Immunofluorescent staining of Akt and PTEN.

<p>A: The LLI effects on immunofluorescence staining of p-PTEN and p-Akt in cultures cells (bottom). Corresponding phase contrast micrographs are shown on top of each fluorescent image. It should be noted that tumor cells normally proliferate in high proportions and p-Akt is often highly expressed in cancer cells of different natures, whereas our result of control group shows low level of p-Akt expression due to haze reduction. Cal.: 100 µm. B: Average fluorescence intensity for p-PTEN or p-Akt normalized to the control value. Asterisks: one-way ANOVA, * p<0.05, ** p<0.01.</p

Effects of LLI, DAPT and combined application of both on cell proliferation.

<p>Proliferation ratios for each group at 24 h and 48 h after LLI for control (white bars), 60 min LLI (light grey bars), DAPT (grey bars) or the combination of LLI and DAPT (dark grey bars) are shown. Asterisks: one-way ANOVA, * p<0.05, ** p<0.01.</p

Effects of LLI on the number of A-172 cells.

<p>A: Sample images of A-172 cells under light microscope. The number of cells increased 48 h post-LLI (right column) after 20, 40, 60 min LLI compared to pre-LLI (left column). Cal.: 100 µm. B: Proliferation ratio (the ratio of cell number at 24 or 48 hours following LLI and cell number before LLI) was normalized to control (no LLI) (n = 12 for each group). C: A summary of colorimetric analysis by MTT staining performed at 48 h after LLI (each group: n = 16). The optical density of each group was normalized to the value of control group (no LLI) at 48 h after initial condition. Asterisks: one-way ANOVA, * p<0.05, ** p<0.01.</p

Effects of LLI on the fluorescence intensities of p-Akt, p-PTEN and Aβ.

<p>A, B: Schematic diagrams of Notch and APP signaling pathways. GS can cleave APP and Notch, making Aβ and ICN, respectively (A). GSI inhibits Aβ expression but also inhibits ICN expression (B), a side-effect against cell survival through PTEN activation (dashed box in B). C: LLI effects on immunofluorescence staining of p-Akt, p-PTEN and Aβ in cells pretreated with GSI. Cal.: 100 µm. D: Average fluorescence intensity for p-PTEN (green bars), p-Akt (pink bars) and Aβ (blue bars) was normalized to control. Asterisks: one-way ANOVA, ** p<0.01.</p

Effects of LLI on ATP level in cell lysates.

<p>The ATP/ADP ratio measured using a luminescence-based assay for control and for 20, 40 and 60 min LLI. The luminescent densities for ATP and ADP were measured 48 h after LLI treatment. The ATP/ADP ratio was calculated and averaged (n = 5). Asterisks: one-way ANOVA, * p<0.05.</p

532 nm Low-Power Laser Irradiation Facilitates the Migration of GABAergic Neural Stem/Progenitor Cells in Mouse Neocortex

<div><p>Background and Objective</p><p>Accumulating evidence has shown that low-power laser irradiation (LLI) affects cell proliferation and survival, but little is known about LLI effects on neural stem/progenitor cells (NSPCs). Here we investigate whether transcranial 532 nm LLI affects NSPCs in adult murine neocortex and in neurospheres from embryonic mice.</p><p>Study Design/Materials and Methods</p><p>We applied 532 nm LLI (Nd:YVO₄, CW, 60 mW) on neocortical surface via cranium in adult mice and on cultured cells from embryonic mouse brains in vitro to investigate the proliferation and migration of NSPCs and Akt expression using immunohistochemical assays and Western blotting techniques.</p><p>Results</p><p>In vivo experiments demonstrated that 532 nm LLI significantly facilitated the migration of GABAergic NSPCs that were induced to proliferate in layer 1 by mild ischemia. In vitro experiments using GABAergic NSPCs derived from embryonic day 14 ganglionic eminence demonstrated that 532 nm LLI for 60 min promoted the migration of GAD67-immunopositive NSPCs with a significant increase of Akt expression. Meanwhile, the LLI induced proliferation, but not migration, of NSPCs that give rise to excitatory neurons.</p><p>Conclusion</p><p>It is concluded that 532 nm LLI promoted the migration of GABAergic NSPCs into deeper layers of the neocortex in vivo by elevating Akt expression.</p></div

LLI effects on pAkt and Akt expression of cultured cells.

<p>A: Western blot analysis of neurosphere lysates from post-LLI (4 hours) of E10 forebrain. B: Quantification of blots. Each blot intensity was normalized to GAPDH of loading control (mean ± SD, n = 3, t-test *p < 0.05).</p

Trans-well migration experiments.

<p>A: Location of brain parts dissected for making neurospheres of NSPCs. Label “c” shows the cortex of E16, which includes NSPCs of excitatory and GABAergic neurons. B: Method of trans-well migration test. Bottom membrane of inserted trans-well has pores of 8 μm diameter. Neurospheres derived from “c” were placed on insert trans-well, and 532 nm laser was irradiated from above the media. Cells were fixed by 4% PFA at 48 h after LLI. C: A cell that moved through the pores stained by GAD67 (red) and Hochest (blue). Holes in the Hochest image are the pores of trans-well. Scale bar: 10 μm. Double positive cells were found only in the LLI experimental group (mean ± SD, n = 4, t-test *p < 0.05).</p

LLI effects on proliferation of NSPC cells from E10 forebrain and E14 MGE.

<p>A: Location of brain parts dissected for making neurosphere of NSPCs. “a” shows cortical wall of E10 forebrain, which generates excitatory neurons, whereas “b” shows E14 MGE, which generates GABAergic neurons. B: CCK-8 assay to show biochemically cell proliferation for different duration of LLI (mean ± SD, n = 4 for each duration, t-test, *p < 0.05). The non-irradiated group was standardized as 100%. C: Cell counting of DAPI staining to clarify the increase in cell number following the dissociation from neurospheres. LLI significantly promoted proliferation (mean ± SD, n = 5, t-test, *p < 0.05).</p

Effects of transcranial LLI on cell migration of NSPCs.

<p>A: Scheme of LLI treatment to adult mouse brain. Auditory cortical area was irradiated on the left side of the cortex through cranium. The opposite hemisphere was used as a control. B: TTC staining for detecting cell death. LLI after the mild occlusion of CCA (CCAO) did not induce cell death (left) compared with sham control (right). C: Protocol for in vivo experiments from CCAO to immunostaining. Brains were fixed at 4 h and 5 days after LLI. D: Photographs of each immunostaining at post-LLI (4 hours) and 5 days after LLI. EdU, GAD67 and Ki67 positive cells (white arrow) were found in layer 1 of post-LLI sections. EdU and GAD67 positive cells but Ki67 negative cells (yellow arrow) were found in deep layer at 5 days after LLI. GAD67 positive, EdU and Ki67 negative cell (arrowhead) is a mature inhibitory neuron. Scale bars: 10 μm. E: The laminar pattern of the cell density (mean ± SEM, n = 4) of EdU and GAD67 positive, Ki67-negative cells from layer 1 to 6 was significantly different between LLI and control groups (KS test, p<0.01). F: Cumulative fraction of total cells for non-parametric KS 2-sample test. Each accumulative curve shows higher distribution in layer 1–4 in control group, while does in layer 5–6 in LLI group. G: Total cell density of EdU and GAD67 positive and Ki67 negative (EdU+/GAD67+/Ki67-) cells did not differ between control and LLI groups.</p