Low-level laser therapy (LLLT) reduces oxidative stress in primary cortical neurons in vitro

Abstract

Low-level laser (light) therapy (LLLT) involves absorption of photons being in the mitochondria of cells leading to improvement in electron transport, increased mitochondrial membrane potential (MMP), and greater ATP production. Low levels of reactive oxygen species (ROS) are produced by LLLT in normal cells that are beneficial. We exposed primary cultured murine cortical neurons to oxidative stressors: hydrogen peroxide, cobalt chloride and rotenone in the presence or absence of LLLT (3 J/cm², CW, 810 nm wavelength laser, 20 mW/cm²). Cell viability was determined by Prestoblue™ assay. ROS in mitochondria was detected using Mito-sox, while ROS in cytoplasm was detected with CellRox™. MMP was measured with tetramethylrhodamine. In normal neurons LLLT elevated MMP and increased ROS. In oxidatively-stressed cells LLLT increased MMP but reduced high ROS levels and protected cultured cortical neurons from death. Although LLLT increases ROS in normal neurons, it reduces ROS in oxidatively-stressed neurons. In both cases MMP is increased. These data may explain how LLLT can reduce clinical oxidative stress in various lesions while increasing ROS in cells in vitro.

Keywords: cobalt chloride; cultured cortical neurons; hydrogen peroxide; low-level laser therapy; oxidative stress; reactive oxygen species.

Representative images of cortical neurons labeled with MitoRox (red) for mitochondrial ROS, Mitotracker green for mitochondrial colocalization and Hoescht (blue) for nuclei (seen in triple overlay).

Figure 1

Cell viability of cortical neurons treated with increasing doses of CoCl₂ for 1 hour with and without 3 J/ cm² 810 nm laser in 1^st 2.5 min. N = 6 wells per group, error bars are SEM and * = p < 0.05 and ** = p < 0.01.

Figure 2

Cell viability of cortical neurons treated with CoCl₂ (10 and 20 μM) for 1 hour with and without 3 J/cm² 810 nm laser in 1^st 2.5 min. N = 6 wells per group, error bars are SEM and * = p < 0.05 and ** = p < 0.01.

Figure 3

Cell viability of cortical neurons treated with increasing doses of rotenone for 1 hour with and without 3 J/ cm² 810 nm laser in 1^st 2.5 min. N = 6 wells per group, error bars are SEM and * = p < 0.05.

Figure 4

(online color at: www.biophotonics-journal.org) Representative images of cortical neurons labeled with MitoRox (red) for mitochondrial ROS, Mitotracker green for mitochondrial colocalization and Hoescht (blue) for nuclei (seen in triple overlay). (A) control cortical neurons (B) cortical neurons with 3 J/cm² 810 nm laser; (C) cortical neurons treated with 500 μM CoCl₂; (D) cortical neurons treated with 500 μM CoCl₂ and 3 J/cm² 810 nm laser; (E) cortical neurons treated with 20 μM H₂O₂; (F) cortical neurons treated with 20 μM H₂O₂ and 3 J/cm² 810 nm laser. (G) cortical neurons treated with 200 nM rotenone; (H) cortical neurons treated with 200 nM rotenone and 3 J/cm² 810 nm laser. Scale bar = 20 μm.

Figure 5

Quantification of fluorescence measurements from the groups shown in Figure 4. N = 6 fields per group, error bars are SEM and * = p < 0.05 and ** = p < 0.01.

Figure 6

(online color at: www.biophotonics-journal.org) Representative images of cortical neurons labeled with Cellrox (deep red) for cytoplasmic ROS, and Hoescht (blue) for nuclei. (A) control cortical neurons (B) cortical neurons with 3 J/cm² 810 nm laser; (C) cortical neurons treated with 500 μM CoCl₂; (D) cortical neurons treated with 500 μM CoCl₂ and 3 J/cm² 810 nm laser; (E) cortical neurons treated with 20 μM H₂O₂; (F) cortical neurons treated with 20 μM H₂O₂ and 3 J/cm² 810 nm laser. (G) cortical neurons treated with 200 nM rotenone; (H) cortical neurons treated with 200 nM rotenone and 3 J/cm² 810 nm laser. Scale bar = 20 μm.

Figure 7

Quantification of fluorescence measurements from the groups shown in Figure 6. N = 6 fields per group, error bars are SEM and * = p < 0.05 and ** = p < 0.01.

Figure 8

(online color at: www.biophotonics-journal.org) Representative images of cortical neurons labeled with TMRM for mitochondrial membrane potential, and Hoescht (blue) for nuclei. (A) control cortical neurons (B) cortical neurons with 3 J/cm² 810 nm laser; (C) cortical neurons treated with 500 μM CoCl₂; (D) cortical neurons treated with 500 μM CoCl₂ and 3 J/cm² 810 nm laser; (E) cortical neurons treated with 20 μM H₂O₂; (F) cortical neurons treated with 20 μM H₂O₂ and 3 J/cm² 810 nm laser. (G) cortical neurons treated with 200 nM rotenone; (H) cortical neurons treated with 200 nM rotenone and 3 J/cm² 810 nm laser. Scale bar = 20 μm.

Figure 9

Quantification of fluorescence measurements from the groups shown in Figure 8. N = 6 fields per group, error bars are SEM and * = p < 0.05 and ** = p < 0.01.