Photobiomodulation with near infrared light mitigates Alzheimer's disease-related pathology in cerebral cortex - evidence from two transgenic mouse models

Sivaraman Purushothuman, Daniel M Johnstone, Charith Nandasena, John Mitrofanis, Jonathan Stone, Sivaraman Purushothuman, Daniel M Johnstone, Charith Nandasena, John Mitrofanis, Jonathan Stone

Abstract

Introduction: Previous work has demonstrated the efficacy of irradiating tissue with red to infrared light in mitigating cerebral pathology and degeneration in animal models of stroke, traumatic brain injury, parkinsonism and Alzheimer's disease (AD). Using mouse models, we explored the neuroprotective effect of near infrared light (NIr) treatment, delivered at an age when substantial pathology is already present in the cerebral cortex.

Methods: We studied two mouse models with AD-related pathologies: the K369I tau transgenic model (K3), engineered to develop neurofibrillary tangles, and the APPswe/PSEN1dE9 transgenic model (APP/PS1), engineered to develop amyloid plaques. Mice were treated with NIr 20 times over a four-week period and histochemistry was used to quantify AD-related pathological hallmarks and other markers of cell damage in the neocortex and hippocampus.

Results: In the K3 mice, NIr treatment was associated with a reduction in hyperphosphorylated tau, neurofibrillary tangles and oxidative stress markers (4-hydroxynonenal and 8-hydroxy-2'-deoxyguanosine) to near wildtype levels in the neocortex and hippocampus, and with a restoration of expression of the mitochondrial marker cytochrome c oxidase in surviving neurons. In the APP/PS1 mice, NIr treatment was associated with a reduction in the size and number of amyloid-β plaques in the neocortex and hippocampus.

Conclusions: Our results, in two transgenic mouse models, suggest that NIr may have potential as an effective, minimally-invasive intervention for mitigating, and even reversing, progressive cerebral degenerations.

Figures

Figure 1
Figure 1
Time course of the natural development of cortical pathology in K3 and APP/PS1 mice. (A), (B), (C), (D), (E), (F) Micrographs of hyperphosphorylated tau labelling (red), using the AT8 antibody, in the neocortex (A to C) and hippocampus (D to F) of untreated K3 mice at 3 months (A, D), 6 months (B, E) and 12 months (C, F) of age. (G), (H), (I) Micrographs of amyloid-beta (Aβ) labelling (green), using the 6E10 antibody, in neocortex of untreated APP/PS1 mice at 3 months (G), 4.5 months (H) and 12 months (I) of age. Arrowheads indicate intraneuronal Aβ labelling, arrows indicate extracellular plaques. Comparable immunolabelling was achieved with the 4G8 antibody. Sections were co-labelled for glial fibrillary acidic protein (red), a marker of astrocytes. For all sections, nuclei were labelled with bisbenzimide (blue). Scale in (H) applies to (A) to (G).
Figure 2
Figure 2
Effect of near-infrared light treatment on hyperphosphorylated tau and neurofibrillary tangles in the neocortex of K3 mice. (A), (B) Quantification of tau AT8 immunolabelling, based on average labelling intensity (A) and labelled area (B). All error bars indicate standard error of mean. *P < 0.05, **P < 0.01. (C), (D), (E) Representative photomicrographs of sections stained with Bielschowsky silver stain to demonstrate neurofibrillary tangles (NFTs). Arrows indicate axonal swellings and NFTs. (F), (G), (H) Representative micrographs of AT8 (red) labelling within neurons of the neocortex retrosplenial area. Nuclei were labelled with bisbenzimide (blue). Scale bars = 50 μm; scale in (E) applies to (C) and (D), scale in (H) applies to (F) and (G). NIr, near-infrared light; WT, wildtype.
Figure 3
Figure 3
Effect of near-infrared light treatment on hyperphosphorylated tau and neurofibrillary tangles in the hippocampus of K3 mice. (A), (B) Quantification of tau AT8 immunolabelling, based on average labelling intensity (A) and labelled area (B). All error bars indicate standard error of mean. **P < 0.01, ***P < 0.001. (C), (D), (E) Representative photomicrographs of sections of the hippocampal subiculum area, stained with Bielschowsky silver stain to demonstrate neurofibrillary tangles (NFTs). Arrows indicate axonal swellings and NFTs. The classical silver stain also labelled axons in the white matter core (WM). (F), (G), (H) Representative micrographs of AT8 (red) labelling within hippocampal CA1 pyramidal neurons. Nuclei were labelled with bisbenzimide (blue). Scale bars = 50 μm; scale in (E) applies to (C) and (D), scale in (H) applies to (F) and (G). NIr, near-infrared light; WT, wildtype.
Figure 4
Figure 4
Effect of near-infrared light treatment on oxidative stress markers in the neocortex of K3 mice. (A), (B), (F), (G) Quantification of immunolabelling of two oxidative stress markers, 4-hydroxynonenal (4-HNE; A, B) and 8-hydroxy-2′-deoxyguanosine (8-OHDG; F, G), based on average labelling intensity (A, F) and labelled area (B, G). All error bars indicate standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.001. (C), (D), (E), (H), (I), (J) Representative micrographs of 4-HNE (red) labelling (C, D, E) and 8-OHDG (red) labelling (H, I, J) within layers IV and V of the neocortical retrosplenial area. Nuclei were labelled with bisbenzimide (blue). Scale bar = 50 μm; scale in (J) applies to all other micrographs. NIr, near-infrared light; WT, wildtype.
Figure 5
Figure 5
Effect of near-infrared light treatment on cytochrome c oxidase labelling in the neocortex and hippocampus of K3 mice. (A), (B), (F), (G) Quantification of immunolabelling of the mitochondrial marker cytochrome c oxidase (COX) in the neocortex retrosplenial area (A, B) and hippocampal CA1 layer (F, G), based on average labelling intensity (A, F) and labelled area (B, G). All error bars indicate standard error of the mean. *P < 0.05, ***P < 0.001. (C), (D), (E), (H), (I), (J) Representative micrographs of COX (red) labelling in the neocortex retrosplenial area (C, D, E) and hippocampal CA1 layer (H, I, J). Nuclei were labelled with bisbenzimide (blue). Scale bar = 50 μm; scale in (J) applies to all other micrographs. NIr, near-infrared light; WT, wildtype.
Figure 6
Figure 6
Effect of near-infrared light on amyloid-beta and plaque pathology in APP/PS1 mice. (A), (B), (C), (D), (E), (F) Quantification of amyloid-beta (Aβ) 4G8 immunolabelling of amyloid plaques in the neocortex (A, B, C) and hippocampus (D, E, F), based on plaque burden (A, D), plaque size (B, E) and number of plaques (C, F). All error bars indicate standard error of the mean. *P < 0.05, ***P < 0.001, ****P < 0.0001. (H), (I), (J), (K), (L), (M) Representative micrographs showing Aβ labelling with the 4G8 antibody (brown) in the neocortex (H, I, J) and hippocampus (K, L, M). Arrows indicate plaques. Scale bar = 100 μm; scale in (M) applies to all other micrographs. DG, dentate gyrus of hippocampus; NIr, near-infrared light; SLM, stratum lacunosum moleculare; WT, wildtype.
Figure 7
Figure 7
Effect of near-infrared light on Congo red-positive plaque numbers in APP/PS1 mice. (A), (B) Quantification of Congo red-positive plaque counts in the neocortex (A) and hippocampus (B). All error bars indicate standard error of the mean. (C), (D), (E), (F), (G), (H) Representative micrographs showing Congo red staining of plaques in the neocortex (C, E, G) and hippocampus (D, F, H). Arrows indicate plaques. Scale bar = 50 μm; scale in (H) applies to all other micrographs. DG, dentate gyrus of hippocampus; NIr, near-infrared light; SLM, stratum lacunosum moleculare; WT, wildtype.

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