A Grant Report: Examining the Efficacy of Remote Photobiomodulation Therapy in Adolescents with Major Depressive Disorder

Abstract

Major depressive disorder (MDD) is a prevalent mental health condition affecting a significant portion of the population worldwide. This condition can impact individuals of all ages, including adolescents, leading to an impact on various aspects of their lives. Adolescence is a crucial phase of human development, characterized by several neurobiological changes. The onset of MDD during this period can result in damage not only to teenagers but also might have long-lasting implications for their future as adults. Notably, the onset of MDD in adolescents is often associated with various biomarkers, such as increased levels of inflammatory cytokines (e.g., IL-6, TNF-α), oxidative stress markers, and alterations in neurotransmitter levels, indicating a complex interplay of biological factors. Therefore, early intervention is essential for addressing MDD during this phase. Photobiomodulation therapy (PBMT) emerges as an innovative and promising approach that utilizes light, especially in the near-infrared (NIR) and red spectra, to trigger biological and therapeutic effects. Notably, targeting the skull and abdomen with PBMT might explore the bidirectional communication between the intestinal system and the central nervous system in a remote and/or systemic way. In this context, we present the rationale and design of an ongoing study aiming to assess the efficacy of PBMT on depressive symptoms and biomarkers associated with oxidative stress and mitochondrial function in adolescents with MDD.

1. Introduction

Mental and behavioral disorders are a range of neuropsychiatric disorders characterized by psychological or behavioral changes, often accompanied by an impairment in a person’s functional activities [1]. These mental health conditions act as barriers that hinder an individual’s engagement with their environment, depriving them of their freedom and the opportunity to interact with others [2].

On a global scale, mental disorders have been gaining significant attention due to their increasing prevalence in both low- and middle-income countries and high-income countries, there being approximately 700 million people worldwide who suffer from mental and neurological disorders [3]. Furthermore, one in every four individuals will develop one of these disorders during their lifetime, making it rare for families not to have a member with a mental disorder [4].

Among mental disorders, major depressive disorder (MDD) is the most prevalent, causing the highest level of disability worldwide, accounting for 40.5% of all disability attributed to mental disorders. During adolescence, the rates of MDD significantly increase between the ages of 13 and 18, primarily due to emotional factors. The underlying incidence in this population has increased significantly in recent years. However, despite its early onset and chronic course, only 1% of young individuals in the United States receive outpatient treatment for MDD each year. These findings highlight adolescence as a critical period of development to identify individuals at high risk of MDD and prevent the onset of this disorder [5].

1.1. MDD in Adolescence

The number of people with mental disorders in the world has been increasing [6]. These conditions are considered ailments of the 21st century, causing various damages not only to the individuals affected but also to healthcare systems [7,8]. Furthermore, it is worth noting that depressive disorders entail high expenditures in public health. In 2021, the World Health Organization (WHO) emphasized, through the new mental health atlas, a global deficit in mental health investment [9].

MDD is a common mental illness, especially in mid and late adolescence, with a prevalence of 4–5%, which, due to its particularities, poses a significant challenge and requires an effective diagnosis. Primary care providers are often the first point of contact for adolescents, playing a crucial role in the diagnosis and management of this condition. Additionally, several entities also recommend screening for MDD during this period [10,11,12].

Adolescence is a very important phase of human development characterized by significant somatic growth and secondary sexual transformations. The WHO defines adolescence as the period between 10 and 19 years of age, spanning almost a decade, beginning with puberty and the transition from childhood. It can be the most intense period within the entire human developmental cycle in terms of self-esteem, autonomy, and intimacy due to the process of maturation and personality [13].

Adolescence is also characterized by dynamic brain development associated with an increasing amount of information and motor, cognitive, and social transformations. Some areas of the brain continue to develop and mature during this period, as explained by the neuroimaging studies from Blakemore [14], which confirmed that the human brain continues to develop throughout adolescence and beyond. More specifically, during and after puberty, modifications occur in specific brain regions such as the prefrontal cortex (PFC), parietal cortex, and superior temporal cortex. These regions are directly involved in the ability to understand other people, playing fundamental roles in cognitive and emotional processes related to social understanding and influencing how young individuals process information, make decisions and handle emotions.

During adolescence, individuals undergo physical development and gain the cognitive, emotional, social, and economic resources that form the foundation for their future health and well-being [15]. This period is crucial for developing knowledge and skills, learning to manage emotions and relationships, and acquiring the attributes and competencies necessary for adulthood [16,17]. However, adolescence is also a period of physical, psychological, and social vulnerability, with complex changes in the human development process. This might result in behaviors and emotions not previously experienced by the individual, their family, friends, and professionals who interact with them. Therefore, due to being a vulnerable period, the experience of adolescence requires efforts and special attention from family members, healthcare professionals, and educators to help the future adult cope with situations and problems that may lead to health-related harm and issues [18].

Most diagnosed mental health issues in adulthood begin during adolescence, with half of diagnosable lifetime mental disorders starting by age 14 due to psychological, emotional, and physiological changes. This percentage increases to 75% by age 24. Mental health problems during this period are more common and often include temporary reactions to life stressors. While these issues are generally less severe and of shorter duration than mental disorders, they can potentially develop into more serious conditions [19].

Approximately 25% of adolescents meet the criteria for MDD [20], which is associated with long-term adverse consequences, including social and economic difficulties, poorer physical health, and an increased risk of recurrent depressive episodes throughout adulthood [21,22]. In particular, early-onset MDD is associated with an increased risk of substance abuse, bipolar disorder, high-risk sexual behaviors, and suicide [23]. The Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5), defines MDD as a depressed mood or loss of interest or pleasure in activities for a minimum period of two weeks. It also considers that adolescents may exhibit irritability instead of sadness [24].

MDD in adolescents has become a subject of great scientific relevance as its prevalence continues to rise. The clinical characteristics of MDD in adolescents are similar to those presented in adults, ranging from no more than a mild symptom of fatigue and sadness to the deepest state of apathy with a complete psychotic disregard for reality [25].

Changes in synaptic transmissions, related to acute stress and altered concentrations of neurotransmitters in the major excitatory and inhibitory systems, may also be associated with MDD. Acute stress has been linked to increased extracellular glutamate in the medial PFC (mPFC) and hippocampus, as well as neuronal atrophy in these brain regions. These findings support the hypothesis of glutamate-mediated excitotoxicity through actions on NMDA receptors [26].

Chronic stress has been associated with decreased glutamate-to-glutamine cycling and metabolism, as well as reduced levels of the neurotransmitter gamma-aminobutyric acid (GABA), primarily due to diminished glial metabolism. Furthermore, chronic stress leads to oxidative stress and mitochondrial dysfunction, which are critical in the pathophysiology of depression. Biomarkers of oxidative stress, such as increased levels of reactive oxygen species (ROS) and reduced antioxidant defenses, along with mitochondrial dysfunction markers, have been found in individuals with depression. These biomarkers are systemic, highlighting the pervasive impact of oxidative stress and mitochondrial dysfunction throughout the body. This evidence suggests that chronic stress not only reduces the structure and function of glutamatergic neurons but also induces systemic oxidative stress and mitochondrial damage. This is consistent with the hypothesis that the atrophy of these neurons, combined with oxidative and mitochondrial stress, contributes to the reduction in the volume of cortical and limbic structures found in individuals with depression [27].

Preclinical and clinical studies have also demonstrated the involvement of energy metabolism, lipid metabolism, and amino acids, as well as the intestinal microbiota, in the pathophysiology of MDD. Using a method of urinary metabolomics [28], metabolites were identified that distinguish individuals with MDD from healthy controls, with five metabolites identified as potential biomarkers, including malonate, formate, N-methylnicotinamide, m-hydroxyphenylacetate, and alanine. These metabolites are primarily involved in energy metabolism, intestinal microbial, and nicotinic acid-tryptophan pathways [28,29].

The damages caused by MDD are not only directed at individuals with the disease but also affect their families and society, which makes it important to create new treatment alternatives [30]. Among the treatment alternatives for this mental health disorder, photobiomodulation therapy (PBMT) emerges as a promising non-invasive approach.

1.2. Photobiomodulation Therapy (PBMT)

PBMT, previously known as low-level laser therapy (LLLT), has been used for many years in the treatment of various neuropsychiatric disorders. The technique is simple, cost-effective, and can be integrated into conventional treatments or used alone in some diseases [31]. PBMT can be defined as the use of non-ionizing electromagnetic energy (low-intensity photonic emissions) at a specific wavelength (commonly 600 to 1000 nm) capable of triggering photochemical changes in photon-sensitive cellular structures, leading to the modulation of cellular and biological processes [32].

Light, coming from any source, such as a laser (light amplification by stimulated emission of radiation) or an LED (light-emitting diode) device, is a form of electromagnetic energy that exists as particles called photons that move in waves at a constant speed. Photon waves move at the speed of light and are defined by two properties: amplitude and wavelength (λ). Amplitude is related to intensity and, in the case of light, to brightness [33,34,35]. High amplitudes correspond to high energy levels and are expressed in joules (J). Meanwhile, the wavelength is an important parameter to assess how light is emitted and how it will interact with the tissue. In the case of lasers used in photomedicine, the wavelength is measured in nanometers (nm) or micrometers (µm) [36]. Wavelength and energy are properties inversely proportional, which means that the smaller the wavelength, the greater the frequency of the waves and the energy of the photons [37].

Transcranial PBM (t-PBM) uses extracranial light, particularly in the near-infrared (NIR) and red spectra, for biological and therapeutic effects [38]. Studies conducted using t-PBM have demonstrated efficacy in the treatment of various neuropsychiatric disorders, including depression [38,39,40]. On the other hand, there are systemic applications of light, such as to the abdominal region. This type of PBMT is also suggested to be beneficial for the treatment of depression. This exposure to light can positively influence subcutaneous adipose tissue and blood vessels by modulating the autonomic nervous system [41]. Additionally by regulating the production of neurotransmitters such as serotonin and impacting sleep patterns, this modality plays a crucial role in maintaining emotional balance. There are many other effects of light interacting with the tissue of the Central Nervous System (CNS) during PBMT. It is essential to reference and describe these effects in more detail, including the influence on synaptic plasticity, the reduction in neural inflammation, and the promotion of neurogenesis, to provide a more accurate and comprehensive understanding of the benefits of PBMT [42,43,44,45,46].

1.3. Remote Photobiomodulation Therapy Application in MDD

The CNS and the gastrointestinal tract communicate through a bidirectional signaling network. In this sense, microbial peptides affect sensory neurons via vagal and spinal pathways, influencing neural circuits in the brain. Additionally, humoral factors can modulate brain function, while gut dysbiosis disrupts normal communication, leading to diseases. The gut hosts trillions of microorganisms, collectively known as the intestinal microbiota, which play a fundamental role in the overall health of the body. This microbiota has a significant influence on regulating the immune system, synthesizing vitamins, and producing neurotransmitters [30]. Intestinal microbes can shape neural development, modulate neurotransmission, and affect behavior, thereby contributing to the pathogenesis and/or progression of many neurodevelopmental, neuropsychiatric, and neurological conditions [47].

In recent years, it has become evident that the intestinal microbiota plays a crucial role in influencing brain functions and behavior [48,49]. Compromises in the gastrointestinal tract not only impair the protective intestinal barrier but also elevate the production of inflammatory mediators, potentially triggering psychiatric disorders like anxiety and depression [50]. Accumulating evidence over the past decade highlights the significant role of this communication in the pathophysiology of MDD [51,52]. The gastrointestinal tract is a key site for the production of important neurotransmitters, including serotonin, which plays a vital role in regulating various physiological functions and behaviors [53].

GABA and dopamine are also produced in the intestine, mostly by enteroendocrine and enterochromaffin cells, while others are influenced by the intestinal flora [54]. Recent research suggests that interventions targeting this communication may have positive effects on mental health, including an improvement in symptoms of depression and anxiety [51,52]. In this context, PBMT emerges as a promising intervention strategy, which can be applied both to the scalp (transcranial) and over the abdominal region. Although this study was not designed to evaluate the gut–brain axis specifically, the rationale behind our approach was motivated by this bidirectional communication’s potential impact on MDD. In Figure 1, a schematic figure is presented, showing the rationale that motivated our choice in applying PBMT to MDD and its implications for the remote application of note. As has and will be mentioned, this study was not designed to evaluate the gut–brain axis due to some limitations.

1.4. Innovation

Disorders of the nervous system, particularly MDD, affect many people worldwide, and the prevalence of mental health disorders has been increasing due to the 2019 coronavirus pandemic [55,56,57,58]. Pharmacotherapy remains the cornerstone of treatment for these disorders [59]. However, despite substantial costs, treatment outcomes within pharmacological interventions often fall short of expectations: nearly half of patients do not respond adequately, 40% of responders experience relapses, and many deteriorate despite ongoing treatment [59]. Recognizing the need for novel therapeutic approaches to address the immense burden of these disorders, our primary contribution lies in providing evidence regarding the efficacy of PBMT in alleviating depressive symptoms and modulating biomarkers associated with oxidative stress and mitochondrial function in adolescents with MDD. LED offers better cost-effectiveness in its use, as it allows the combination of wavelengths and a matrix of various sizes, meaning it can irradiate larger areas than a laser with lower energy expenditure [60,61]. These advancements enable numerous applications of LED today, such as in flat devices like armbands, blankets, and caps. Previous studies have explored abdominal applications of photobiomodulation in the context of Alzheimer’s disease and Parkinson’s disease, demonstrating its potential benefits in preclinical and clinical settings [62,63]. Our study represents one of the pioneering investigations into the potential of PBMT for this specific population.

2. Aims

The primary aim of this study is to assess the impact of PBMT on depressive symptoms and biomarkers associated with oxidative stress and mitochondrial function in adolescents with MDD. Additionally, we aim to analyze various secondary outcomes including levels of MDD, anxiety, stress and resilience, quality of life, sleep patterns, as well as catalase and superoxide dismutase enzyme activity levels, serum concentrations of thiobarbituric acid, malondialdehyde, and carbonylated proteins, and activities of mitochondrial complexes I, II, and IV.

Specific Aims and Hypotheses

Aim 1: To investigate the impact of PBMT on depressive symptoms, as measured by the Hamilton Depression Scale, and associated biomarkers, including catalase enzyme (CAT) and superoxide dismutase enzyme (SOD) activity levels, serum concentrations of thiobarbituric acid malondialdehyde carbonylated proteins, and activities of mitochondrial complexes I, II, and IV, in adolescents with MDD. Hypothesis 1: PBMT will lead to a decrease in depressive symptoms among adolescents, as evidenced by changes in depression scores and biomarkers of oxidative stress and mitochondrial function before and after intervention through pre- and post-intervention results in the depression level questionnaire and biomarkers of oxidative stress and mitochondrial function.

Aim 2: To assess the effect of PBMT on anxiety symptoms, as measured by the Hamilton Anxiety Scale, in adolescents with MDD. Hypothesis 2: PBMT will lead to a decrease in anxiety symptoms among adolescents, as indicated by pre- and post-intervention assessments of anxiety levels.

Aim 3: To examine the impact of PBMT on stress and resilience, utilizing the Hamilton Anxiety Scale and Resilience Scale adapted for Brazil by Pesce et al. [61], in adolescents with MDD. Hypothesis 3: PBMT will lead to an improvement in adolescents’ ability to be resilient to stressors, as reflected in changes observed in the Perceived Stress Scale (PSS) validated by Nielsen et al. [64], before and after intervention.

3. Study Design

This is a double-blind, sham-controlled, parallel study to test the biological effects of PBMT in adolescents with MDD. We aim to enroll a total of 72 individuals aged 15 to 17 diagnosed with MDD based on the DSM-V criteria. Medications, augmentative devices, and other interventions are recorded upon entry and during the study. Later, participants are divided into three groups: Experimental Group 1 (EG1), Experimental Group 2 (EG2), and Control Group (CG), each one composed of 24 participants.

Sample Size Calculation: The sample size calculation was estimated to employ multivariate analysis of variance (MANOVA) for repeated measures with interactions over time and between groups. For this purpose, G*Power software (version 3.1.9.2) was used for the calculation. The study design was established with 3 groups and 2 measurements over time (3 × 2). The Type I error was set at 0.05 (alpha), while the Type II error was set at 0.85 (power). An effect size of 0.4 was determined as the magnitude of the effect to be observed. As a result, the inclusion of 72 participants in the sample appears robust enough to ensure sufficient power in the results, with 24 participants in the Control Group (CG), 24 in Experimental Group 1 (EG1), and 24 in Experimental Group 2 (EG2).

Inclusion criteria: The inclusion criteria involve the following: 1. Individuals of both sexes aged between 15 and 17 years old; 2. Diagnosis of MDD made by a psychiatrist according to DSM-V criteria; 3. Be a student of the 8th Regional Education Management (GERED) of Campos Novos, Santa Catarina (SC), Brazil.

Exclusion criteria: The exclusion criteria consist of five conditions: 1. Diagnosis of epilepsy; 2. Having any cognitive impairment; 3. Changes in medications, augmentative devices, and other interventions that may interfere with the study conduct; 4. Participation in other clinical research trials that may affect primary outcomes or adherence to the proposed study; 5. History of migraine with aura in the last six months. Additionally, all measures will be taken to avoid exposure to the thyroid region in the neck, areas with open wounds, or areas that may result in direct irradiation to the eye.

3.1. Source of Subjects

Patient data will be obtained from the records of Dr. José Athanázio General Hospital and the Psychosocial Care Center (CAPS) in Campos Novos, SC, Brazil. Recruitment efforts will focus on referrals from healthcare professionals and educational institutions, as well as the distribution of flyers, radio broadcasts, and the use of social media platforms. Additionally, outreach will be directed toward local schools in Campos Novos, SC, through targeted announcements on radio and social media channels. During the recruitment process, it will be emphasized that study participation is limited to individuals with a confirmed diagnosis, either in writing or in a digitally signed document, by a psychiatrist or neurologist.

3.2. Subject Enrollment

This research was approved by the Research Ethics Committee of the University of Southern Santa Catarina under number 6.210.964. The study protocol was registered and approved in the Brazilian Clinical Trials Registry, part of the International Clinical Trials Registry Platform, under number U1111-1299-4634.

Individuals interested in participating in the study will contact the study team through designated phone numbers. Moreover, patients can be referred to the study via the CAPS. All interested individuals will undergo screening by a trained study team member to ensure eligibility and, most importantly, their ability for informed and voluntary consent. The study team member will describe the study and go through the informed consent procedures. Participants will read the consent form (CF) without any time constraints and will have the opportunity to ask any questions related to the study procedures. Individuals will be officially enrolled upon signing the CF and will be randomized into three different groups: EG1 (n = 24), EG2 (n = 24), or CG (n = 24). By nature, the randomization protocol will evenly distribute age and sex among the groups.

Individuals interested in participating will be first personally assessed for eligibility before signing the CF, a procedure conducted by the Principal Investigator (PI) or Co-Investigators (Co-Is). Individuals will have the opportunity to consider participation before signing the CF, and they will be officially enrolled upon signing it. The PI or other licensed Co-Is will review and co-sign the CF for any study subjects.

3.3. Study Procedures

The timeline for the study procedure is summarized in Figure 2. Further details about each study step are described in the following subsections.

3.4. Study Visits

Pre-screening (Week-1): Before study enrollment, potential participants will be contacted by a trained study team member for screening purposes. The study’s trained team members will review study details and procedures and ask a series of questions used to assess eligibility for the study. Before collecting any information, individuals will be asked to provide verbal authorization to record their responses and will have the option not to answer any questions if they choose not to. If the individual is eligible for the study, they will be invited for a screening visit. If the individual is not eligible or decides not to enroll, any information collected during the call will be destroyed to protect privacy. If an individual is eligible for the study and enrolls, the information collected during the call will be added to their research file. Individuals will receive a copy of the CF for review, and the screening visit will be scheduled.

Screening (Week 0): Participants will review the CF with the PI of the study and will have the opportunity to ask questions. Subjects will be accompanied by an adult (e.g., parents or respective legal guardians of the teenager), and both will complete an ICS (Informed Consent Survey) assessing their understanding of the study procedures through yes/no, true/false, and open-ended questions. If an incorrect response is given, the study researcher will clarify the item. If the individual cannot comprehend the correct answer, they will not be able to enroll. The screening visit will be in person, and the consent form will be collected on paper. Subjects will be considered enrolled in the study upon signing the CF. Participants will respond to a series of questions to assess eligibility for the study, including demographic information and concurrent medications and therapies. If eligible, they will be invited for the initial visit; otherwise, they will not be able to continue in the study.

Initial Visit (Week 1): Participants will complete a battery of neuropsychological tests, including the following: Hamilton Depression Rating Scale (HAM-D): Assesses the severity of depressive symptoms with an emphasis on somatic symptoms. The scale ranges from 17 to 21 items, with scores classified as normal (0–7), mild depression (8–13), moderate (14–18), severe (19–22), and very severe (>23) [65]. Validated for Brazilian adolescents by Freire et al. (2014) [66]; Hamilton Anxiety Rating Scale (HAM-A): Semi-structured, assesses physical and somatic symptoms of anxiety. Highly reliable, applicable to adults, adolescents, and children [67]. The Brazilian version was adapted by Pesce et al. (2005) [61]; Resilience Scale, along with the PSQI, all translated and validated in Portuguese [68] Pittsburgh Sleep Quality Index (PSQI): Uses the translated and validated Portuguese version [69]; 12-Item Short-Form Health Survey (SF-12): Assesses quality of life [70,71]; Physical Activity Questionnaire for Adolescents (APAQ): Measures the level of physical activity [72]. Additionally, 10 mL of blood will be collected from the cubital vein of each participant using 5 mL BD Vacutainer gel tubes (lot: 367812, 0.80 × 25 mm needles, brand SR Lot 5292198) and FirstLab vacuum needles of 23G and 25G. This procedure will be performed in order to assess oxidative stress and antioxidant enzyme levels. As mentioned previously, participants will then be randomized into three groups: CG, EG1, and EG2. Participants from the CG will undergo a PBM cap (t-PBM regimen) with a power of 1 mW, solely to simulate irradiation, for the same treatment period as the experimental groups. The EG1 will undergo a PBM cap, 3 days/week for 12 weeks, and the EG2 will be subjected to a PBM cap and blanket for 3 days a week for 12 weeks.

Outcome methodologies: The results of the neuropsychological assessments, sleep quality, quality of life, and physical activity levels will be analyzed to determine the treatment’s efficacy. Blood collection will allow for biochemical analyses, providing additional data on participants’ health. These data will be used to correlate the effects of the interventions with changes in mental and physical health parameters.

Treatment (Weeks 2–13): Participants will take the equipment home and undergo at least three treatments per week over twelve weeks, with a minimum interval of 24 h between each session. They will complete a minimum of 36 treatment sessions, each lasting at least 20 min, using both the cap and the blanket. The blanket will be applied to the abdominal region while the participant is in the supine position on a flat surface, simultaneously with the use of the cap. There will be in-person follow-up visits every two weeks. The study team will complete an intervention monitoring form at each weekly treatment session to ensure continuous and correct use of the equipment.

Follow-up visit for the final study (Week 14): The final study follow-up visit takes place one week after the final treatment session. Participants will complete the same battery of tests conducted at the beginning of the study, which includes the HAM-D, HAM-A, (PSS), Resilience Scale, along with the PSQI, all translated and validated in Portuguese [68], SF-12 [70,71], and APAQ [72]. Additionally, 10 mL of blood will be withdrawn from the cubital vein of each participant as described for Week 1. CG participants who were subjected to t-PBM (PBM cap) with a power of 1 mW, solely to simulate irradiation, will have the option to receive the same treatments as the EG1 and EG2 groups for up to 12 weeks at no cost to the participant.

3.5. PBM Administration

We will use two PBM devices, one for t-PBM administration and another for abdominal administration. For t-PBM, the device consists of a cap (Infrallux^©, São Paulo, Brazil) with an inner space composed of 198 LEDs covering the entire head. For the abdominal administration, we will use a blanket (Sportllux Advanced Pro Back^©, São Paulo, Brazil), consisting of 264 LEDs in total. The PBM parameters for both devices (cap and blanket) are described in

Table 1. PBM parameters for transcranial (cap) and abdominal (blanket) administrations.

	Cap	Blanket
Wavelengths	850 nm	830 and 620 nm
Total number of diodes	198	264
Quantity of red diodes	-	132
Quantity of infrared diodes	198	132
Power per LED	8 mW	1 mW
Optical spot diameter	5 mm	5 mm
Light emission angle (20 1/2)	120°	120°
Irradiance	29 W/cm2	44.3 W/cm2
Fluence	35 J/cm2	33.6 J/cm2
Total energy delivered per session	27.6 J	21 J
Total energy delivered for the study period	993.6 J	756 J

.

PBM treatment on the abdominal region will be administered using a blanket composed of 264 special LEDs (Sportllux Advanced Pro Back^®, São Paulo, Brazil). The blanket measures 15 × 25 cm and is mounted on a belt approximately 1.20 m long, equipped with Velcro at the ends for secure fastening. The LED blanket is divided into two types of wavelengths: 132 LEDs operate in the red wavelength range (620 nm) and 132 LEDs in the infrared wavelength range (830 nm). Each LED has a total power output of 35 mW, with a peak power of 8 mW per LED. The irradiance provided by the blanket is 44.3 mW/cm², ensuring effective energy delivery. The total energy output per session is 21 J, and the fluence delivered is 33.6 J/cm². Over the course of the study, with treatments administered three times per week for three months, the total energy delivered will amount to 756 J. The treatment area occupied by the LEDs measures 20 cm in length and 12 cm in width, allowing for comprehensive coverage of the targeted abdominal region.

We chose to use a low-power light of only 1 mW to simulate irradiation in the control group. This decision was based on a study by Salehpour et al. [73], which highlights that photobiomodulation can activate neuroprotective mechanisms through intracellular and molecular modulation, increasing cerebral blood flow and balancing cellular metabolism. However, most studies use higher power levels, generally in the range of 100 mW or more. Another study by Farazi et al. [74] addresses photobiomodulation protocols with specific light intensities and their effects on patients with mild cognitive impairment and dementia. This study also reinforces that treatments with higher intensities tend to be more effective. Therefore, the use of a low power of 1 mW in the control group serves to simulate irradiation without triggering the robust therapeutic effects observed at higher intensities, thus allowing for a more accurate comparison of the effects of photobiomodulation at different intensities.

The participant will lie on their back on a flat surface to undergo the treatment. The photobiomodulation blanket will be positioned around the abdominal area, firmly secured around the waist, and covering the central region of the abdomen. The participant will be in a supine position (on their back), with legs extended and arms relaxed along the body. The photobiomodulation blanket will be adjusted so that the embedded LED lights make direct contact with the skin of the treated area.

3.6. Assessments

Hamilton Depression Scale (HAM-D): To measure depressive symptoms, the instrument used will be the Hamilton Depression Scale (HAM-D), developed in 1960 to assess the severity of depressive symptoms with a greater emphasis on somatic symptoms. It consists of versions ranging from 17 to 21 questions that should be answered by the evaluator [65]. The scores resulting from the HAM-D application will be subclassified according to the original 1960 publication. It has been validated for Brazilian adolescents by Freire et al. [65]. This questionnaire (Annex A) has 14 questions, seven to investigate the likelihood of anxiety (questions 1, 3, 5, 7, 9, 11, and 13) and seven for depression (questions 2, 4, 6, 8, 10, 12, and 14). Each question has four alternatives with a scoring possibility from 0 to 3 points each. After marking the answers, it is necessary to add up the values obtained for the anxiety questions separately from the questions that assess the level of depression. For the classification of depression and anxiety symptoms, the following separate scores will be used: 0–7 points = unlikely; 8–11 points = possible (questionable or doubtful); 12–21 points = probable.

Hamilton Anxiety Scale (HAM-A): The initial scale designed for evaluating anxiety is a semi-structured tool that emphasizes both psychical and somatic symptoms. It exhibits high reliability and is applicable to adults, adolescents, and children [64]. The scale comprises 14 groups of symptoms, subdivided into two groups: seven related to psychic anxiety, associated with anxious mood symptoms and seven related to somatic anxiety, associated with physical symptoms of anxiety. Each item is assessed on a scale ranging from 0 to 4, according to the symptom’s intensity (0 = absent, 2 = mild, 3 = moderate, 4 = severe). The sum of the scores obtained for each item results in a total score ranging from 0 to 56. Its development was based on the principle that the more severe the manifestation of a pathology, the greater the number of characteristic symptoms that are presented [67].

Perceived Stress Scale (PSS): To measure the stress level, the PSS will be used, which evaluates the perception of stressful experiences in the previous month using a five-point scale. The tool consists of 14 items, seven positive and seven negative, with response options ranging from 0 to 4 (0 = never; 1 = almost never; 2 = sometimes; 3 = fairly often; 4 = very often). The items assess the occurrence of negative and positive feelings, the ability to cope with stressful situations, as well as core components of stress in individuals [64].

Resilience Scale: The only resilience scale adapted for Brazil by Pesce et al. (2005) will also be used, and this scale has been demonstrated to have good internal validity indicating good internal consistency of the instrument [61,64].

Pittsburgh Sleep Quality Index: In the translated and validated version for Portuguese, this tool assesses sleep quality and disturbances over a one-month period. It is a standardized, straightforward questionnaire well-accepted by individuals [66]. Comprising 19 self-reported questions and five questions directed to the spouse or room companion, the last five questions are solely used for clinical practice and do not contribute to the total score of the index. The 19 questions are categorized into seven components, graded on scores from zero (no difficulty) to three (severe difficulty). The questionnaire’s components include C1 subjective sleep quality, C2 sleep latency, C3 sleep duration, C4 habitual sleep efficiency, C5 sleep disturbances, C6 use of sleep medications, and C7 daytime dysfunction due to sleep issues. The sum of values assigned to these seven components ranges from zero to twenty-one in the total questionnaire score, meaning that higher numbers indicate poorer sleep quality. A total score exceeding five suggests significant dysfunction in at least two components or moderate dysfunction in at least three components [69].

Short-Form Health Survey (SF-12): Quality of life will be assessed using the Portuguese version of the 12-Item Short-Form Health Survey (SF-12) [70,71], consisting of 12 items that evaluate functional capacity, physical aspects, pain, general health status, vitality, social aspects, emotional aspects, and mental health, considering the individual’s perception of their life over the past four weeks. Each of the 12 items has a set of possible responses distributed on a Likert-type scale. Through the application of a specific algorithm in the questionnaire, two domains can be calculated: the physical (Physical Component Summary or PCS) and the mental (Mental Component Summary or MCS). These scores range on a scale from zero to one hundred, with higher values correlated with better quality of life [71].

Physical Activity Questionnaire for Adolescents (QAFA): The Physical Activity Level will be assessed using the Physical Activity Questionnaire for Adolescents (QAFA) validated by Farias Júnior et al. [72]. The questionnaire consists of a list of 24 moderate to vigorous intensity physical activities (≥3 METs), with the possibility for the adolescent to add two more activities. When completing the questionnaire, adolescents report the frequency (days/week) and duration (hours/min/day) of physical activities practiced in the last week. To determine the level of physical activity, the sum of the product of the time spent on each physical activity by their respective frequencies of practice is calculated, following the procedure described in the annex. Adolescents engaging in physical activity equal to or greater than 300 min/week are considered sufficiently active, while others are classified as insufficiently active according to Biddle et al. [75].

3.7. Data Management

A comprehensive web-based data acquisition and management system, REDCap, was programmed to process, edit, and store all study data in a centralized database.

3.8. Statistical Analyses

The data will be inputted into platforms and analyzed using GraphPad Prism software (v.9.0). Numerical variable analyses will begin by observing the data distribution through a Shapiro–Wilk test. Parametric data will be compared using a one-way or two-way analysis of variance (ANOVA), followed by a Student–Newman–Keuls or Bonferroni test. Non-parametric results will be statistically analyzed between groups using a Kruskal–Wallis test. In all analyses, p-values less than 0.05 will be considered statistically significant.

To test Aim 1, we will compare the groups in terms of the changes in depressive symptoms, measured by associated biomarkers, including the activity levels of the enzymes catalase (CAT) and superoxide dismutase (SOD), serum concentrations of carbonylated proteins, malondialdehyde, and thiobarbituric acid, as well as the activities of mitochondrial complexes I, II, and IV in adolescents with MDD, before and after all sessions of PBMT. To test Aim 2, we will compare the groups in terms of the changes in anxiety symptoms, measured by the Hamilton Anxiety Rating Scale, before and after all PBMT sessions in adolescents with MDD. To test Aim 3, we will compare the groups regarding the impact of PBMT on stress and resilience, using the Hamilton Anxiety Rating Scale and the Resilience Scale adapted for Brazil by Pesce et al. [61], before and after all PBMT sessions in adolescents with MDD.

3.9. Risks and Discomforts

There are risks and discomforts associated with assessments of depression, anxiety, and stress levels, as with any neuropsychological test, and also with other tests, where participants may become frustrated due to performance difficulty or boredom during longer sessions. Participants may refuse to answer questions that make them uncomfortable.

The PBM device emits light with a wavelength longer than the human eye can see. The team will receive training on the basic safety procedures related to the use of the device. The team administering PBM will take care not to operate the LED unless it is in direct contact with the subject’s skin. Protective eyewear is not required as the device is an LED. The failure of the LED device, resulting in the interruption of the investigative therapy, cannot cause any adverse events, as far as we know. Delivering NIR-LED energy to an inappropriate location, such as directly over an open eye, would not pose a risk to the subject, given the use of divergent light rays. Based on previous observations with similar LED devices, the application of LEDs may result in a slight sensation of warmth during use. However, the skin temperature is maintained well below the level of thermal damage, based on the experience of clinical trials in humans to date and the sale of PBM devices for the intended use.

3.10. Demographic Data on Existing Participants

Participants are recruited from all areas of the city of Campos Novos, SC. On 16 October 2023, a total of 72 participants were recruited for this study. Fifty-four participants self-identify as women (75%), and eighteen participants self-identify as men (25%). The participants range in age from 15 to 17 years old. The study is ongoing.

3.11. Monitoring and Quality Assurance

Upon enrolling in the study, each participant will receive a unique identification number that will be used in all study forms and documents in place of the participant’s name. These documents will be stored separately from other materials in a locked file, accessible only to the study team. Participants can request the withdrawal of their data by contacting the Principal Investigator (PI), whose contact information is provided in the consent form. Experienced doctors with doctoral-level qualifications will be available to participants, both by phone and in person if necessary, to discuss any concerns during the study. This will be clearly communicated to participants, both verbally and in writing.

The data collected in the project will include neuropsychological information, sleep quality, quality of life, physical activity level, and blood data. The information collected will be used exclusively for research purposes, and there are no plans to share it with anyone outside the study team. The data will not be sent to anyone outside the study team and will not be collected outside the research sites, ensuring that no external data is received. The study team will meet weekly throughout the duration of the project to review progress, discuss any safety concerns that arise, and make recommendations to improve safety procedures if necessary.

4. Discussion

Existing research provides evidence suggesting that the use of PBMT in treating MDD may yield positive outcomes in alleviating depressive symptoms for certain individuals. These studies have highlighted improvements in mood, memory, and cognition [73,74,75]. Building upon this foundation, we propose a hypothesis that t-PBM plus the abdominal administration of PBMT could potentially alleviate depression and comorbid anxiety symptoms in adolescents with MDD. Furthermore, employing PBMT through a remote application may enhance its efficacy due to the bidirectional communication between the enteric nervous system of the gut and the CNS in the brain.

In a study performed by Cassano et al. [76], the antidepressant effect of t-PBM with near-infrared light was investigated in individuals with MDD. The results indicated an antidepressant effect of t-PBM when delivered to the bilateral dorsolateral PFC twice a week for eight weeks. This approach exhibited a medium to large effect size in patients with MDD.

Disner, Beevers, and Lima [77] tested the hypothesis that LLLT enhanced the effects of attention bias modification (ABM) in adults with elevated symptoms of depression and concluded that, when applied to the right PFC, LLLT had beneficial effects of ABM in treating depression symptoms in adults with elevated depression symptoms. Based on the results of this study, LLLT may alleviate depression by modulating the cognitive response to negative attention bias. LLLT acts as a form of neuroenhancement, which, when combined with ABM, may enhance its efficacy in the treatment of depression.

Although human studies demonstrate the effectiveness of t-PBM, defining the ideal dosage remains a challenge, as indicated by Iosifescu et al. [48], who tested the efficacy of t-PBM with low irradiance and energy per session in individuals with MDD. Despite variations in treatment parameters, both animal and human studies suggest the potential of t-PBM as an antidepressant treatment. Such an effect might be due to increased mitochondrial energy production and regional blood flow promoted by t-PBM administration (for a review, see Askalsky and Iosifescu [78]). Larger studies are needed to confirm its role, and a crucial next step involves a comparative study to determine the optimal parameters for t-PBM as an antidepressant treatment.

Caldieraro, Silva, and Cassano [79] conducted a comprehensive review of the literature on t-PBM for depression management, reinforcing the evidence regarding its mechanism of action. Animal and human studies support its potential impact on depression, and although larger-scale studies are necessary, the available data suggest that t-PBM is a safe and easily administrable option. The next crucial phase involves a comparative study to determine the optimal parameters for t-PBM as an antidepressant treatment.

In the context of anxiety, studies show a combined anxiolytic and antidepressant effect of t-PBM. Schiffer et al. treated individuals with comorbid MDD and anxiety using t-PBM, resulting in a significant improvement in anxiety. T-PBM, with its innovative approach to stimulate neural activity through low-intensity light irradiation, presents a potential therapeutic option for both MDD and anxiety [80]. Additionally, a study by the same author analyzed the effects of t-PBM with 945 nm LED in university students with anxiety and depression, concluding that it enhances brain activity and may clinically reduce anxiety and depression.

Concurrently, Maiello et al. [81] explored the effects of t-PBM on individuals with generalized anxiety, acknowledging t-PBM as an experimental and non-invasive treatment for mood and anxiety disorders. Their study aimed to test the anxiolytic effect of t-PBM with NIR in individuals suffering from generalized anxiety disorder (GAD). The conclusion was that t-PBM with NIR holds promise as a treatment alternative for GAD.

Fernandes et al. [82] investigated the effects of PBM on auriculotherapy points for sleep disorders, anxiety, and temporomandibular dysfunctions. The study concluded that it was effective in treating anxiety. In animal studies, Farazi et al. [74] explored the application of transcranial t-PBM on the depressive and anxious behaviors in a noise stress model in mice, suggesting a reduction in anxiety. This ongoing study aims to validate and expand upon these findings based on existing literature.

This study protocol aims to address the treatment of depression in adolescents through transcranial and systemic/abdominal photobiomodulation. Photobiomodulation has shown promise in treating this condition, and in this study, even greater effectiveness is anticipated due to the consideration of a remote application. Several studies indicate the relevance of this connection in depression [83,84,85]. This study is innovative because it is the first to employ transcranial and systemic/abdominal photobiomodulation in the treatment of depression. Additionally, it stands out for being a minimally invasive method. However, the lack of a longer-term follow-up of patients may be considered a limitation of this study, as monitoring participants after treatment allows for assessing whether the treatment benefits are maintained in the long term or if there is a regression of the positive effects. It is also important to note that measuring the gut–brain axis was not the primary purpose of this study, which remains as a limitation. Future research is recommended to include a longer-term follow-up, not limited to the months of PBMT but extending over several months after treatment completion. Additionally, future studies need to address the gut–brain axis to further understand its role and potential impact in relation to treatment outcomes.