule neuron (right).
Fig 3.
DCX+ cell size, migration, and dendritic arborization are partially rescued by PBM treatment in 3xTg mice.
A. Representative images of DCX+ newborn neurons. (Scale bars = 10µm) B. Quantification of DCX cell size shows no significant differences between groups, but DCX migration into the GCL is significantly reduced in 3xTg Sham, but not 3xTg + PBM compared to WT (One-way ANOVA, *p < 0.05), C. Simple linear regression shows a positive correlation between DCX cell size and NSC number. D. Sholl concentric sphere analysis of DCX+ cells shows dystrophic sprouting of multiple dendrites in close proximity to the soma in 3xTg mice, and severely diminished dendritic complexity at increasing Sholl sphere radii. Left: two-way repeated-measures ANOVA shows significant effect of Sholl radius on the number of intersections and a significant interaction between Sholl radius and treatment, but no overall effect of PBM treatment on number of Sholl intersections. Right: Multiple comparisons between groups show significant differences between WT ctrl and 3xTg Sham, but not 3xTg + PBM in the number of intersections between 85 and 95µm Sholl radii.
Fig 4.
One month of PBM therapy led to increased Aβ plaque deposition, and larger average size of non-microglia associated plaques.
A. Representative images of whole hippocampal sections showing extracellular Aβ deposits, labeled with 6E10 primary antibody, and microglia labeled with Iba1 (Dapi labeled in blue, scale bar = 500μm). Enlarged panel showing microglia associated plaques (MAP; arrowheads) and non-microglia associated plaques (non-MAP; asterisks) in the subiculum (scale bar = 100μm). B. PBM appears to increase the overall deposition of extracellular Aβ plaques and average plaque size. C. Quantitative analysis shows no change in the frequency of microglia association with plaques. Microglia-associated plaques (MAP) are on average similar in size between groups, whereas non-MAP are significantly larger in 3xTg + PBM animals. (Unpaired t-test. *p < 0.05).
Fig 5.
PBM induces focal activation of microglial phagocytosis around Aβ plaques.
A. Representative confocal images of Aβ plaques (6E10), microglia (Iba1) and CD68 showing diffuse microglial phagocytosis away from plaques in sham compared to localized phagocytosis around plaques in PBM-treated animals. B. Quantitative analysis of total and activated microglia show no overall differences in the neuroinflammatory environment surrounding Aβ plaques. (Unpaired t-test). C. Analysis of the distribution of CD68+ microglia shows a trend of higher concentration of phagocytic cells in contact with plaques, and decreasing phagocytosis away from plaques. (Two-way ANOVA).
Fig 6.
Intracellular tau hyperphosphorylation in the hippocampus is not significantly altered by PBM treatment in aged 3xTg-AD mice.
A. Representative images of hippocampal sections showing extensive intracellular accumulation of pTau (AT8, red; BIII-tubulin, green; DAPI, blue). (Scale bar = 200µm) B. Regional quantifications of AT8+ cells show significant accumulation of intracellular pTau throughout the hippocampus of 3xTg-AD mice with no apparent effect of PBM treatment (One-way ANOVA. *p<0.05, **p<0.01, ***p<0.001).
Fig 7.
PBM significantly reduces hyperphosphorylation of tau with no effect on total tau expression.
A. Western blots of total hippocampus detergent-soluble protein extract show decreased hyperphosphorylated tau (AT8: pSer202, pThr205) in 3xTg + PBM compared to Sham with no corresponding decrease in total tau expression. B. Quantitative analysis of AT8 and Tau5 western blots normalized to total protein and pTau to total tau ratio. (One-way ANOVA. *p<0.05, ****p<0.0001). C. Simple linear regression analysis shows a negative correlation between pTau/Tau ration and neural stem cell number.