Low-level laser therapy (810 nm) protects primary cortical neurons against excitotoxicity in vitro

Abstract

Excitotoxicity describes a pathogenic process whereby death of neurons releases large amounts of the excitatory neurotransmitter glutamate, which then proceeds to activate a set of glutamatergic receptors on neighboring neurons (glutamate, N-methyl-D-aspartate (NMDA), and kainate), opening ion channels leading to an influx of calcium ions producing mitochondrial dysfunction and cell death. Excitotoxicity contributes to brain damage after stroke, traumatic brain injury, and neurodegenerative diseases, and is also involved in spinal cord injury. We tested whether low level laser (light) therapy (LLLT) at 810 nm could protect primary murine cultured cortical neurons against excitotoxicity in vitro produced by addition of glutamate, NMDA or kainate. Although the prevention of cell death was modest but significant, LLLT (3 J/cm(2) delivered at 25 mW/cm(2) over 2 min) gave highly significant benefits in increasing ATP, raising mitochondrial membrane potential, reducing intracellular calcium concentrations, reducing oxidative stress and reducing nitric oxide. The action of LLLT in abrogating excitotoxicity may play a role in explaining its beneficial effects in diverse central nervous system pathologies.

Keywords: cultured cortical neurons; excitotoxicity; kainic acid; low-level laser therapy; mitochondrial membrane potential glutamate. NMDA; reactive oxygen species.

Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figures

Figure 1. Effect of 810-nm laser on MTT cell viability in cortical neurons with excitotoxicity

Neurons were treated with excitotoxins for 1 hour with and without 3 J/cm2 810 nm laser in first 3 min. Points are means of 6 wells per group, error bars are SEM and *p < 0.05; ** = p<0.01 vs sham (dark).

Figure 2. Effect of 810-nm laser on ATP generation in cortical neurons with excitotoxicity

Quantification by luminescence plate reader of the relative light unit values per mg cell protein using Cell Titer Glo ATP assay in cortical neurons as control and with 3 excitotoxins, with and without LLLT (3 J/cm2 810 nm laser). Points are means from nine wells. Error bars are SEM. * = p <0.05; ** = p<0.01; *** = p<0.001 vs sham (dark).

Figure 3. Effect of 810-nm laser on intracellular calcium release in cortical neurons with excitotoxicity

(A) Fluo-4 AM (green) fluorescence for calcium and Hoechst (blue) fluorescence for nuclei in cortical neurons as control and with 3 excitotoxins, with and without LLLT (3 J/cm2 810 nm laser). Scale bar = 20 µm. (B) Quantification by Image J of mean fluorescence values. N= 6 fields per group. Error bars are SEM and * = p <0.05; ** = p<0.01; *** = p<0.001 vs sham (dark).

Figure 4. Effect of 810-nm laser on mitochondrial membrane potential (MMP) in cortical neurons with excitotoxicity

(A) TMRM (red) fluorescence for MMP and Hoechst (blue) fluorescence for nuclei in cortical neurons as control and with 3 excitotoxins, with and without LLLT (3 J/cm2 810 nm laser). Scale bar = 20 µm. (B) Quantification by Image J of mean fluorescence values. N= 6 fields per group. Error bars are SEM and * = p <0.05; ** = p<0.01 vs sham (dark).

Figure 5. Effect of 810-nm laser on intracellular reactive oxygen species (ROS) in cortical neurons with excitotoxicity

(A) CellRox Red (red) fluorescence for cytoplasmic ROS and Hoechst (blue) fluorescence for nuclei in cortical neurons as control and with 3 excitotoxins, with and without LLLT (3 J/cm2 810 nm laser). Scale bar = 20 µm. (B) Quantification by Image J of mean fluorescence values. N= 6 fields per group. Error bars are SEM and * = p <0.05 vs sham (dark).

Figure 6. Effect of 810-nm laser on nitric oxide (NO) production in cortical neurons with excitotoxicity

(A) DAF-FM (green) fluorescence for NO in cortical neurons as control and with 3 excitotoxins, with and without LLLT (3 J/cm2 810 nm laser). Scale bar = 20 µm. (B) Quantification by Image J of mean fluorescence values. N= 6 fields per group. Error bars are SEM and * = p <0.05; ** = p <0.01 vs sham (dark).