The geniculate ganglion is a collection of sensory neurons of the facial nerve, wherein half the neurons innervate the tastebuds and the other half innervate the outer ear [1]. The sensory innervation provided by the geniculate ganglion appears necessary for forming taste bud cells, not only during embryogenesis but also through adult life [2]. Geniculate ganglion neuronal cells (GGNs) that innervate the tastebuds receive inputs from tongue taste receptor cells that selectively transduce one of five taste qualities: sweet, sour, salty, bitter, and umami [3]. Damage to GGNs could significantly affect taste perception and quality of life. The sense of taste dramatically impacts the quality of life by preparing our body to ingest food, encouraging eating through pleasure and satisfaction, and helping us detect toxins in our foods to keep us safe. It links our external environment to our internal needs; hunger and thirst [4].

Photobiomodulation therapy (PBMT) is a non-invasive treatment that uses low-level light therapy to stimulate cellular function and promote tissue healing [5]. The therapeutic effect of PBMT for treating or managing diseases and injuries has gained significant popularity among researchers and clinicians, especially for managing oral complications of cancer therapy, such as oral mucositis [6,7] or taste alterations [8,9]. In literature, red or near-infrared (NIR) light with 600 to 1,000 nm wavelength has shown beneficial effects to neuronal growth [10,11] and support nerve regeneration [12,13]. One of the effects of PBMT using NIR is related to the chromophore cytochrome C oxidase (COX) containing both heme and copper centers and absorbs light into the NIR region, which affects the creation of adenosine triphosphate (ATP). Based on studies, the photons dissociate inhibitory nitric oxide (NO) from the enzyme, increasing electron transport, mitochondrial membrane potential, and ATP production [14]. Although PBMT mechanisms of action have been identified, validation and comprehensive analysis of its effects and biosafety are lacking.

Herein, we investigated the effects of 850 nm PBMT on the primary culture of GGNs with a particular role in taste function. The goal is to determine the potential protective or therapeutic effect of PBMT on sensory neurons such as GGNs regarding cell viability, proliferation, and ATP production.

Cell isolation, culture, and characterization

GGNs were isolated from neonatal rats (P1; Orientbio Corp.), seeded in plates coated with 0.1 mg/mL Poly-D-Lysine (PDL; Sigma-Aldrich), and cultured in Neurobasal Medium (Gibco^TM) supplemented with 1% Ampicillin (Sigma-Aldrich) to prevent bacterial contamination, 2% B27-A0 (Gibco^TM) to support neuronal survival and differentiation, and 0.5 mM Glutamax (Gibco^TM) to provide an additional source of energy and nutrients. The GGNs were cultured at 37°C in a humidified incubator with 5% CO₂, and the culture media was changed every three days. The cells were allowed to grow for three days before they were used for the subsequent experiments.

Immunofluorescence staining characterized isolated GGNs with neuronal and geniculate ganglion cell-specific markers. In brief, GGNs were fixed with 4% paraformaldehyde (PFA) for 15 minutes and permeabilized with 0.1% phosphate-buffered saline (PBS)-Triton X-100 for 5 minutes 3 times. Blocking was performed with 3% bovine serum albumin (BSA) and incubated in primary antibodies: microtubule-associated protein 2 (MAP2; Abcam), beta III tubulin (1:100; Abcam), Nestin (1:100; Novus Biologicals), glial fibrillary acidic protein (GFAP; Abcam), myelin basic protein (MBP; BioLegend) and Phox2b (1:100; Abcam) overnight at 4°C. After washing with 1X PBS, the corresponding secondary antibody (Alexa Fluor 488; Invitrogen) was applied. VECTASHIELD with DAPI (H-1200; Vector Laboratories) was used to mount and stain the cell nucleus. Stained samples were observed, and images were acquired by confocal microscopy (FV-3000; Olympus).

PBMT

GGNs were seeded in 24-well culture plates coated with PDL and exposed to PBMT using an 850 nm light-emitting diode (LED) panel with a power setting of 9.70 mW. Irradiation was performed in an incubator set at 37°C and 5% CO₂ (Fig. 1). Treatment groups were divided into four: control (no treatment), PBMT of 1 J (103 seconds), 5 J (515 seconds), and 10 J (1,031 seconds). The PBMT was performed once on the third day after cell seeding.

Figure 1. Photobiomodulation (PBM) treatment scheme. PBM therapy performed using 850 nm light-emitting diode device panel with 9.70 mW power output at varying durations equivalent to 1, 5, and 10 J energy treatments.

Cell viability assay

MTT assay was performed to assess the effects of PBMT on cell viability. The MTT assay measures the activity of mitochondrial enzymes, which reflects the metabolic activity of cells. MTT assay, the cells were incubated with 0.5 mg/ml MTT (Thiazolyl Blue Tetrazolium Bromide; Sigma-Aldrich) solution for 4 hours at 37°C. The formazan crystals were solubilized with dimethyl sulfoxide (DMSO; BioShop), and the absorbance was measured at 570 nm using a microplate reader (Infinite 200 Pro; Tecan).

Adenosine diphosphate/adenosine triphosphate assay

The adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratio assay measures the relative levels of ATP and ADP in cells, reflecting the energy status of cells. Non-treated and treated cells were treated with an ATP monitoring enzyme. The ADP/ATP ratio was calculated following the ADP/ATP Ratio Assay Kit (BioVision, Abcam) manufacturer’s instructions based on the bioluminescent detection of ADP and ATP levels. The assay rapidly screens apoptosis, necrosis, growth arrest, and cell proliferation simultaneously in mammalian cells.

Statistical analysis

The data were analyzed using a one-way ANOVA followed by Tukey’s post hoc test. The results were expressed as mean±standard deviation with p < 0.05 values considered statistically significant.

In this study, we successfully isolated and cultured GGNs in vitro. The cultured GGNs exhibited characteristics of mature neuronal cells, as indicated by the expression of neuronal markers MAP2 and Phox2b, a marker of neuronal cells related to taste function (Fig. 2) [15]. Moreover, the absence of astrocyte marker GFAP and oligodendrocyte marker MBP in the cultured cells suggested the purity of the neuronal population (Fig. 3). Additionally, the low expression of neural progenitor cell marker Nestin indicated that most cells maintained in culture were mature neurons. These findings demonstrate the successful isolation and culture of GGNs, providing a valuable foundation for further investigations into the therapeutic potential of these neurons in various neurologic and taste-related conditions.

Figure 2. Isolated and cultured geniculate ganglion neuronal cells express MAP2 neuronal marker and Phox2b marker associated with taste function.

Figure 3. Geniculate ganglion neuronal cells maintained in culture for three days consist of mature neurons expressing beta III tubulin, minimal neural progenitor cells (Nestin), with no detection of astrocyte (GFAP) and oligodendrite marker (MBP) positive cells.

The MTT assay results demonstrated that the cell viability in the PBM-treated cells was not significantly different from the control group on day 1 (Fig. 4). Our results showed that PBMT at a dose of 10 J significantly increased cell viability compared to other treatment groups on day 3 and day 7, as demonstrated by the MTT assay (p < 0.05). However, PBMT at doses of 1 and 5 J did not show a significant increase in cell viability compared to the control group. These findings suggest a certain energy level is required to enhance cell viability with PBMT. Importantly, no detrimental or cytotoxic effects were observed for any of the PBMT treatments, indicating the safety of low-dose PBM for GGNs. However, further studies are needed to determine the optimal parameters for PBMT in the GGNs and to elucidate the underlying mechanisms of PBMT.

Figure 4. Cell viability via MTT assay after photobiomodulation treatment using 850 nm light-emitting diode device panel with 9.70 mW power output at varying durations equivalent to 1, 5, and 10 J energy treatments. **p < 0.01 and ****p < 0.0001.

The ADP/ATP assay results showed a significant difference in the ADP/ATP ratio between the PBM-treated cells and the control group on the third day after PBM treatment (Fig. 5). PBMT at doses of 1 and 5 J showed a significant increase in the ADP/ATP ratio compared with the control group, while the 10 J group exhibited a marked difference relative to other groups. Similar to the MTT assay result, these findings indicate that the effects of PBMT become evident three days after treatment, suggesting enhanced metabolic properties and improved cell proliferation. Therefore, PBMT in these types of cells does not present immediately but enhances the cells’ metabolic properties as proliferation improves in the latter days.

Figure 5. Adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratio apoptosis analysis after photobiomodulation treatment using 850 nm light-emitting diode device panel with 9.70 mW power output at varying durations equivalent to 1, 5, and 10 J energy treatments. *p < 0.5, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

The study aimed to investigate the effects of PBMT on the viability of primary GGNs cultures. PBM is relatively safe at low doses for GGNs, and it has the potential to be a non-invasive approach to improving cell viability. However, further investigations are required to determine the optimal parameters for PBMT in GGNs and elucidate its effects’ underlying mechanisms. Based on existing literature, PBM induces an increase in available electrons for molecular oxygen reduction in the catalytic center of Cox, thereby increasing mitochondrial membrane potential and levels of ATP [16]. Consequently, ATP synthesis changes reactive oxygen species (ROS), calcium ions (Ca²⁺), and NO concentrations. These changes in ATP levels may be correlated with the observed effects on cell viability and proliferation, as PBMT can alter the concentrations of ROS, Ca²⁺, and NO. Thus, the effects of PBMT on ATP levels were analyzed and correlated with the effects on viability or proliferation of GGNs via MTT assay.

PBMT was found to prevent GGN apoptosis and positively affect cell proliferation. However, it is essential to note that the effects of the 10 J treatment decreased significantly at day 7 compared to the 5 J treatment. This result may be attributed to the phenomenon known as hormesis, wherein a biphasic dose-response curve is observed. In this case, too low or too high doses, irradiance, delivery time, or the number of repetitions can lead to no significant effect or unwanted inhibitory effects [17,18]. The biphasic response follows the “Arndt-Schulz Law,” wherein weak stimuli slightly accelerate activity, and more potent stimuli increase it further until a peak is reached. However, more potent stimuli inhibit the activity until a negative response is achieved. This response has been demonstrated several times in low-level light works [14,19-21]. Further studies are needed to explore the underlying mechanisms of hormesis in PBMT and its implications for low-level light therapy.

The study provides valuable insight into the potential therapeutic applications of PBMT at an appropriate energy level for taste or neurological diseases involving geniculate ganglion tissue. However, certain limitations should be acknowledged. First, in vitro, cultured cells may not fully represent the complex physiological environment of GGNs in vivo. Second, the study focused solely on cell viability and ATP generation mechanisms, warranting further research to unravel the underlying mechanisms of PBMT. Moreover, future studies need to address the long-term effects of PBMT, and determining optimal parameters, such as wavelength, power density, and treatment duration, necessitates future investigations. These factors can significantly influence the therapeutic efficacy of PBM in clinical applications.

In conclusion, our findings suggest that PBM may be a safe and non-invasive approach for treating the geniculate ganglion, potentially for addressing taste dysfunction. Our results showed that the PBM treatment of 10 J significantly improved the cell viability in the GGNs and prevented its apoptosis. However, further studies are needed to optimize the parameters and investigate the underlying mechanisms and potential side effects of PBM therapy in the GGNs. Finally, we did not investigate the potential side effects of PBM therapy, such as oxidative stress and DNA damage, which may occur at high doses or prolonged exposure to PBMT.

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Conceptualization: PSC. Data curation: HYL. Formal analysis: SYP. Funding acquisition: PSC. Investigation: SHW. Methodology: SHW. Project administration: SHW. Software: AP. Validation: HSR. Visualization: CA. Writing–original draft: HK. Writing–review & editing: all authors.

Seung Hoon Woo is the Editor-in-Chief of the journal and Celine Abueva, Andrew Padalhin, and Phil-Sang Chung are editorial board members of the journal, but they were not involved in the review process of this manuscript. Otherwise, there is no conflict of interest to declare.

This work was supported by the Dankook Institute of Medicine and Optics in 2023. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (RS-2023-00247651 and NRF-2020R1A6A1A03043283), Leading Foreign Research Institute Recruitment Program through NRF funded by the Ministry of Science and ICT (NRF-2023K1A4A3A02057280), Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health & Welfare (HI20C2088), Korea Medical Device Development Fund grant funded by the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety (KMDF_PR_20200901_0027-03), Republic of Korea. The present research was supported by the research fund of Dankook University Research and Business Development Foundation in 2023.

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