Photobiomodulation with a 660-Nanometer Light-Emitting Diode Promotes Cell Proliferation in Astrocyte Culture

Astrocytes act as neural stem cells (NSCs) that have the potential to self-renew and differentiate into other neuronal cells. The protein expression of these astrocytes depends on the stage of differentiation, showing sequential expression of multiple proteins such as octamer-binding transcription factor 4 (Oct4), nestin, glial fibrillary acidic protein (GFAP), and aldehyde dehydrogenase 1 family member L1 (aldh1L1). Photobiomodulation (PBM) affects cell apoptosis, proliferation, migration, and adhesion. We hypothesized that astrocyte proliferation and differentiation would be modulated by PBM. We used an optimized astrocyte culture method and a 660-nanometer light-emitting diode (LED) to enhance the biological actions of many kinds of cells. We determined that the 660-nanometer LED promoted the biological actions of cultured astrocytes by increasing the reactive oxygen species levels. The overall viability of the cultured cells, which included various cells other than astrocytes, did not change after LED exposure; however, astrocyte-specific proliferation was observed by the increased co-expression of GFAP and bromodeoxyuridine (BrdU)/Ki67. Furthermore, the 660-nanometer LED provides evidence of differentiation, as shown by the decreased Oct4 and GFAP co-expression and increased nestin and aldh1L1 expression. These results demonstrate that a 660-nanometer LED can modify astrocyte proliferation, which suggests the efficacy of the therapeutic application of LED in various pathological states of the central nervous system.


Introduction
The brain contains the following two kinds of cell: neurons and glial cells. Glial cells include astrocytes, microglia, and oligodendrocytes. Among glia cells, microglia belong to the monocyte/macrophage lineage, while astrocytes, belong to neural lineage. Astrocytes are the most abundant and have crucial roles in the brain.
Protein expression determines the characteristics of cells. Astrocytes differentiate from neural stem cells (NSCs) into mature cells, and protein expression starts with the embryonic rosette (octamer-binding transcription factor 4, Oct4) and goes through neural progenitors (nestin) to astrocytes (glial fibrillary acidic protein, GFAP) [1]. Oct4 is a neural progenitor transcription factor. Nestin is a cytoskeletal intermediate filament. It has been used as a marker for progenitor cells in the CNS. During maturation, nestin is gradually downregulated and replaced by tissue-or cell-specific intermediate filament proteins, such as GFAP and microtubule-associated protein 2 (MAP2) [2]. GFAP expression increases again in reactive astrocytes when an immune response occurs [3]. Therefore, GFAP is not From DIV0 to DIV6, cells were incubated in DMEM+ (DMEM, FBS, P/S [P = 5000 U/mL, S = 5000 μg/mL]). On DIV6, cells were subcultured for isolation of astrocytes. Cells were incubated in NB+ medium (Neurobasal, B27, GlutaMAX, P/S (P = 5000 U/mL, S = 5000 μg/mL)). The LED irradiation protocol was divided into the following two methods: irradiation on DIV7 only, and irradiation on both DIV7 and DIV10. Each colored circle denotes the sampling time for several experiments. The red circle indicates verification for cell differentiation evidence, and the blue and green circles indicate cell proliferation and ROS assay, respectively.

Cell Culture
Cultured astrocytes were prepared as reported previously [25] with minor modifications. Briefly, brains were extracted from Sprague Dawley rats at embryonic day 17. Cortices were separated from extracted brain tissues and incubated in a water bath at 37 °C with 0.25% trypsin for 20 min, followed by gentle trituration.
Dissociated cells were counted and plated on 0.04% polyethylenimine (PEI)-coated culture dishes (4,000,000 cells/60 mm dish) with culture media (10% fetal bovine serum, 5000 U/mL penicillin, and 5000 μg/mL streptomycin in Dulbecco's modified Eagle's medium; Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Six days after the culture, the dishes were shaken at 110 rpm for 6 h. After shaking, cells were treated with 0.25% trypsin and plated on a 0.04% PEI-coated microplate or 18-mm coverslip in a 12-well plate (20,000 From DIV0 to DIV6, cells were incubated in DMEM+ (DMEM, FBS, P/S [P = 5000 U/mL, S = 5000 µg/mL]). On DIV6, cells were subcultured for isolation of astrocytes. Cells were incubated in NB+ medium (Neurobasal, B27, GlutaMAX, P/S (P = 5000 U/mL, S = 5000 µg/mL)). The LED irradiation protocol was divided into the following two methods: irradiation on DIV7 only, and irradiation on both DIV7 and DIV10. Each colored circle denotes the sampling time for several experiments. The red circle indicates verification for cell differentiation evidence, and the blue and green circles indicate cell proliferation and ROS assay, respectively.

Cell Culture
Cultured astrocytes were prepared as reported previously [25] with minor modifications. Briefly, brains were extracted from Sprague Dawley rats at embryonic day 17. Cortices were separated from extracted brain tissues and incubated in a water bath at 37 • C with 0.25% trypsin for 20 min, followed by gentle trituration.

Cell Viability Assay
A Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) was used for analyzing cell viability. Cells were prepared in a 96-well microplate at a density of 1 × 10 4 cells/well. A CCK-8 reagent was added to cells 24 h after 660-nanometer LED irradiation and incubated for 2 h at 37 • C. After 2 h, the absorbance of cells was measured at 450 nm with a microplate reader. Cell viability (%) was calculated by determining the optical density of LED-irradiated cells compared to control cells.

ROS Assay
For the reactive oxygen species (ROS) assay, the DCFDA/H2DCFDA Cellular ROS Assay Kit (ab113851; Abcam, Cambridge, UK) was used. Cells were plated on an 18-mm coverslip in a 12-well plate at a density of 2 × 10 4 cells/well and irradiated with the 660-nanometer LED on DIV7 and DIV10. On the last day of irradiation, the medium was removed immediately after 660-nanometer LED irradiation and the cells were washed with 1× supplemented buffer (equilibrated at 37 • C before use) at least twice. Then, 20 mM DCFDA solution was diluted in 1× supplemented buffer to a final concentration of 20 µM. Diluted DCFDA solution was added to stain the cells. Cells were incubated for 45 min at 37 • C in the dark and then washed with 1× buffer once or twice. Live cell fluorescent microscopy was performed. Oxidation derivative of DCFDA (2 ,7 -dichlorofluorescein, DCF) is fluorescent and it is detected by ex/em at 485 nm/535 nm, as it is FITC. To avoid photobleaching, low-light or dark conditions were maintained.

Statistical Analysis
Images were analyzed with ImageJ software (NIH). To analyze the expression pattern of proteins related to NSCs, astrocytes, and cell proliferation, cells were manually counted in the region of interest. Statistical analyses were performed using Prism v7.0 (GraphPad, La Jolla, CA, USA). All experimental and control data are presented as means ± standard errors of the mean. The Shapiro-Wilk normality test was used to determine whether the data followed a Gaussian distribution. When it did, two-tailed unpaired t-tests were performed; otherwise, the Mann-Whitney U test was used.

Verification of the Astrocyte Culture
A protocol was used for culturing astrocytes that establishes in vivo morphology and gene expression [25,26], and allows astrocytes to be cultured in a two-dimensional in vivo environment [26]. The astrocytes were cultured according to a previous study with minor modifications ( Figure 1B) [25]; the cultured astrocytes are shown in Figure 2A. To verify the culture of astrocytes, they were immunostained with markers for astrocytes (GFAP and aldh1L1), neurons (NeuN and MAP2), and microglia (Iba1). Dual immunostained images showed that most of the cells expressed the astrocyte markers GFAP and aldh1L1. Cultured astrocytes have a star shape, which is considered in vivo-like morphology. A few cells expressed other markers, such as NeuN, MAP2, and Iba1 ( Figure 2B-E). In total, 60-80 of every 100 cells expressed astrocyte markers (aldh1L1 or GFAP), and 20 expressed neuronal cell markers (NeuN or MAP2). The remaining cells expressed a microglia marker (Iba1) ( Figure 2F). In the case of microglia, it accounts for 8.6%, and this amount can release large quantities of agents and affect the astrocytes. Furthermore, there are previous studies in which pro-inflammatory cytokines' (e.g., TNFα, IL-6, IL-1β) release of microglia is decreased by PBM [27,28]. Therefore, to confirm that microglia had no effect on this experiment, the level of cytokines was measured through Western blot (Supplementary Figures S1 and S2). As a result, there were no significance differences, confirming that the effect of inflammatory response by microglia is negligible. In addition, in the triple staining, astrocytes (GFAP) and neurons (MAP2) showed similar results to dual staining, but microglia (Iba1) were not detected ( Figure 2G,H). These results showed that astrocytes were cultured successfully.

Astrocyte Proliferation was Exclusively Enhanced by 660-nm LED Irradiation
PBM at 660 nm regulates the biological actions of many types of cells, including diabetic wounded cells, fibroblasts, and human adipose-derived stem cells [24,[29][30][31]. In the astrocyte culture, DIV7-DIV14 is considered the developmental stage, and astrocytes before DIV14 still have neural stem cell (NSC) features [26]. Therefore, irradiation was conducted on DIV7 and DIV10 to determine whether the 660-nanometer LED affected the developmental stage of the astrocyte culture ( Figure 1B). Two exposures were the most appropriate conditions for a period between DIV7 and DIV14 in order to expose the maximum amount of light at regular intervals. To determine whether the 660-nanometer LED causes toxicity or an increase in overall cell proliferation, a cell viability assay was performed in an astrocyte culture, which includes various cell types other than astrocytes (n = nine wells). The cells were irradiated with a total density ranging from 3 to 18 J/cm 2 . The results showed that the 660-nanometer LED had no effects on the overall cell density at any power density ( Figure 3A). Once the cells were stimulated by PBM, cytochrome c oxidase (CCO) in the mitochondria respiratory chain was activated, leading to a membrane potential (MMP) increase. It leads to dissociation of inhibitory nitric oxide from CCO. As a result, enzyme activity, ATP production, and reactive oxygen species (ROS) were increased [32,33]. In this study, to evaluate whether LED affects cellular activity, an ROS assay, in which ROS expression was altered by LED irradiation [34], was performed. The cells were irradiated by the 660-nanometer LED on DIV7 and DIV10.

Astrocyte Proliferation was Exclusively Enhanced by 660-nm LED Irradiation
PBM at 660 nm regulates the biological actions of many types of cells, including diabetic wounded cells, fibroblasts, and human adipose-derived stem cells [24,[29][30][31]. In the astrocyte culture, DIV7-DIV14 is considered the developmental stage, and astrocytes before DIV14 still have neural stem cell (NSC) features [26]. Therefore, irradiation was conducted on DIV7 and DIV10 to determine whether the 660-nanometer LED affected the developmental stage of the astrocyte culture ( Figure 1B). Two exposures were the most appropriate conditions for a period between DIV7 and DIV14 in order to expose the maximum amount of light at regular intervals. To determine whether the 660-nanometer LED causes toxicity or an increase in overall cell proliferation, a cell viability assay was performed in an astrocyte culture, which includes various cell types other than astrocytes (n = nine wells). The cells were irradiated with a total density ranging from 3 to 18 J/cm 2 . The results showed that the 660-nanometer LED had no effects on the overall cell density at any power density ( Figure 3A). Once the cells were stimulated by PBM, cytochrome c oxidase (CCO) in the mitochondria respiratory chain was activated, leading to a mem- On DIV10, 20 µM DCFDA solution was treated immediately after irradiation and incubated for 45 min at 37 • C in the dark. ROS expression was significantly increased by 660-nanometer LED irradiation (Mann-Whitney U test; control vs. 660 nm; n = 48 vs. 57; ** p < 0. 01; p = 0.0083; U = 960; Figure 3B,C). To evaluate cell-specific proliferation, BrdU staining was performed after 660-nanometer LED irradiation, as BrdU labels proliferating cells. Cells were fixed after 2 h of BrdU treatment. The percentage of BrdU-positive astrocytes (BrdU + /GFAP + ) was significantly increased after 660-nanometer LED irradiation (Mann-Whitney U test; control vs. 660 nm; n = 58 vs. 55; **** p < 0.0001; U = 635; Figure 4A,B). To confirm this cell-specific proliferation, Ki67 staining was also performed. The percentage of Ki67-positive astrocytes (Ki67 + /GFAP + ) was significantly increased On DIV10, 20 μM DCFDA solution was treated immediately after irradiation and incubated for 45 min at 37 °C in the dark. ROS expression was significantly increased by 660-nanometer LED irradiation (Mann-Whitney U test; control vs. 660 nm; n = 48 vs. 57; ** p < 0. 01; p = 0.0083; U = 960; Figure 3B,C). To evaluate cell-specific proliferation, BrdU staining was performed after 660-nanometer LED irradiation, as BrdU labels proliferating cells. Cells were fixed after 2 h of BrdU treatment. The percentage of BrdU-positive astrocytes (BrdU + /GFAP + ) was significantly increased after 660-nanometer LED irradiation (Mann-Whitney U test; control vs. 660 nm; n = 58 vs. 55; **** p < 0.0001; U = 635; Figure  4A,B). To confirm this cell-specific proliferation, Ki67 staining was also performed. The percentage of Ki67-positive astrocytes (Ki67 + /GFAP + ) was significantly increased (Mann-Whitney U test; n = 45; *** p < 0.001; p = 0.0005; U = 597; Figure 4C,D). These results suggest that 660-nanometer LED irradiation specifically increases astrocyte proliferation.

Possibility of Astrocyte Differentiation by 660-nm LED Irradiation
The enhancement of cell proliferation by the 660-nanometer LED was confirmed. Not only cell proliferation but also cell differentiation, especially stem cell differentiation, can be regulated by PBM [35][36][37][38][39][40]. The protein expression pattern of cells differs according to the differentiation stage. In the case of astrocytes, GFAP, nestin, and Pax6, among other markers, are expressed in progenitor cells. In mature astrocytes, aldh1L1, aldolase, fruc-

Possibility of Astrocyte Differentiation by 660-nm LED Irradiation
The enhancement of cell proliferation by the 660-nanometer LED was confirmed. Not only cell proliferation but also cell differentiation, especially stem cell differentiation, can be regulated by PBM [35][36][37][38][39][40]. The protein expression pattern of cells differs according to the differentiation stage. In the case of astrocytes, GFAP, nestin, and Pax6, among other markers, are expressed in progenitor cells. In mature astrocytes, aldh1L1, aldolase, fructose-bisphosphate C, cluster of differentiation 44, and excitatory amino acid transporter 1 are expressed [1]. Cells were immunostained with pluripotent and NSC markers (Oct4 and nestin, respectively), developmental marker (GFAP), and maturity marker (aldh1L1) after twice being irradiated with the 660-nanometer LED, to determine whether the LED regulated the differentiation of astrocytes. The co-expression of Oct4 or nestin with GFAP or aldh1L1 (markers of immature or mature astrocytes; Table 2) were analyzed. The expression of cells co-expressing GFAP (developing astrocytes) and Oct4 (pluripotent stem cells) was significantly decreased by the 660-nanometer LED irradiation (Mann-Whitney U test; n = 35; * p < 0.05; p = 0.0240; U = 421; Figure 5A,B) ( Table 2). The number of cells co-expressing aldh1L1 (mature astrocytes) and Oct4 (pluripotent stem cells) was non-significantly decreased (p > 0.05; Figure 5C,D; Table 2). Another NSC marker with more widespread expression during differentiation, nestin, was used to confirm possibility of differentiation by 660-nanometer LED irradiation; only nestin-positive cells were significantly increased by irradiation (Mann-Whitney U test; (GFAP − /Nestin + ) and (aldh1L1 − /Nestin + ); n = 38 and 39; * p < 0.05, ** p < 0.01; p = 0.0057 and 0.0335; U = 462.5 and 640.5; Figure 6A-D). The expression of cells positive for both nestin and GFAP was decreased, but not significantly (p > 0.05; Table 2). In the case of mature astrocytes, aldh1L1-positive cell expression tended to increase, but not significantly (p > 0.05; Table 2). Overall, these results show the decreased co-expression of Oct4 and increased expression of nestin, suggesting the possibility of differentiation. Western blot analysis was also performed to confirm the final differentiation possibility. The results showed that 660-nanometer LED irradiation did not change Oct4 expression, while the expression of aldh1L1 and nestin was significantly increased (two-tailed unpaired t-test and Mann-Whitney U test; n = 4; ** p < 0.01 and * p < 0.05; p = 0.0087 and 0.0286; t = 3.822; df = 6 and U = 0, respectively; Supplementary Figures S3 and S4). More cells differentiated into mature astrocytes after 660-nanometer LED irradiation compared to the control group ( Figures 5 and 6), suggesting that 660-nanometer LED irradiation can provide evidence for cell differentiation. In addition, an abundance of astrocyte processes was seen postirradiation (Figures 5 and 6). Astrocyte processes wrap around individual synapses, and an increased number of processes better supports neuronal cell structures via synaptogenesis and neurotransmitter recycling [41][42][43][44]. However, there is possibility that these results are induced by the proliferation of specific cell types not by differentiation.  The number of cells co-expressing aldh1L1 (mature astrocytes) and Oct4 (pluripotent stem cells) was non-significantly decreased (p > 0.05; Figure 5C,D; Table2). Another NSC marker with more widespread expression during differentiation, nestin, was used to confirm possibility of differentiation by 660-nanometer LED irradiation; only nestin-positive cells were significantly increased by irradiation (Mann-Whitney U test; (GFAP − /Nestin + ) and (aldh1L1 − /Nestin + ); n = 38 and 39; * p < 0.05, ** p < 0.01; p = 0.0057 and 0.0335; U = 462.5

Alteration of Neurotrophic Factors in Astrocyte Culture by 660-nm LED Irradiation
LED irradiation can increase the release of growth factors, which in turn promotes cell proliferation and differentiation [45]. Several neurotrophic factors are expressed in the brain, including NGF, BDNF, and neurotrophin-3/-4. Neurotrophic factors promote cell survival and differentiation [46]. Therefore, we hypothesized that the regulation of cell proliferation by 660-nanometer LED irradiation might occur through the release of neurotrophic factors. To identify key neurotrophic factors for cell proliferation and differentiation, cells were fixed for 2 h after irradiation, where 2 h is the optimal reaction time of the neurotrophic factors tested.
Immunostaining of representative neurotrophic factor markers, NGF and BDNF, was then performed. To confirm the increase in neurotrophic factors, the marker intensity was analyzed. The results showed that the intensity of NGF was significantly increased by 660-nanometer LED irradiation (Mann-Whitney U test; n = 30; ** p < 0.01; p = 0.0053; U = 263.5; Figure 7A,C), while the intensity of BDNF was non-significantly decreased (Mann-Whitney U test; n = 30; p > 0.05; p = 0.7412; U = 427; Figure 7B,D). To confirm the immunostaining results, Western blot analysis was performed. No difference in the protein expression of the precursor pro-BDNF and mature BDNF was observed between the control cells and the 660-nanometer LED-irradiated cells. However, the protein level of NGF was significantly increased by LED irradiation (Mann-Whitney U test; n = 4; * p < 0.05; p = 0.0286; U = 0; Figure 7E, Supplementary Figure S5). These results indicate that the LED-mediated increase in cell proliferation was regulated by the increased NGF level, suggesting that upregulation of the biological activity of astrocytes after 660-nanometer LED irradiation might occur through NGF.
performed to confirm the final differentiation possibility. The results showed that 660-nanometer LED irradiation did not change Oct4 expression, while the expression of aldh1L1 and nestin was significantly increased (two-tailed unpaired t-test and Mann-Whitney U test; n = 4; ** p < 0.01 and * p < 0.05; p = 0.0087 and 0.0286; t = 3.822; df = 6 and U = 0, respectively; Supplementary Figures S3 and S4). More cells differentiated into mature astrocytes after 660-nanometer LED irradiation compared to the control group (Figures 5 and 6), suggesting that 660-nanometer LED irradiation can provide evidence for cell differentiation. In addition, an abundance of astrocyte processes was seen post-irradiation ( Figures 5 and 6). Astrocyte processes wrap around individual synapses, and an increased number of processes better supports neuronal cell structures via synaptogenesis and neurotransmitter recycling [41][42][43][44]. However, there is possibility that these results are induced by the proliferation of specific cell types not by differentiation.  the 660-nanometer LED-irradiated cells. However, the protein level of NGF was significantly increased by LED irradiation (Mann-Whitney U test; n = 4; * p < 0.05; p = 0.0286; U = 0; Figure 7E, Supplementary Figure S5). These results indicate that the LED-mediated increase in cell proliferation was regulated by the increased NGF level, suggesting that upregulation of the biological activity of astrocytes after 660-nanometer LED irradiation might occur through NGF.

Discussion
This study showed that ROS expression was increased by 660-nanometer LED irradiation. Immunostaining showed that cell proliferation and the possibility of differentiation were also increased by irradiation, which was confirmed using Western blot analyses. To determine the underlying mechanisms, we focused on the representative neurotrophic factors (BDNF and NGF) that can be increased by LED irradiation [45,47]. To this end, Western blotting and immunostaining were performed, and the results showed that the NGF protein expression was significantly increased by 660-nanometer LED irradiation; this suggests that the irradiation affected cell proliferation and differentiation possibility by upregulating the neurotrophic factor, NGF. Alternatively, PBM promotes DNA and RNA synthesis, and also increases protein production. Moreover, it plays a role in regulating the enzymatic activity and the pH inside and outside cells, and promotes cellular metabolism [48,49].
These effects of PBM might be related to the proliferation and differentiation possibility of astrocytes by PBM.
This study confirmed that PBM increased astrocyte proliferation, while the viability of all cells did not differ between the control and 660-nanometer groups. Contrary to some studies reporting that PBM increases cell viability, we found no difference in cell viability. This might have been because our culture system was dominated by astrocytes; therefore, the other cells might not have been healthy, and most neuron cell-related research has focused on pathological situations and mainly used 800-nanometer LEDs [50,51].
Cell proliferation is driven by cytochrome c oxidase (CCO), the first photoreceptor of red and near-infrared light. However, a previous study showed that near-infrared, not red, light predominantly affected mitochondrial cytochrome oxidase [52]. Another recent study suggested that 660-nanometer PBM does not require CCO to enhance cell proliferation [53]. In our study, a 660-nanometer LED significantly increased both astrocytespecific proliferation and the ROS level. Therefore, we speculate that cell proliferation was increased via ROS, suggesting that ROS production is involved in the mechanism related to the effects of a red LED on cell proliferation. More research is needed to answer these questions.
In addition to cell proliferation and migration, the expression of various genes associated with cytokines and growth factors is induced by PBM [54,55]. We hypothesized that the growth factors involved in cell survival and differentiation mediate the proliferation and differentiation induced by PBM [46,56]. In this study, the expression of growth factors was altered by PBM; in particular, NGF protein expression was significantly increased by 660-nanometer LED irradiation, consistent with a previous study showing that PBM increases cell proliferation and NGF gene expression in human Schwann cells [57]. In addition, a previous study showing that NGF increases the number of cultured astrocytes [58] also posited that PBM-induced increases in NGF expression affect astrocyte proliferation. However, since the receptors and roles of NGF are diverse, more studies are needed regarding the specific mechanism underlying PBM-induced astrocyte expression.
Furthermore, we examined changes in astrocytes induced by PBM. The astrocyte culture contained HB-EGF to induce astrocytes in vivo and aid cell growth. Previous studies have suggested that HB-EGF can cause de-differentiation in the cell culture [25,26]. Therefore, it was unclear whether the evidence of cell differentiation in this study was related to HB-EGF. Further studies need to develop an improved culture technique without HB-EGF.
In conclusion, this study demonstrated that 660-nanometer LED irradiation affects the proliferation of cultured astrocytes and provides evidence of differentiation. The results suggested that astrocyte characteristics can be affected by 660-nanometer LED irradiation. Based on our findings, PBM can be expected to be a useful treatment for brain disorders and brain cell damage due to its interaction with astrocytes. In addition, this study provides further insight into the effects of PBM on astrocytes.