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. 2016 Feb 17;6:21172.
doi: 10.1038/srep21172.

Blue Light Modulates Murine Microglial Gene Expression in the Absence of Optogenetic Protein Expression

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Free PMC article

Blue Light Modulates Murine Microglial Gene Expression in the Absence of Optogenetic Protein Expression

Kevin P Cheng et al. Sci Rep. .
Free PMC article

Abstract

Neural optogenetic applications over the past decade have steadily increased; however the effects of commonly used blue light paradigms on surrounding, non-optogenetic protein-expressing CNS cells are rarely considered, despite their simultaneous exposure. Here we report that blue light (450 nm) repetitively delivered in both long-duration boluses and rapid optogenetic bursts gene-specifically altered basal expression of inflammatory and neurotrophic genes in immortalized and primary murine wild type microglial cultures. In addition, blue light reduced pro-inflammatory gene expression in microglia activated with lipopolysaccharide. These results demonstrate previously unreported, off-target effects of blue light in cells not expressing optogenetic constructs. The unexpected gene modulatory effects of blue light on wild type CNS resident immune cells have novel and important implications for the neuro-optogenetic field. Further studies are needed to elucidate the molecular mechanisms and potential therapeutic utility of blue light modulation of the wild type CNS.

Figures

Figure 1
Figure 1. Blue light dose-dependently increases basal gene expression in naïve microglia.
Blue light (450 nm) was delivered for 1 second per minute for 6 hrs at the indicated energy doses. Data are graphed as means + 1 SEM of light-induced % change in gene expression relative to that observed in the absence of light (dotted line). (a) Expression of pro-inflammatory and (b) anti-inflammatory/growth factor genes in N9 microglia (n = 6 each light condition). (c) Expression of pro-inflammatory and (d) anti-inflammatory/growth factor genes in primary microglia (n = 3 light doses 1, 10, 25, 50 mJ/cm2; n = 6 light dose 0, 100 mJ/cm2). *P < 0.05, **P < 0.01, ***P < 0.001 vs. no light control by Holm-Sidak test.
Figure 2
Figure 2. Blue light dose-dependently alters LPS-stimulated microglial inflammatory gene expression.
Blue light (450 nm) was delivered for 1 second per minute for 6 hrs at the indicated energy doses, to cells treated with vehicle or 1 μg/mL LPS. Data are graphed as means + 1 SEM of light-induced % change in LPS-stimulated gene expression relative to that observed in the presence of LPS and absence of light (dotted line). (a) Expression of pro-inflammatory and (b) anti-inflammatory/growth factor genes in N9 microglia (n = 6 each light condition). (c) Expression of pro-inflammatory and (d) anti-inflammatory/growth factor genes in primary microglia (n = 3 light doses 1, 10, 25, 50 mJ/cm2; n = 6 light dose 0, 100 mJ/cm2). *P < 0.05, **P < 0.01, ***P < 0.001 vs. no light control by Holm-Sidak test or Tukey test (LPS-treated: Tgfβ, Igf-1, Cox-2).
Figure 3
Figure 3. Blue light exposure reduces IL-1β cytokine levels in the culture medium of primary microglia treated with LPS.
Primary microglia were exposed to blue light (450 nm) for 1 second per min at an intensity of 100 mW/cm2 and treated with either vehicle or LPS (1 μg/mL) for 6 hours. Data are graphed as means + 1 SEM of arbitrary units normalized to the LPS only condition. IL-1β levels were below the detection threshold of the assay and are designated as not detectable (N.D.). n = 3, ***P < 0.001 student’s t-test.
Figure 4
Figure 4. Blue light does not decrease microglial cell viability or promote apoptosis.
N9 microglia were exposed to blue light (450 nm) for 1 second per minute for 6 hrs at the indicated energy doses and assayed immediately following or 18 hours post-exposure. Viability (left panel) and apoptosis (right panel) are graphed as means + 1 SEM assessed relative to the no light controls (0 mJ/cm2). No statistically significant differences in viability or apoptosis were detected.
Figure 5
Figure 5. Blue light does not promote DNA damage in microglia.
N9 microglia were exposed to no light (0 mJ/cm2; control, top row) or blue light (450 nm) at 50 mJ/cm2 (middle row) and 100 mJ/cm2 (bottom row) for 1 sec/min for 6 hours, after which time they were stained with anti-γ-H2AX antibodies (left column). Brightfield images of the same field-of-view are shown in the middle column. As a positive control for DNA strand breaks, cells were also exposed to 100 μM etoposide and stained with anti-γ-H2AX antibodies (right column). No dose of light tested induced DNA damage, and blue light did not further increase etoposide-induced DNA damage. Scale bar = 50 μm.
Figure 6
Figure 6. Blue light delivered in an optogenetic pattern decreases LPS-stimulated microglial inflammatory gene expression.
The effect of optogenetic light delivery on the basal expression of (a) pro-inflammatory and (b) anti-inflammatory/growth factor genes (n = 6), or LPS-induced expression of (c) pro-inflammatory and (d) anti-inflammatory/growth factor genes (n = 6) is shown. 15 ms pulses of blue light (450 nm) was delivered at 10 Hz, (~25 mW/cm2) for 6 hrs to primary microglia treated with vehicle (a,b) or 1 μg/mL LPS (c,d). Data are graphed as means + 1 SEM of light-induced % change in gene expression relative to the no light controls (dotted line; a,b) or light-induced % change in LPS-stimulated gene expression relative to LPS alone in the absence of light (dotted line; c,d). *P < 0.05, **P < 0.01, ***P < 0.001 vs. respective to no light controls by Holm-Sidak test.

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