No Effects of Photobiomodulation on Prefrontal Cortex and Hippocampal Cytochrome C Oxidase Activity and Expression of c-Fos Protein of Young Male and Female Rats

Abstract

The role of light in our biological processes and systems is extensively known. In addition, the use of light devices has been introduced in the field of healthcare as an opportunity to administer power light at specific wavelengths to improve our body functions and counteract light deficiency. One of these techniques is photobiomodulation (PBM), which uses red to infrared light in a non-invasive way to stimulate, heal, regenerate, and protect tissue. The main proposed mechanism of action is the stimulation of the cytochrome c oxidase (CCO), the terminal enzyme in the mitochondrial electron transport chain. PBM has achieved positive effects on brain activity and behavioral function of several adult animal models of health and disease, the potential use of this technique in developing stages is not surprising. This research aims to examine the effects of PBM on the prefrontal cortex and hippocampus of 23 day-old healthy male (n = 31) and female (n = 30) Wistar rats. Three groups of each sex were used: a PBM group which received 5 days of PBM, a device group submitted to the same conditions but without light radiation, and a control basal group. CCO histochemistry and c-Fos immunostaining were used to analyze brain metabolic activity and immediate early genes activation, respectively. Results displayed no metabolic differences between the three groups in both sexes. The same results were found in the analysis of c-Fos positive cells, reporting no differences between groups. This research, in contrast to the PBM consequences reported in healthy adult subjects, showed a lack of PBM effects in the brain markers we examined in young healthy rat brains. At this stage, brain function, specifically brain mitochondrial function, is not disturbed so it could be that the action of PBM in the mitochondria may not be detectable using the analysis of CCO activity and c-Fos protein expression. Further studies are needed to examine in depth the effects of PBM in brain development, cognitive functions and postnatal disorders, along with the exploration of the optimal light parameters.

Keywords: brain stimulation; development; low-level light therapy; nervous system; photobiomodulation.

FIGURE 1

CCO results (mean ± SEM). (A) CCO values in 29-day-old male groups (PBM, PBMD, C). There were no differences between the groups in the assessed areas (p > 0.05). (B) CCO values in 29 day-old female groups (PBM, PBMD, C). No significant differences were found in any of the areas (p > 0.05). Groups: C, control group; PBMD, photobiomodulation device group; PBM, photobiomodulation group. Areas: CG, Cingulate cortex; PL, Prelimbic cortex; IL, Infralimbic cortex; CA1, field CA1 of hippocampus; CA3, field CA3 of hippocampus; DG, Dentate Gyrus. PND, postnatal day; CCO, cytochrome c oxidase.

FIGURE 2

CCO samples. Representative photograph of the CCO optical density of the three groups (C, PBMD, and PBM) in PFC and HPC. There were no differences between groups in any of the studied regions in male or female samples. Groups: C, control group; PBMD, photobiomodulation device group; PBM, photobiomodulation group. Areas: CG, Cingulate cortex; PL, Prelimbic cortex; IL, Infralimbic cortex; CA1, field CA1 of hippocampus; CA3, field CA3 of hippocampus; DG, Dentate Gyrus. PFC, prefrontal cortex; HPC, hippocampus; PND, postnatal day; CCO, cytochrome c oxidase.

FIGURE 3

CCO differences between sexes in the three groups (PBM, PBMD, and C) in each region of interest (mean ± SEM). Female groups showed a general pattern of higher metabolic activity in all the studied areas (*p < 0.05). Groups: C, control group; PBMD, photobiomodulation device group; PBM, photobiomodulation group. Areas: CG, Cingulate cortex; PL, Prelimbic cortex; IL, Infralimbic cortex; CA1,field CA1 of hippocampus; CA3, field CA3 of hippocampus; DG, Dentate Gyrus. PFC, prefrontal cortex; HPC, hippocampus; PND, postnatal day; CCO, cytochrome c oxidase.

FIGURE 4

c-Fos-positive cells (positive cells of c-Fos/μm²) results in the regions of interest. (A) c-Fos results in 29 day-old male groups (C, PBMD, and PBM). No significant differences were found in any of the areas (p > 0.05). (B) c-Fos results in 29 day-old female groups (C, PBMD, PBM). There were no differences between the groups in the assessed areas (p > 0.05). Groups: C, control group; PBMD, photobiomodulation device group; PBM, photobiomodulation group. Areas: CG, Cingulate cortex; PL, Prelimbic cortex; IL, Infralimbic cortex; CA1, field CA1 of hippocampus; CA3, field CA3 of hippocampus; DG, Dentate Gyrus. PND, postnatal day.

FIGURE 5

c-Fos samples. Representative microphotograph of the c-Fos immunostaining in the IL cortex. (A) c-Fos sample of the control group. (B) c-Fos sample of the photobiomodulation device group. (C) c-Fos sample of the photobiomodulation group. No differences in c-Fos protein expression were found between the three groups in the male or female groups. IL, Infralimbic cortex.

FIGURE 6

c-Fos positive cells (positive cells of c-Fos/μm²) differences between sexes in the three groups (PBM, PBMD, and C) in each region of interest (mean ± SEM). Male groups showed more c-Fos positive cells in the prefrontal cortex (CG, IL, and PL) (*p < 0.05) while female groups showed higher c-Fos expression in CA3 (*p < 0.05). Groups: C, control group; PBMD, photobiomodulation device group; PBM, photobiomodulation group. Areas: CG, Cingulate cortex; PL, Prelimbic cortex; IL, Infralimbic cortex; CA1, field CA1 of hippocampus; CA3, field CA3 of hippocampus; DG, Dentate Gyrus.