Photobiomodulation as a Therapeutic Approach for Attention-Deficit/Hyperactivity Disorder in Model Rats

Photobiomodulation as a Therapeutic Approach for Attention-Deficit/Hyperactivity Disorder in Model Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Photobiomodulation as a Therapeutic Approach for Attention-Deficit/Hyperactivity Disorder in Model Rats Yu-Jui Huang, Yi-Tien Li, Min-Lan Tsai, Feng-Chin Lee, Akira Niwa, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6990648/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Oct, 2025 Read the published version in Lasers in Medical Science → Version 1 posted 11 You are reading this latest preprint version Abstract Significance: Current treatment options for Attention-deficit/hyperactivity disorder (ADHD) primarily involve pharmacological and behavioral interventions, but concerns about side effects, long-term safety, and limited efficacy in certain populations necessitate alternative therapies. This study investigates the therapeutic potential of Photobiomodulation therapy (PBMT) in an ADHD animal model, contributing to the growing field of nonpharmacological interventions. The study aims to evaluate the effects of PBMT on ADHD symptoms using a spontaneously hypertensive rat (SHR) model. Specifically, it examines whether PBMT can reduce impulsivity and hyperactivity by modulating neuroinflammation, preserving neuronal integrity, and enhancing structural brain connectivity. Methods: SHRs, a well-established ADHD animal model, were used to assess PBMT’s effects. The experimental design included three groups: a normal control group (WKY), an ADHD model group receiving PBMT (SHR25), and an ADHD model group without PBMT intervention (SHR). PBMT was administered daily for 25 seconds at a wavelength of 808 nm over 21 consecutive days. Behavioral assessments (open field test), diffusion tensor imaging (DTI), and immunohistochemical analysis were conducted to evaluate neuroinflammation, neuronal integrity, and myelination. Results: PBMT significantly reduced impulsivity and hyperactivity in SHRs, as indicated by decreased total track length and average speed in the open field test. DTI analysis revealed improved neural connectivity in ADHD-associated brain regions, particularly the prefrontal cortex, striatum, and hippocampus. Immunohistochemical staining demonstrated a significant reduction in Iba-1 positive cells, indicating decreased neuroinflammation. Additionally, PBMT preserved myelin integrity in ADHD-related brain regions, suggesting neuroprotective effects. Conclusions: The findings suggest that PBMT is an effective nonpharmacological intervention for ADHD, reducing impulsivity and hyperactivity while preserving neuronal structure and function. By mitigating neuroinflammation and improving neuronal connectivity, PBMT presents a promising therapeutic alternative to conventional ADHD treatments. Further studies are needed to explore its clinical applicability and long-term safety in human populations. Photobiomodulation therapy (PBMT) Attention-deficit/hyperactivity disorder (ADHD) Neuroinflammation Neuronal connectivity Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by persistent symptoms of inattention, hyperactivity, and impulsivity that disrupt normal functioning and development [1]. This disorder affects individuals across all stages of life, with symptoms often persisting from childhood through adolescence and into adulthood [2]. Management strategies for ADHD typically involve a combination of behavioral interventions, education, and pharmacological treatments. Advances in neuroscience, psychology, and pharmacology have improved the understanding of ADHD and its treatment. Contemporary ADHD treatment strategies often include pharmacotherapy and behavioral interventions [3]. For decades, stimulant medications such as methylphenidate have remained central in pharmacological management of ADHD [4]. These stimulants enhance attention, focus, and impulse control by increasing the levels of neurotransmitters, such as dopamine and norepinephrine, in the brain [5]. However, stimulant medications are associated with side effects such as appetite suppression, insomnia, irritability, and, in some cases, cardiovascular complications [6]. Additionally, concerns have been raised regarding their long-term effects on growth and development in children and the potential for their misuse, particularly among adolescents and young adults seeking stimulants for cognitive enhancement or recreational purposes [7]. Nonstimulant medications, such as atomoxetine, guanfacine, and clonidine, offer alternative treatment options for patients who either cannot tolerate stimulants or do not achieve adequate symptom control by using them. Nonstimulant medications target different neurotransmitter systems, such as those involving norepinephrine and α2-adrenergic receptors, to alleviate ADHD symptoms [8] . Unlike stimulants, nonstimulants require several weeks of consistent use to produce therapeutic effects, which can present challenges for patients seeking immediate relief from symptoms. Common side effects of nonstimulant medications include sedation and gastrointestinal disturbances. Additionally, one such medication, atomoxetine, has been associated with occasional increases in liver enzyme levels, and therefore, regular monitoring is required during treatment with the drug. In ADHD treatment, medication is frequently employed to manage impulsivity. However, for preschool-aged children, pharmacological interventions are typically avoided. Clinical guidelines often recommend psychological therapies as the primary approach for this population [9]. In consideration of this, nonpharmacological treatment options, particularly for patients aged <6 years, must be developed that are able to reduce impulsivity in individuals with ADHD. Photobiomodulation therapy (PBMT), a type of low-level light therapy, holds promise as a treatment for ADHD. ADHD is a neurodevelopmental disorder characterized by persistent symptoms of inattention, hyperactivity, and impulsivity, which substantially impair daily functioning. PBMT involves the use of specific wavelengths of light to stimulate biological processes at the cellular level through modulation of mitochondrial function and oxidative metabolism. Studies have demonstrated the potential of PBMT in modulating neurochemical pathways [10,11]. PBMT may influence neurotransmitter systems, such as those involving dopamine and norepinephrine, which play critical roles in the regulation of attention and impulse control [12]. Additionally, PBMT has been demonstrated to mitigate neuroinflammation and oxidative stress [11,13], both of which have increasingly been recognized as contributors to ADHD pathogenesis. The current study investigated the potential of PBMT as a treatment for ADHD by using spontaneously hypertensive rat (SHR) animal models. The findings were compelling. PBMT demonstrated efficacy in reducing impulsivity, potentially through the suppression of inflammation in the prefrontal cortex, striatum, and hippocampus, thereby preserving cellular integrity. Furthermore, continuous PBMT exposure over 21 consecutive days of irradiation appeared to sustain neuronal connectivity, suggesting that PBMT may exert long-term beneficial effects on brain structure and function. Materials and Methods ADHD animal model The use of animals in this study adhered to established animal research guidelines for the reporting of in vivo experiments and the principles outlined in the Basel Declaration, with careful consideration of the 3R principle (Replacement, Reduction, and Refinement). Postnatal day 42 rats were procured from BioLASCO (Taiwan). SHRs, which are widely recognized as a robust animal model for ADHD, were employed in this study. Wistar–Kyoto rats (WKY) were used as controls [14]. Transcranial PBMT The laser apparatus used in this study was designed by Transverse Industries Co., Ltd. (Taiwan) and was described in detail in another study [15]. Briefly, the device employs a gallium aluminum arsenide (GaAlAs) diode laser with an output wavelength of 808 nm and a power output of 110 mW per laser. The laser operates in continuous mode, with the beam collimated by a lens measuring 11 mm in height. The apparatus comprises laser light sources fitted with single lens hoods. A schematic of the laser structure and lens hood is provided in Fig. 1A and B. The elliptical laser beam emitted at the horizontal plane in front of the lens hood has a major axis of 3.5 mm and a minor axis of 3.0 mm (Fig. 1C), resulting in a beam area of 0.0825 cm 2 (Fig. 1D). Because the rat’s scalp was positioned in close contact with the lens hood during PBMT, the calculated power density at the scalp surface was approximately 1.333 W/cm 2 . Each session of PBMT lasted 25 s, with a radiant exposure of approximately 33.3 J/cm 2 and a total radiant energy of 2.75 J delivered per rat. At 15 minutes prior to irradiation, each rat’s scalp hair was removed using a depilatory cream, and the scalp was marked for precise alignment. An Eppendorf tube (internal diameter: 12 mm) was used to facilitate the attachment of the front of a lens hood. The prominence of the skull at the Lambda was identified through a tactile assessment and by observing the contour of the skull through the scalp. The center of the Eppendorf tube was aligned with the Lambda (−6 mm from Bregma) and adjusted to the midline of the scalp. After their scalps were marked, the rats’ bodies were gently wrapped in towels, and the scalp markings were aligned with the inner edge of the lens hood for irradiation (Fig. 1E–G). The experimental rats were divided into three groups: a normal control group (WKY), an ADHD model group receiving PBMT (SHR25), and an ADHD model group receiving no PBMT intervention (SHR). In the SHR25 group, each rat received a single 25-s session of PBMT. The SHR group underwent sham irradiation with the power supply turned off, whereas the WKY group received no intervention. For all groups, the open field test was conducted to evaluate ADHD-related behavioral symptoms. Additionally, diffusion tensor imaging (DTI) was performed to assess neural connectivity, and immunohistochemistry was performed to analyze tissue-level changes. Experiment protocol The experimental animals were divided into three groups: WKY (normal control), SHR25 (ADHD model group receiving PBMT), and SHR (ADHD model group not receiving PBMT). The groups were further subdivided on the basis of the number of PBMT treatment days. DTI was conducted on days 0, 1, 7, 14, and 21, and an open field test and immunostaining were conducted on days 7, 14, and 21 (Fig. 1H). Open field test (behavioral evaluation) An open field test was employed to evaluate hyperactivity and locomotor activity in the experimental animals. Behavioral evaluations were conducted at 7, 14, and 21 days following PBMT. For the field tests, each rat was placed at the center of a black acrylic box measuring 48 × 48 × 42 cm 3 . The center point of the rat’s body was tracked to record the total track length (in meters), and the average speed of movement (in meters per second) was calculated. Magnetic resonance imaging data acquisition Longitudinal magnetic resonance imaging (MRI) was performed on each animal prior to laser treatment and at day 1, week 2, and week 3 after laser treatment by using a PharmaScan 7T system (Bruker Biospin). During imaging, the animals were anesthetized with approximately 1.2% isoflurane to ensure immobilization and minimize motion artifacts. A stereotaxic headpiece and holder equipped with ear and tooth bars were used to stabilize the head throughout the procedure. DTI was performed using echo planar imaging with a b value of 1200 s/mm 2 applied along 30 noncollinear and noncoplanar diffusion directions and 5 b 0 images. The imaging parameters were as follows: repetition time (TR)/echo time (TE) = 3000/37 ms, flip angle = 90°, δ/Δ = 5/15 ms, acquisition matrix = 128 × 128, and resolution = 0.156 × 0.156 mm 2 . A total of 16 slices were acquired, with each being 1-mm thick, with two averages. Additionally, T 2 -weighted images were obtained for the coregistration and normalization of DTI data by using the following parameters: TR/TE = 3600/40 ms, flip angle = 90°, voxel size = 0.078 × 0.078 × 1 mm 3 , matrix = 256 × 256 × 16, and number of averages = 4. DTI analysis DTI data were preprocessed using FMRIB Software Library (FSL) 5.0.10 (FMRIB, Oxford, UK) and MRtrix 3.0.2 (Brain Research Institute, Melbourne, VIC, Australia). Raw images were preprocessed through denoising, Gibbs ringing artifact removal, and bias field correction, which were performed using MRtrix. Subsequently, the data were processed through the standard FSL pipeline, including brain mask estimation, as well as eddy-current and motion correction. Fixel-based analysis of fiber density was performed using the MRtrix multishell (two unique high b values: 1000 and 2500 s/mm2) multitissue (three tissue classes: gray matter, white matter, and cerebrospinal fluid) constrained spherical deconvolution method [16] in combination with the probabilistic streamlining method [17]. DTI-derived fractional anisotropy maps were generated from the processed data. These maps were subsequently spatially normalized to the template space by using the MRtrix framework [18,19]. Immunofluorescence staining Immunofluorescence staining was performed following established protocols [13]. Briefly, brain sections were transferred to room temperature (RT) and were permeabilized in acetone precooled to 4°C for 1 min. After undergoing air drying, the sections were blocked with 5% skim milk (Sigma-Aldrich) for 1 h at RT, after which they were rinsed under running tap water. Subsequently, the sections were incubated overnight at 4°C in a humid chamber with a primary antibody (GTX11427, GeneTex, Taiwan) diluted at a ratio of 1:200 in skim milk. The following day, the sections were brought to RT and rinsed with PBST. They were then incubated in the dark for 1 h in a humid chamber with a secondary antibody, donkey antimouse Cy3 (715-165-151, Jackson ImmunoResearch) diluted at a ratio of 1:600 in PBS at 4°C. After incubation, the sections were rinsed again with PBST to remove unbound antibodies. Results Significant PBMT-Related Reduction in Total Track Length and Average Speed in the SHR25 Group To evaluate the therapeutic effects of PBMT on ADHD symptoms, we conducted behavioral assessments on the SHR (disease model), SHR25 (PBMT-treated ADHD model), and WKY (normal control) groups. Behavioral activity was assessed through total track length (TTL) and average speed (AS), which were used as indicators of hyperactivity and impulsivity. The movement patterns of the SHR25 group indicated notably less hyperactivity than those of the SHR group did, aligning more closely with the patterns WKY group (Fig. 2A and Supplementary Video). A quantitative analysis revealed that TTL was significantly reduced in the SHR25 group relative to in the SHR group beginning on day 1 after PBMT treatment (Fig. 2B and 2C). The SHR25 group exhibited TTL values of 26.51 ± SD, whereas those in the SHR group were 28.65 ± SD ( P < 0.01). This significant reduction persisted at subsequent time points, including days 14 and 21 ( P < 0.01 for all comparisons). AS measurements corroborated the reduction in hyperactivity and impulsivity in the SHR25 group. A significant decrease in AS was observed in the SHR25 group starting from day 1 following PBMT treatment (0.048 ± SD in the SHR25 group versus 0.044 ± SD in the SHR group, P < 0.01). This trend continued until day 21. Improvements in Behavior and Microstructural Changes in the Brain In addition to behavioral changes, we investigated microstructural changes in the brain following PBMT. Our analysis focused on the prefrontal cortex, striatum, and hippocampus, regions known to play critical roles in ADHD pathogenesis. DTI was employed to assess neural tract integrity and connectivity in these areas postirradiation, and the results indicated significant differences in microstructural integrity between the SHR25 and SHR groups (Fig. 3A and 3B). Notably, the SHR25 group exhibited structural connectivity patterns that more closely resembled those observed in the WKY group than those in the SHR group. Significant PBMT-Related Reduction in the Number of Iba-1 Positive Cells, an Inflammation Marker In addition to the behavioral improvements and structural changes observed in the brain post-PBMT irradiation, we investigated cellular-level changes that occurred in response to PBMT. Specifically, we examined the number of Iba-1 positive cells in each group by using Iba-1 staining, which indicates the inflammatory state (Fig. 4A–4D). Our results revealed a significant reduction in the number of Iba-1 positive cells in the PBMT-treated SHR25 group relative to in the untreated SHR group. In the prefrontal cortex, Iba-1 positive cell counts were significantly decreased on days 7, 14, and 21 ( P < 0.05). In the striatum, a significant reduction was observed on day 21 ( P < 0.05), and in the hippocampus, reductions were significant on day 14 ( P < 0.05). PBMT-Related Preservation of Neuronal Myelination and Integrity To evaluate the neuroprotective effects of PBMT, we assessed the number of myelin basic protein (MBP) positive cells in each group (Fig. 4A–C). The results revealed a significant increase in the number of MBP-positive cells in the PBMT-treated SRH25 group compared with in the untreated SHR group. This phenomenon was observed in the prefrontal cortex, striatum, and hippocampus, with the most pronounced protective effect observed in the prefrontal cortex. Specifically, in the prefrontal cortex, MBP positive cell counts were significantly higher on days 7, 14, and 21 ( P < 0.05). Discussion In this study, we investigated the potential of PBMT as a novel treatment for ADHD. This study applied PBMT at a wavelength of 808 nm to the local head region of SHRs, a well-established animal model of ADHD. The PBMT was administered for 25 s daily for 21 consecutive days. The results indicated a significant reduction in impulsivity in the PBMT-treated SHR group. Furthermore, neuroanatomical analysis conducted using DTI revealed that PBMT effectively preserved neuronal connectivity in ADHD-related brain regions, particularly the prefrontal cortex, striatum, and hippocampus. Notably, the observed structural connectivity in these regions closely resembled that of normal rats. Additionally, we examined the mechanism underlying these effects and noted that PBMT reduced neuroinflammatory states in the brain and helped to maintain the integrity of myelin in the prefrontal cortex, striatum, and hippocampus. These findings were confirmed through immunohistochemical analysis. Neuroinflammation refers to a phenomenon in the central nervous system (CNS) wherein immune cells become activated and release inflammatory mediators in response to various stimuli. Traditionally, ADHD has been primarily attributed to dysfunctions in neurotransmitter systems, particularly those involving dopamine and norepinephrine [20,21]. However, emerging research suggests that neuroinflammation may also contribute to the onset and manifestation of ADHD symptoms. Studies have reported elevated levels of proinflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), in the blood and cerebrospinal fluid of patients with ADHD (Ref). These cytokines, which are markers of immune system activation, indicate the presence of inflammatory responses in the brain and their potential involvement in ADHD pathogenesis [22,23]. Research has further suggested a strong association between neuroinflammation in the CNS and ADHD development [24-26]. Positron emission tomography imaging studies have revealed microglial activation, a hallmark of CNS inflammation, in ADHD-associated brain regions such as the prefrontal cortex and striatum [27,28]. Additionally, both animal and human studies have reported increased Iba-1 immunoreactivity indicative of activated microglia, which in turn release proinflammatory cytokines and reactive oxygen species. These mediators contribute to neurotoxicity and impair neural function. These findings collectively imply that microglial activation and the resulting neuroinflammation may contribute to ADHD pathogenesis [29,30]. These findings underscore the potential therapeutic value of targeting neuroinflammation, particularly microglial activation, in the treatment of ADHD. PBMT, in which red or near-infrared light is used to promote tissue repair and reduce inflammation, is increasingly being used in clinical settings. Among the various wavelengths that have been studied, near-infrared light (600–800 nm) has demonstrated notable anti-inflammatory effects [11,31]. In the present study, SHRs exposed to local PBMT at 808 nm exhibited a substantial reduction in the number of Iba-1-positive microglial cells in ADHD-associated brain regions, including the prefrontal cortex, striatum, and hippocampus [32,33]. Microglia, the resident immune cells of the CNS, play crucial roles in surveillance, phagocytosis, and the regulation of immune responses. Upon activation by inflammatory signals, microglia induce neuroinflammation, which may exacerbate ADHD-related neural dysfunction. The findings of the current study suggest that localized PBMT effectively reduces microglial numbers and inflammation in ADHD model animals. The neuroprotective effects of PBMT were evident in this study. Immunohistochemical analysis revealed that the myelination marker MBP was preserved at levels resembling those observed in normal controls in the PBMT-treated group. These findings suggest that PBMT suppresses neuroinflammation in the CNS of ADHD model animals, thereby preserving neuronal integrity and promoting more typical neural function. PBMT acts by interacting with cellular chromophores, particularly within mitochondria, thereby enhancing mitochondrial function and initiating a range of biological effects, including the following: Increased Adenosine Triphosphate Production: Photons stimulate the synthesis of ATP, which provides energy for critical cellular processes. Reduced Oxidative Stress: PBMT lowers the levels of reactive oxygen species and strengthens antioxidant defenses, thus reducing oxidative damage. Modulated Inflammatory Pathways: PBMT influences cellular signaling pathways, modulating the production of cytokines and chemokines involved in immune regulation. Considering these effects, PBMT holds promise as a therapeutic approach for ADHD. In this study, localized PBMT effectively suppressed neuroinflammation in brain regions implicated in ADHD, preserved neuronal connectivity, and alleviated ADHD symptoms. This study further highlighted that continuous application of PBMT over several days can induce structural changes in the brain. DTI is a valuable tool for visualizing and confirming neural connectivity in the CNS. Although PBMT is traditionally recognized for its relatively immediate effects, the present study demonstrated that continuous application of PBMT for 21 days induced changes observable through DTI, suggesting improvements in structural connectivity. These findings imply that the therapeutic effects of PBMT may be long-lasting. Limitations The generalizability of the current finding regarding PBMT’s effects to primates, particularly humans, remains uncertain. Human skulls are thicker than those of rats, and this may attenuate the intended effects of PBMT. Additionally, the optimal parameters for PBMT application in humans have yet to be fully explored. The practicality of ensuring that children, a primary target group for ADHD treatment, can remain still during daily PBMT sessions remains a consideration that must be addressed. Although this study demonstrated structural improvements through DTI following continuous PBMT, further research is required to determine the long-term durability of these effects. Future Directions Additional research must be conducted to elucidate the broad effects of PBMT on neuroinflammatory processes in ADHD. A deeper understanding of the interactions between neuroinflammation, neurotransmitter systems, and genetic factors would provide more comprehensive insights into ADHD pathogenesis and treatment responsiveness. Integrating PBMT into ADHD management strategies could significantly enhance symptom control and improve patient quality of life. Conclusion This study demonstrated that PBMT alleviates ADHD symptoms through the reduction of CNS inflammation. Moreover, PBMT exerts effects beyond neurotransmitter modulation, inducing structural changes in neuronal connectivity. These findings support the potential of PBMT as a novel therapeutic approach for ADHD. Declarations Data availability statement All data in support of the findings of this paper are available within the article or as supplementary material. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethical Approval The study protocol was approved by the Laboratory Animal Center of Taipei Medical University (No. LAC-2019-0262). Funding This study was supported by the Grant-in-Aid for Scientific Research (108TMUH-NE-05 and 110TMUH-SP-01) from Taipei Medical University Hospital and the Grant-in-Aid for Scientific Research (113-2314-B-038 -119) from the National Science and Technology Council, Taiwan. Acknowledgments We thank Chih-Chuan Li for their valuable contributions in refining the description of PBMT. References Shah A, Banner N, Heginbotham C, Fulford B. 7. American Psychiatric Association (2013) Diagnostic and Statistical Manual of Mental Disorders, 5th edn. American Psychiatric Publishing, Arlington, VA. 8. Bechara, A., Dolan, S. and Hindes, A.(2002) Decision-making and addiction (Part II): myopia for the future or hypersensitivity to reward? Neuropsychologia, 40, 1690–1705. 9. Office of Public Sector Information (2005) The Mental Capacity Act 2005. http://www. Substance Use and Older People 2014; 21(5):9. Barkley RA, Fischer M. Hyperactive child syndrome and estimated life expectancy at young adult follow-up: the role of ADHD persistence and other potential predictors. Journal of Attention Disorders 2019; 23(9):907-923. Peterson BS, Trampush J, Maglione M, Bolshakova M, Rozelle M, Miles J, Pakdaman S, Brown M, Yagyu S, Motala A. Treatments for ADHD in Children and Adolescents: A Systematic Review. Pediatrics 2024; 153(4):e2024065787. Huss M, Duhan P, Gandhi P, Chen C-W, Spannhuth C, Kumar V. Methylphenidate dose optimization for ADHD treatment: review of safety, efficacy, and clinical necessity. Neuropsychiatric disease and treatment 2017:1741-1751. Arnsten AF. Stimulants: therapeutic actions in ADHD. Neuropsychopharmacology 2006; 31(11):2376-2383. Cortese S, Holtmann M, Banaschewski T, Buitelaar J, Coghill D, Danckaerts M, Dittmann RW, Graham J, Taylor E, Sergeant J. Practitioner review: current best practice in the management of adverse events during treatment with ADHD medications in children and adolescents. Journal of Child Psychology and Psychiatry 2013; 54(3):227-246. Harstad EB, Weaver AL, Katusic SK, Colligan RC, Kumar S, Chan E, Voigt RG, Barbaresi WJ. ADHD, stimulant treatment, and growth: a longitudinal study. Pediatrics 2014; 134(4):e935-e944. Arnsten AF, Pliszka SR. Catecholamine influences on prefrontal cortical function: relevance to treatment of attention deficit/hyperactivity disorder and related disorders. Pharmacology Biochemistry and Behavior 2011; 99(2):211-216. Wolraich ML, Hagan JF, Allan C, Chan E, Davison D, Earls M, Evans SW, Flinn SK, Froehlich T, Frost J. Clinical practice guideline for the diagnosis, evaluation, and treatment of attention-deficit/hyperactivity disorder in children and adolescents. Pediatrics 2019; 144(4). Amaroli A, Pasquale C, Zekiy A, Benedicenti S, Marchegiani A, Sabbieti MG, Agas D. Steering the multipotent mesenchymal cells towards an anti-inflammatory and osteogenic bias via photobiomodulation therapy: How to kill two birds with one stone. Journal of Tissue Engineering 2022; 13:20417314221110192. Hamblin MR. Mechanisms and applications of the anti-inflammatory effects of photobiomodulation. AIMS biophysics 2017; 4(3):337-361. Huang Y-Y, Chen AC-H, Carroll JD, Hamblin MR. Biphasic dose response in low level light therapy. Dose-response 2009; 7(4):dose-response. 09-027. Hamblin. Tsai CM, Chang SF, Li CC, Chang H. Transcranial photobiomodulation (808 nm) attenuates pentylenetetrazole-induced seizures by suppressing hippocampal neuroinflammation, astrogliosis, and microgliosis in peripubertal rats. Neurophotonics 2022; 9(1):015006. Sagvolden T, Johansen EB, Wøien G, Walaas SI, Storm-Mathisen J, Bergersen LH, Hvalby O, Jensen V, Aase H, Russell VA, Killeen PR, Dasbanerjee T, Middleton FA, Faraone SV. The spontaneously hypertensive rat model of ADHD--the importance of selecting the appropriate reference strain. Neuropharmacology 2009; 57(7-8):619-626. Kou YT, Liu HT, Hou CY, Lin CY, Tsai CM, Chang H. A transient protective effect of low-level laser irradiation against disuse-induced atrophy of rats. Lasers Med Sci 2019; 34(9):1829-1839. Dhollander T, Connelly A. Generating a T1-like contrast using 3-tissue constrained spherical deconvolution results from single-shell (or multi-shell) diffusion MR data. 2016. Tournier JD, Calamante F, Connelly A. Improved probabilistic streamlines tractography by 2nd order integration over fibre orientation distributions. 2010. John Wiley & Sons, Inc New Jersey, NJ. Paxinos G, Watson C. The rat brain in stereotaxic coordinates: hard cover edition: Elsevier. 2006. Valdés-Hernández PA, Sumiyoshi A, Nonaka H, Haga R, Aubert-Vásquez E, Ogawa T, Iturria-Medina Y, Riera JJ, Kawashima R. An in vivo MRI template set for morphometry, tissue segmentation, and fMRI localization in rats. Frontiers in neuroinformatics 2011; 5:26. Cortese S, Kelly C, Chabernaud C, Proal E, Di Martino A, Milham MP, Castellanos FX. Toward systems neuroscience of ADHD: a meta-analysis of 55 fMRI studies. American Journal of Psychiatry 2012; 169(10):1038-1055. del Campo N, Fryer TD, Hong YT, Smith R, Brichard L, Acosta-Cabronero J, Chamberlain SR, Tait R, Izquierdo D, Regenthal R. A positron emission tomography study of nigro-striatal dopaminergic mechanisms underlying attention: implications for ADHD and its treatment. Brain 2013; 136(11):3252-3270. Saccaro LF, Schilliger Z, Perroud N, Piguet C. Inflammation, Anxiety, and Stress in Attention-Deficit/Hyperactivity Disorder. Biomedicines 2021; 9(10):1313. Ferencova N, Visnovcova Z, Ondrejka I, Hrtanek I, Bujnakova I, Kovacova V, Macejova A, Tonhajzerova I. Peripheral Inflammatory Markers in Autism Spectrum Disorder and Attention Deficit/Hyperactivity Disorder at Adolescent Age. International Journal of Molecular Sciences 2023; 24(14):11710. Wong RSY. Psychopathology of attention deficit/hyperactivity disorder: from an inflammatory perspective. The Egyptian Journal of Neurology, Psychiatry and Neurosurgery 2022; 58(1):123. Leffa DT, Torres IL, Rohde LA. A review on the role of inflammation in attention-deficit/hyperactivity disorder. Neuroimmunomodulation 2019; 25(5-6):328-333. Corona JC. Role of Oxidative Stress and Neuroinflammation in Attention-Deficit/Hyperactivity Disorder. Antioxidants (Basel) 2020; 9(11). Yokokura M, Takebasashi K, Takao A, Nakaizumi K, Yoshikawa E, Futatsubashi M, Suzuki K, Nakamura K, Yamasue H, Ouchi Y. In vivo imaging of dopamine D1 receptor and activated microglia in attention-deficit/hyperactivity disorder: a positron emission tomography study. Molecular psychiatry 2021; 26(9):4958-4967. Yamamoto M, Inada T. Positron emission tomography studies in adult patients with attention-deficit/hyperactivity disorder. Japanese Journal of Radiology 2023; 41(4):382-392. Furlan VA, MacAuslan D, Ha K, Patel N, Adam S, Zanagar B, Venugopal S. Microglial Dyshomeostasis: A Common Substrate in Neurodevelopmental and Neurodegenerative Diseases. Neuroglia 2024; 5(2):119-128. Woodburn SC, Bollinger JL, Wohleb ES. The semantics of microglia activation: neuroinflammation, homeostasis, and stress. Journal of Neuroinflammation 2021; 18(1):258. Tsai SR, Hamblin MR. Biological effects and medical applications of infrared radiation. J Photochem Photobiol B 2017; 170:197-207. Bush G, Valera EM, Seidman LJ. Functional neuroimaging of attention-deficit/hyperactivity disorder: a review and suggested future directions. Biological psychiatry 2005; 57(11):1273-1284. Seidman LJ, Valera EM, Makris N. Structural brain imaging of attention-deficit/hyperactivity disorder. Biological psychiatry 2005; 57(11):1263-1272. Additional Declarations No competing interests reported. Supplementary Files SupplimentWKY0.mp4 Supplementary Video Results of the behavioral evaluation conducted on the SHR (disease model), SHR25 (PBMT-treated), and WKY (normal control) groups. SupplimentSHR0s.mp4 Supplementary Video Results of the behavioral evaluation conducted on the SHR (disease model), SHR25 (PBMT-treated), and WKY (normal control) groups. SupplimentSHR25s.mp4 Supplementary Video Results of the behavioral evaluation conducted on the SHR (disease model), SHR25 (PBMT-treated), and WKY (normal control) groups. Cite Share Download PDF Status: Published Journal Publication published 10 Oct, 2025 Read the published version in Lasers in Medical Science → Version 1 posted Editorial decision: Revision requested 16 Aug, 2025 Reviews received at journal 16 Aug, 2025 Reviewers agreed at journal 14 Aug, 2025 Reviewers agreed at journal 08 Aug, 2025 Reviewers agreed at journal 08 Aug, 2025 Reviews received at journal 25 Jul, 2025 Reviewers agreed at journal 25 Jul, 2025 Reviewers invited by journal 24 Jul, 2025 Editor assigned by journal 24 Jul, 2025 Submission checks completed at journal 08 Jul, 2025 First submitted to journal 27 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6990648","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":490816432,"identity":"7c1bb1c6-4af5-4ef1-8c65-9bf3cbe86d06","order_by":0,"name":"Yu-Jui Huang","email":"","orcid":"","institution":"Taipei Medical University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yu-Jui","middleName":"","lastName":"Huang","suffix":""},{"id":490816433,"identity":"d060e4ed-58ea-4336-9150-2c745cf37c0b","order_by":1,"name":"Yi-Tien Li","email":"","orcid":"","institution":"Taipei Medical University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yi-Tien","middleName":"","lastName":"Li","suffix":""},{"id":490816434,"identity":"db09b68e-5be9-43b4-a651-ff7c7d1eb683","order_by":2,"name":"Min-Lan Tsai","email":"","orcid":"","institution":"Taipei Medical University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Min-Lan","middleName":"","lastName":"Tsai","suffix":""},{"id":490816435,"identity":"c48aec48-7d7a-41c5-bbe9-58e6184df652","order_by":3,"name":"Feng-Chin Lee","email":"","orcid":"","institution":"Taipei Medical University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Feng-Chin","middleName":"","lastName":"Lee","suffix":""},{"id":490816436,"identity":"0968add4-d303-4820-8d8d-c4e68939a2df","order_by":4,"name":"Akira Niwa","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Akira","middleName":"","lastName":"Niwa","suffix":""},{"id":490816437,"identity":"aa807de1-0e90-447a-8ec4-04432edbebad","order_by":5,"name":"Chiung-Hui Yen","email":"","orcid":"","institution":"Taipei Medical University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chiung-Hui","middleName":"","lastName":"Yen","suffix":""},{"id":490816438,"identity":"a239946e-4430-411d-9bed-1545ac30a764","order_by":6,"name":"Cheng-Ying Chu","email":"","orcid":"","institution":"Taipei Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cheng-Ying","middleName":"","lastName":"Chu","suffix":""},{"id":490816439,"identity":"9f6afb15-ab9a-4969-8310-0106bb31e260","order_by":7,"name":"Hsi Chang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACxgYwZZMA5TMTrSUtgYENpoWNOMsOk6CFuf104sMfNefzGOS70yQYKqwTG+R7DPA7rCd3szHPsdvFDGy82yQYzqQnNrDxENDSkLtNmrHhNlAlUAtj22EQYwN+Lf1vt//82XAOquUfMVpm5G5j4G04ANXSQJSWt5uleY4lJ7ax5W62SDiWbtzGlv8BrxbD/tyNH3/U2CX2M5/deONDjbVsP/OxBPxaGqAMcGwkMBARk/KEFIyCUTAKRsEoYAAAOcZD1AoRgV0AAAAASUVORK5CYII=","orcid":"","institution":"Taipei Medical University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Hsi","middleName":"","lastName":"Chang","suffix":""}],"badges":[],"createdAt":"2025-06-27 10:23:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6990648/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6990648/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10103-025-04653-y","type":"published","date":"2025-10-10T15:57:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87830968,"identity":"db037cae-92ae-44f4-aee7-031e6963781d","added_by":"auto","created_at":"2025-07-29 12:24:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":437568,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhotobiomodulation therapy device and experimental protocol. \u003c/strong\u003e(A) Overview of the PBMT device used in this study. (B) Target irradiation site on the rat’s head. (C) Irradiation area, with a beam diameter of 1.8 cm. (D) Illustration of the beam diameter. (E) Preparation of the irradiation site, including shaving of the frontal area prior to treatment. (F) Marking of the target irradiation site. (G) Experimental setup during irradiation treatment. (H) Experimental protocol: DTI was performed on days 0 and 1. PBMT sessions, open field tests, and immunohistochemical (IHC) analyses were conducted on days 7, 14, and 21\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6990648/v1/33222605a39c1ad3fce4009f.png"},{"id":87830970,"identity":"485e3601-3d42-4da8-aefd-e3fe0fe07716","added_by":"auto","created_at":"2025-07-29 12:24:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":412666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePBMT significantly reduced the total track length and average speed in the SHR25 group. \u003c/strong\u003eBehavioral evaluation of PBMT’s effects on ADHD symptoms by using three groups: SHR (disease model), SHR25 (PBMT-treated), and WKY (normal control). (A) Movement tracks demonstrated that the SHR25 group exhibited reduced activity compared with the SHR group, with patterns resembling those observed in the WKY group. (B) An analysis of the total track length (TTL) revealed a significant reduction in the SHR25 group compared with in the SHR group beginning on day 1 post-PBMT treatment (28.65 ± SD vs. 26.51 ± SD, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01), with this effect sustained through day 14 and day 21. (C) An evaluation of the average speed (AS) indicated a significant reduction in the SHR25 group, indicating improved impulsivity control, from day 1 post-PBMT treatment (0.048 ± SD vs. 0.044 ± SD, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01) and persisting through day 21.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6990648/v1/5571c023f5886269dc518cad.png"},{"id":87830965,"identity":"5264ab98-2fb7-49ec-94b9-6579899b9b09","added_by":"auto","created_at":"2025-07-29 12:24:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":315592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePBMT induced improvements in behavioral and microstructural changes in the brain. \u003c/strong\u003eDiffusion tensor imaging (DTI) analysis revealed microstructural changes in ADHD-associated brain regions post-PBMT treatment. (A) DTI data revealed significant improvements in neural tract connectivity in the PBMT-treated group compared with in the untreated SHR group. (B) In the PBMT-treated group, the structural connectivity in ADHD-associated regions (prefrontal cortex, striatum, and hippocampus) more closely resembled that of the normal control (WKY) group than that of the SHR group. These findings suggest that PBMT improved microstructural connectivity, which is consistent with the observed behavioral improvements.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6990648/v1/47335615b76d41b7c740e514.png"},{"id":87830989,"identity":"faa3c151-7b10-48a5-b091-02a53ad1669d","added_by":"auto","created_at":"2025-07-29 12:24:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":971014,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePBMT significantly reduced the number of Iba-1 positive cells, an inflammation marker. \u003c/strong\u003eCellular-level evaluation of neuroinflammation post-PBMT treatment, assessed through Iba-1 staining. (A–D) Quantitative analysis revealed a significant reduction in Iba-1-positive cells, indicative of decreased neuroinflammation, in the PBMT-treated SHR25 group relative to in the untreated SHR group. Reductions in neuroinflammation were observed in ADHD-associated brain regions—the prefrontal cortex: significant reductions were noted at day 7 (±SD), day 14 (±SD), and day 21 (±SD; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05); the striatum: a significant reduction was noted at day 21 (±SD; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and the hippocampus: a significant reduction was noted at day 14 (±SD; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). These findings suggest that PBMT effectively mitigates neuroinflammatory states in ADHD model animals.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6990648/v1/4e4db918cccfe2f3945b0070.png"},{"id":93419636,"identity":"128a145d-9711-44ca-aefe-466ba3b22711","added_by":"auto","created_at":"2025-10-13 16:04:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2587197,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6990648/v1/64d4baf5-9877-4246-9f60-359ed0a0bf5c.pdf"},{"id":87830991,"identity":"700c4989-0e8d-4e42-9cd5-377160d5192b","added_by":"auto","created_at":"2025-07-29 12:24:09","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18436510,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Video\u003c/p\u003e\n\u003cp\u003eResults of the behavioral evaluation conducted on the SHR (disease model), SHR25 (PBMT-treated), and WKY (normal control) groups.\u003c/p\u003e","description":"","filename":"SupplimentWKY0.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6990648/v1/73df4377274dce0acda4a02b.mp4"},{"id":87830984,"identity":"4f1f5dfc-fbe4-48de-825b-593238b66cb6","added_by":"auto","created_at":"2025-07-29 12:24:07","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14941281,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Video\u003c/p\u003e\n\u003cp\u003eResults of the behavioral evaluation conducted on the SHR (disease model), SHR25 (PBMT-treated), and WKY (normal control) groups.\u003c/p\u003e","description":"","filename":"SupplimentSHR0s.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6990648/v1/40c476e386b1aa2c43056b21.mp4"},{"id":87830969,"identity":"e1de76cc-2dc6-434b-accd-7038f4816826","added_by":"auto","created_at":"2025-07-29 12:24:06","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":16425688,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Video\u003c/p\u003e\n\u003cp\u003eResults of the behavioral evaluation conducted on the SHR (disease model), SHR25 (PBMT-treated), and WKY (normal control) groups.\u003c/p\u003e","description":"","filename":"SupplimentSHR25s.mp4","url":"https://assets-eu.researchsquare.com/files/rs-6990648/v1/f34c4fe3f2d889bbf546d65d.mp4"}],"financialInterests":"No competing interests reported.","formattedTitle":"Photobiomodulation as a Therapeutic Approach for Attention-Deficit/Hyperactivity Disorder in Model Rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAttention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder characterized by persistent symptoms of inattention, hyperactivity, and impulsivity that disrupt normal functioning and development [1]. This disorder affects individuals across all stages of life, with symptoms often persisting from childhood through adolescence and into adulthood [2]. Management strategies for ADHD typically involve a combination of behavioral interventions, education, and pharmacological treatments.\u003c/p\u003e\n\u003cp\u003eAdvances in neuroscience, psychology, and pharmacology have improved the understanding of ADHD and its treatment. Contemporary ADHD treatment strategies often include pharmacotherapy and behavioral interventions [3]. For decades, stimulant medications such as methylphenidate have remained central in pharmacological management of ADHD [4]. These stimulants enhance attention, focus, and impulse control by increasing the levels of neurotransmitters, such as dopamine and norepinephrine, in the brain [5]. However, stimulant medications are associated with side effects such as appetite suppression, insomnia, irritability, and, in some cases, cardiovascular complications [6]. Additionally, concerns have been raised regarding their long-term effects on growth and development in children and the potential for their misuse, particularly among adolescents and young adults seeking stimulants for cognitive enhancement or recreational purposes [7].\u003c/p\u003e\n\u003cp\u003eNonstimulant medications, such as atomoxetine, guanfacine, and clonidine, offer alternative treatment options for patients who either cannot tolerate stimulants or do not achieve adequate symptom control by using them. Nonstimulant medications target different neurotransmitter systems, such as those involving norepinephrine and α2-adrenergic receptors, to alleviate ADHD symptoms [8]\u003cstrong\u003e.\u003c/strong\u003e Unlike stimulants, nonstimulants require several weeks of consistent use to produce therapeutic effects, which can present challenges for patients seeking immediate relief from symptoms. Common side effects of nonstimulant medications include sedation and gastrointestinal disturbances. Additionally, one such medication, atomoxetine, has been associated with occasional increases in liver enzyme levels, and therefore, regular monitoring is required during treatment with the drug.\u003c/p\u003e\n\u003cp\u003eIn ADHD treatment, medication is frequently employed to manage impulsivity. However, for preschool-aged children, pharmacological interventions are typically avoided. Clinical guidelines often recommend psychological therapies as the primary approach for this population [9]. In consideration of this, nonpharmacological treatment options, particularly for patients aged \u0026lt;6 years, must be developed that are able to reduce impulsivity in individuals with ADHD.\u003c/p\u003e\n\u003cp\u003ePhotobiomodulation therapy (PBMT), a type of low-level light therapy, holds promise as a treatment for ADHD. ADHD is a neurodevelopmental disorder characterized by persistent symptoms of inattention, hyperactivity, and impulsivity, which substantially impair daily functioning. PBMT involves the use of specific wavelengths of light to stimulate biological processes at the cellular level through modulation of mitochondrial function and oxidative metabolism. Studies have demonstrated the potential of PBMT in modulating neurochemical pathways [10,11]. PBMT may influence neurotransmitter systems, such as those involving dopamine and norepinephrine, which play critical roles in the regulation of attention and impulse control [12]. Additionally, PBMT has been demonstrated to mitigate neuroinflammation and oxidative stress [11,13], both of which have increasingly been recognized as contributors to ADHD pathogenesis.\u003c/p\u003e\n\u003cp\u003eThe current study investigated the potential of PBMT as a treatment for ADHD by using spontaneously hypertensive rat (SHR) animal models. The findings were compelling. PBMT demonstrated efficacy in reducing impulsivity, potentially through the suppression of inflammation in the prefrontal cortex, striatum, and hippocampus, thereby preserving cellular integrity. Furthermore, continuous PBMT exposure over 21 consecutive days of irradiation appeared to sustain neuronal connectivity, suggesting that PBMT may exert long-term beneficial effects on brain structure and function.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eADHD animal model\u003c/p\u003e\n\u003cp\u003eThe use of animals in this study adhered to established animal research guidelines for the reporting of in vivo experiments and the principles outlined in the Basel Declaration, with careful consideration of the 3R principle (Replacement, Reduction, and Refinement). Postnatal day 42 rats were procured from BioLASCO (Taiwan). SHRs, which are widely recognized as a robust animal model for ADHD, were employed in this study. Wistar\u0026ndash;Kyoto rats (WKY) were used as controls [14].\u003c/p\u003e\n\u003cp\u003eTranscranial PBMT\u003c/p\u003e\n\u003cp\u003eThe laser apparatus used in this study was designed by Transverse Industries Co., Ltd. (Taiwan) and was described in detail in another study [15]. Briefly, the device employs a gallium aluminum arsenide (GaAlAs) diode laser with an output wavelength of 808 nm and a power output of 110 mW per laser. The laser operates in continuous mode, with the beam collimated by a lens measuring 11 mm in height. The apparatus comprises laser light sources fitted with single lens hoods. A schematic of the laser structure and lens hood is provided in Fig. 1A and B. The elliptical laser beam emitted at the horizontal plane in front of the lens hood has a major axis of 3.5 mm and a minor axis of 3.0 mm (Fig. 1C), resulting in a beam area of 0.0825 cm\u003csup\u003e2\u003c/sup\u003e (Fig. 1D). Because the rat\u0026rsquo;s scalp was positioned in close contact with the lens hood during PBMT, the calculated power density at the scalp surface was approximately 1.333 W/cm\u003csup\u003e2\u003c/sup\u003e. Each session of PBMT lasted 25 s, with a radiant exposure of approximately 33.3 J/cm\u003csup\u003e2\u003c/sup\u003e and a total radiant energy of 2.75 J delivered per rat. At 15 minutes prior to irradiation, each rat\u0026rsquo;s scalp hair was removed using a depilatory cream, and the scalp was marked for precise alignment. An Eppendorf tube (internal diameter: 12 mm) was used to facilitate the attachment of the front of a lens hood. The prominence of the skull at the Lambda was identified through a tactile assessment and by observing the contour of the skull through the scalp. The center of the Eppendorf tube was aligned with the Lambda (\u0026minus;6 mm from Bregma) and adjusted to the midline of the scalp. After their scalps were marked, the rats\u0026rsquo; bodies were gently wrapped in towels, and the scalp markings were aligned with the inner edge of the lens hood for irradiation (Fig. 1E\u0026ndash;G). The experimental rats were divided into three groups: a normal control group (WKY), an ADHD model group receiving PBMT (SHR25), and an ADHD model group receiving no PBMT intervention (SHR). In the SHR25 group, each rat received a single 25-s session of PBMT. The SHR group underwent sham irradiation with the power supply turned off, whereas the WKY group received no intervention. For all groups, the open field test was conducted to evaluate ADHD-related behavioral symptoms. Additionally, diffusion tensor imaging (DTI) was performed to assess neural connectivity, and immunohistochemistry was performed to analyze tissue-level changes.\u003c/p\u003e\n\u003cp\u003eExperiment protocol\u003c/p\u003e\n\u003cp\u003eThe experimental animals were divided into three groups: WKY (normal control), SHR25 (ADHD model group receiving PBMT), and SHR (ADHD model group not receiving PBMT). The groups were further subdivided on the basis of the number of PBMT treatment days. DTI was conducted on days 0, 1, 7, 14, and 21, and an open field test and immunostaining were conducted on days 7, 14, and 21 (Fig. 1H).\u003c/p\u003e\n\u003cp\u003eOpen field test (behavioral evaluation)\u003c/p\u003e\n\u003cp\u003eAn open field test was employed to evaluate hyperactivity and locomotor activity in the experimental animals. Behavioral evaluations were conducted at 7, 14, and 21 days following PBMT. For the field tests, each rat was placed at the center of a black acrylic box measuring 48 \u0026times; 48 \u0026times; 42 cm\u003csup\u003e3\u003c/sup\u003e. The center point of the rat\u0026rsquo;s body was tracked to record the total track length (in meters), and the average speed of movement (in meters per second) was calculated.\u003c/p\u003e\n\u003cp\u003eMagnetic resonance imaging data acquisition\u003c/p\u003e\n\u003cp\u003eLongitudinal magnetic resonance imaging (MRI) was performed on each animal prior to laser treatment and at day 1, week 2, and week 3 after laser treatment by using a PharmaScan 7T system (Bruker Biospin). During imaging, the animals were anesthetized with approximately 1.2% isoflurane to ensure immobilization and minimize motion artifacts. A stereotaxic headpiece and holder equipped with ear and tooth bars were used to stabilize the head throughout the procedure.\u003c/p\u003e\n\u003cp\u003eDTI was performed using echo planar imaging with a \u003cem\u003eb\u0026nbsp;\u003c/em\u003evalue of 1200 s/mm\u003csup\u003e2\u003c/sup\u003e applied along 30 noncollinear and noncoplanar diffusion directions and 5 \u003cem\u003eb\u003c/em\u003e0 images. The imaging parameters were as follows: repetition time (TR)/echo time (TE) = 3000/37 ms, flip angle = 90\u0026deg;, \u0026delta;/\u0026Delta; = 5/15 ms, acquisition matrix = 128 \u0026times; 128, and resolution = 0.156 \u0026times; 0.156 mm\u003csup\u003e2\u003c/sup\u003e. A total of 16 slices were acquired, with each being 1-mm thick, with two averages. Additionally, T\u003csub\u003e2\u003c/sub\u003e-weighted images were obtained for the coregistration and normalization of DTI data by using the following parameters: TR/TE = 3600/40 ms, flip angle = 90\u0026deg;, voxel size = 0.078 \u0026times; 0.078 \u0026times; 1 mm\u003csup\u003e3\u003c/sup\u003e, matrix = 256 \u0026times; 256 \u0026times; 16, and number of averages = 4.\u003c/p\u003e\n\u003cp\u003eDTI analysis\u003c/p\u003e\n\u003cp\u003eDTI\u0026nbsp;data were preprocessed using FMRIB Software Library (FSL) 5.0.10 (FMRIB, Oxford, UK) and MRtrix 3.0.2 (Brain Research Institute, Melbourne, VIC, Australia). Raw images were preprocessed through denoising, Gibbs ringing artifact removal, and bias field correction, which were performed using MRtrix. Subsequently, the data were processed through the standard FSL pipeline, including brain mask estimation, as well as eddy-current and motion correction. Fixel-based analysis of fiber density was performed using the MRtrix multishell (two unique high \u003cem\u003eb\u003c/em\u003e values: 1000 and 2500 s/mm2) multitissue (three tissue classes: gray matter, white matter, and cerebrospinal fluid) constrained spherical deconvolution method [16] in combination with the probabilistic streamlining method [17]. DTI-derived fractional anisotropy maps were generated from the processed data. These maps were subsequently spatially normalized to the template space by\u0026nbsp;using the MRtrix framework\u0026nbsp;[18,19].\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining was performed following established protocols [13]. Briefly, brain sections were transferred to room temperature (RT) and were permeabilized in acetone precooled to 4\u0026deg;C for 1 min. After undergoing air drying, the sections were blocked with 5% skim milk (Sigma-Aldrich) for 1 h at RT, after which they were rinsed under running tap water. Subsequently, the sections were incubated overnight at 4\u0026deg;C in a humid chamber with a primary antibody (GTX11427, GeneTex, Taiwan) diluted at a ratio of 1:200 in skim milk. The following day, the sections were brought to RT and rinsed with PBST. They were then incubated in the dark for 1 h in a humid chamber with a secondary antibody, donkey antimouse Cy3 (715-165-151, Jackson ImmunoResearch) diluted at a ratio of 1:600 in PBS at 4\u0026deg;C. After incubation, the sections were rinsed again with PBST to remove unbound antibodies.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eSignificant PBMT-Related Reduction in Total Track Length and Average Speed in the SHR25 Group\u003c/p\u003e\n\u003cp\u003eTo evaluate the therapeutic effects of PBMT on ADHD symptoms, we conducted behavioral assessments on the SHR (disease model), SHR25 (PBMT-treated ADHD model), and WKY (normal control) groups. Behavioral activity was assessed through total track length (TTL) and average speed (AS), which were used as indicators of hyperactivity and impulsivity. The movement patterns of the SHR25 group indicated notably less hyperactivity than those of the SHR group did, aligning more closely with the patterns WKY group (Fig. 2A and Supplementary Video). A quantitative analysis revealed that TTL was significantly reduced in the SHR25 group relative to in the SHR group beginning on day 1 after PBMT treatment (Fig. 2B and 2C). The SHR25 group exhibited TTL values of 26.51 \u0026plusmn; SD, whereas those in the SHR group were 28.65 \u0026plusmn; SD (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). This significant reduction persisted at subsequent time points, including days 14 and 21 (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01 for all comparisons). AS measurements corroborated the reduction in hyperactivity and impulsivity in the SHR25 group. A significant decrease in AS was observed in the SHR25 group starting from day 1 following PBMT treatment (0.048 \u0026plusmn; SD in the SHR25 group versus 0.044 \u0026plusmn; SD in the SHR group, \u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.01). This trend continued until day 21.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImprovements in Behavior and Microstructural Changes in the Brain\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to behavioral changes, we investigated microstructural changes in the brain following PBMT. Our analysis focused on the prefrontal cortex, striatum, and hippocampus, regions known to play critical roles in ADHD pathogenesis. DTI was employed to assess neural tract integrity and connectivity in these areas postirradiation, and the results indicated significant differences in microstructural integrity between the SHR25 and SHR groups (Fig. 3A and 3B). Notably, the SHR25 group exhibited structural connectivity patterns that more closely resembled those observed in the WKY group than those in the SHR group.\u003c/p\u003e\n\u003cp\u003eSignificant PBMT-Related Reduction in the Number of Iba-1 Positive Cells, an Inflammation Marker\u003c/p\u003e\n\u003cp\u003eIn addition to the behavioral improvements and structural changes observed in the brain post-PBMT irradiation, we investigated cellular-level changes that occurred in response to PBMT. Specifically, we examined the number of Iba-1 positive cells in each group by using Iba-1 staining, which indicates the inflammatory state (Fig. 4A\u0026ndash;4D). Our results revealed a significant reduction in the number of Iba-1 positive cells in the PBMT-treated SHR25 group relative to in the untreated SHR group. In the prefrontal cortex, Iba-1 positive cell counts were significantly decreased on days 7, 14, and 21 (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). In the striatum, a significant reduction was observed on day 21 (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05), and in the hippocampus, reductions were significant on day 14 (\u003cem\u003eP\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePBMT-Related Preservation of Neuronal Myelination and Integrity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the neuroprotective effects of PBMT, we assessed the number of myelin basic protein (MBP) positive cells in each group (Fig. 4A\u0026ndash;C). The results revealed a significant increase in the number of MBP-positive cells in the PBMT-treated SRH25 group compared with in the untreated SHR group. This phenomenon was observed in the prefrontal cortex, striatum, and hippocampus, with the most pronounced protective effect observed in the prefrontal cortex. Specifically, in the prefrontal cortex, MBP positive cell counts were significantly higher on days 7, 14, and 21 (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the potential of PBMT as a novel treatment for ADHD. This study applied PBMT at a wavelength of 808 nm to the local head region of SHRs, a well-established animal model of ADHD. The PBMT was administered for 25 s daily for 21 consecutive days. The results indicated a significant reduction in impulsivity in the PBMT-treated SHR group. Furthermore, neuroanatomical analysis conducted using DTI revealed that PBMT effectively preserved neuronal connectivity in ADHD-related brain regions, particularly the prefrontal cortex, striatum, and hippocampus. Notably, the observed structural connectivity in these regions closely resembled that of normal rats. Additionally, we examined the mechanism underlying these effects and noted that PBMT reduced neuroinflammatory states in the brain and helped to maintain the integrity of myelin in the prefrontal cortex, striatum, and hippocampus. These findings were confirmed through immunohistochemical analysis.\u003c/p\u003e\n\u003cp\u003eNeuroinflammation refers to a phenomenon in the central nervous system (CNS) wherein immune cells become activated and release inflammatory mediators in response to various stimuli. Traditionally, ADHD has been primarily attributed to dysfunctions in neurotransmitter systems, particularly those involving dopamine and norepinephrine [20,21]. However, emerging research suggests that neuroinflammation may also contribute to the onset and manifestation of ADHD symptoms. Studies have reported elevated levels of proinflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), in the blood and cerebrospinal fluid of patients with ADHD (Ref). These cytokines, which are markers of immune system activation, indicate the presence of inflammatory responses in the brain and their potential involvement in ADHD pathogenesis [22,23]. Research has further suggested a strong association between neuroinflammation in the CNS and ADHD development [24-26]. Positron emission tomography imaging studies have revealed microglial activation, a hallmark of CNS inflammation, in ADHD-associated brain regions such as the prefrontal cortex and striatum [27,28]. Additionally, both animal and human studies have reported increased Iba-1 immunoreactivity indicative of activated microglia, which in turn release proinflammatory cytokines and reactive oxygen species. These mediators contribute to neurotoxicity and impair neural function. These findings collectively imply that microglial activation and the resulting neuroinflammation may contribute to ADHD pathogenesis [29,30]. These findings underscore the potential therapeutic value of targeting neuroinflammation, particularly microglial activation, in the treatment of ADHD.\u003c/p\u003e\n\u003cp\u003ePBMT, in which red or near-infrared light is used to promote tissue repair and reduce inflammation, is increasingly being used in clinical settings. Among the various wavelengths that have been studied, near-infrared light (600–800 nm) has demonstrated notable anti-inflammatory effects [11,31]. In the present study, SHRs exposed to local PBMT at 808 nm exhibited a substantial reduction in the number of Iba-1-positive microglial cells in ADHD-associated brain regions, including the prefrontal cortex, striatum, and hippocampus [32,33]. Microglia, the resident immune cells of the CNS, play crucial roles in surveillance, phagocytosis, and the regulation of immune responses. Upon activation by inflammatory signals, microglia induce neuroinflammation, which may exacerbate ADHD-related neural dysfunction. The findings of the current study suggest that localized PBMT effectively reduces microglial numbers and inflammation in ADHD model animals.\u003c/p\u003e\n\u003cp\u003eThe neuroprotective effects of PBMT were evident in this study. Immunohistochemical analysis revealed that the myelination marker MBP was preserved at levels resembling those observed in normal controls in the PBMT-treated group. These findings suggest that PBMT suppresses neuroinflammation in the CNS of ADHD model animals, thereby preserving neuronal integrity and promoting more typical neural function.\u003c/p\u003e\n\u003cp\u003ePBMT acts by interacting with cellular chromophores, particularly within mitochondria, thereby enhancing mitochondrial function and initiating a range of biological effects, including the following:\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eIncreased Adenosine Triphosphate Production: Photons stimulate the synthesis of ATP, which provides energy for critical cellular processes.\u003c/li\u003e\n \u003cli\u003eReduced Oxidative Stress: PBMT lowers the levels of reactive oxygen species and strengthens antioxidant defenses, thus reducing oxidative damage.\u003c/li\u003e\n \u003cli\u003eModulated Inflammatory Pathways: PBMT influences cellular signaling pathways, modulating the production of cytokines and chemokines involved in immune regulation.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eConsidering these effects, PBMT holds promise as a therapeutic approach for ADHD. In this study, localized PBMT effectively suppressed neuroinflammation in brain regions implicated in ADHD, preserved neuronal connectivity, and alleviated ADHD symptoms.\u003c/p\u003e\n\u003cp\u003eThis study further highlighted that continuous application of PBMT over several days can induce structural changes in the brain. DTI is a valuable tool for visualizing and confirming neural connectivity in the CNS. Although PBMT is traditionally recognized for its relatively immediate effects, the present study demonstrated that continuous application of PBMT for 21 days induced changes observable through DTI, suggesting improvements in structural connectivity. These findings imply that the therapeutic effects of PBMT may be long-lasting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations\u003c/strong\u003e\u003c/p\u003e\n\u003col start=\"1\" type=\"1\"\u003e\n \u003cli\u003eThe generalizability of the current finding regarding PBMT’s effects to primates, particularly humans, remains uncertain. Human skulls are thicker than those of rats, and this may attenuate the intended effects of PBMT. Additionally, the optimal parameters for PBMT application in humans have yet to be fully explored.\u003c/li\u003e\n \u003cli\u003eThe practicality of ensuring that children, a primary target group for ADHD treatment, can remain still during daily PBMT sessions remains a consideration that must be addressed.\u003c/li\u003e\n \u003cli\u003eAlthough this study demonstrated structural improvements through DTI following continuous PBMT, further research is required to determine the long-term durability of these effects.\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cstrong\u003eFuture Directions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAdditional research must be conducted to elucidate the broad effects of PBMT on neuroinflammatory processes in ADHD. A deeper understanding of the interactions between neuroinflammation, neurotransmitter systems, and genetic factors would provide more comprehensive insights into ADHD pathogenesis and treatment responsiveness. Integrating PBMT into ADHD management strategies could significantly enhance symptom control and improve patient quality of life.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrated that PBMT alleviates ADHD symptoms through the reduction of CNS inflammation. Moreover, PBMT exerts effects beyond neurotransmitter modulation, inducing structural changes in neuronal connectivity. These findings support the potential of PBMT as a novel therapeutic approach for ADHD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data in support of the findings of this paper are available within the article or as supplementary material.\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthical Approval\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the Laboratory Animal Center of Taipei Medical University (No. LAC-2019-0262).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Grant-in-Aid for Scientific Research (108TMUH-NE-05 and 110TMUH-SP-01) from Taipei Medical University Hospital and the Grant-in-Aid for Scientific Research (113-2314-B-038 -119) from the National Science and Technology Council, Taiwan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Chih-Chuan Li for their valuable contributions in refining the description of PBMT.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eShah A, Banner N, Heginbotham C, Fulford B. 7. American Psychiatric Association (2013) Diagnostic and Statistical Manual of Mental Disorders, 5th edn. American Psychiatric Publishing, Arlington, VA. 8. Bechara, A., Dolan, S. and Hindes, A.(2002) Decision-making and addiction (Part II): myopia for the future or hypersensitivity to reward? Neuropsychologia, 40, 1690\u0026ndash;1705. 9. Office of Public Sector Information (2005) The Mental Capacity Act 2005. http://www. Substance Use and Older People 2014; 21(5):9.\u003c/li\u003e\n \u003cli\u003eBarkley RA, Fischer M. Hyperactive child syndrome and estimated life expectancy at young adult follow-up: the role of ADHD persistence and other potential predictors. Journal of Attention Disorders 2019; 23(9):907-923.\u003c/li\u003e\n \u003cli\u003ePeterson BS, Trampush J, Maglione M, Bolshakova M, Rozelle M, Miles J, Pakdaman S, Brown M, Yagyu S, Motala A. Treatments for ADHD in Children and Adolescents: A Systematic Review. Pediatrics 2024; 153(4):e2024065787.\u003c/li\u003e\n \u003cli\u003eHuss M, Duhan P, Gandhi P, Chen C-W, Spannhuth C, Kumar V. Methylphenidate dose optimization for ADHD treatment: review of safety, efficacy, and clinical necessity. Neuropsychiatric disease and treatment 2017:1741-1751.\u003c/li\u003e\n \u003cli\u003eArnsten AF. Stimulants: therapeutic actions in ADHD. Neuropsychopharmacology 2006; 31(11):2376-2383.\u003c/li\u003e\n \u003cli\u003eCortese S, Holtmann M, Banaschewski T, Buitelaar J, Coghill D, Danckaerts M, Dittmann RW, Graham J, Taylor E, Sergeant J. Practitioner review: current best practice in the management of adverse events during treatment with ADHD medications in children and adolescents. Journal of Child Psychology and Psychiatry 2013; 54(3):227-246.\u003c/li\u003e\n \u003cli\u003eHarstad EB, Weaver AL, Katusic SK, Colligan RC, Kumar S, Chan E, Voigt RG, Barbaresi WJ. ADHD, stimulant treatment, and growth: a longitudinal study. Pediatrics 2014; 134(4):e935-e944.\u003c/li\u003e\n \u003cli\u003eArnsten AF, Pliszka SR. Catecholamine influences on prefrontal cortical function: relevance to treatment of attention deficit/hyperactivity disorder and related disorders. Pharmacology Biochemistry and Behavior 2011; 99(2):211-216.\u003c/li\u003e\n \u003cli\u003eWolraich ML, Hagan JF, Allan C, Chan E, Davison D, Earls M, Evans SW, Flinn SK, Froehlich T, Frost J. Clinical practice guideline for the diagnosis, evaluation, and treatment of attention-deficit/hyperactivity disorder in children and adolescents. Pediatrics 2019; 144(4).\u003c/li\u003e\n \u003cli\u003eAmaroli A, Pasquale C, Zekiy A, Benedicenti S, Marchegiani A, Sabbieti MG, Agas D. Steering the multipotent mesenchymal cells towards an anti-inflammatory and osteogenic bias via photobiomodulation therapy: How to kill two birds with one stone. Journal of Tissue Engineering 2022; 13:20417314221110192.\u003c/li\u003e\n \u003cli\u003eHamblin MR. Mechanisms and applications of the anti-inflammatory effects of photobiomodulation. AIMS biophysics 2017; 4(3):337-361.\u003c/li\u003e\n \u003cli\u003eHuang Y-Y, Chen AC-H, Carroll JD, Hamblin MR. Biphasic dose response in low level light therapy. Dose-response 2009; 7(4):dose-response. 09-027. Hamblin.\u003c/li\u003e\n \u003cli\u003eTsai CM, Chang SF, Li CC, Chang H. Transcranial photobiomodulation (808 nm) attenuates pentylenetetrazole-induced seizures by suppressing hippocampal neuroinflammation, astrogliosis, and microgliosis in peripubertal rats. Neurophotonics 2022; 9(1):015006.\u003c/li\u003e\n \u003cli\u003eSagvolden T, Johansen EB, W\u0026oslash;ien G, Walaas SI, Storm-Mathisen J, Bergersen LH, Hvalby O, Jensen V, Aase H, Russell VA, Killeen PR, Dasbanerjee T, Middleton FA, Faraone SV. The spontaneously hypertensive rat model of ADHD--the importance of selecting the appropriate reference strain. Neuropharmacology 2009; 57(7-8):619-626.\u003c/li\u003e\n \u003cli\u003eKou YT, Liu HT, Hou CY, Lin CY, Tsai CM, Chang H. A transient protective effect of low-level laser irradiation against disuse-induced atrophy of rats. Lasers Med Sci 2019; 34(9):1829-1839.\u003c/li\u003e\n \u003cli\u003eDhollander T, Connelly A. Generating a T1-like contrast using 3-tissue constrained spherical deconvolution results from single-shell (or multi-shell) diffusion MR data. 2016.\u003c/li\u003e\n \u003cli\u003eTournier JD, Calamante F, Connelly A. Improved probabilistic streamlines tractography by 2nd order integration over fibre orientation distributions. 2010. John Wiley \u0026amp; Sons, Inc New Jersey, NJ.\u003c/li\u003e\n \u003cli\u003ePaxinos G, Watson C. The rat brain in stereotaxic coordinates: hard cover edition: Elsevier. 2006.\u003c/li\u003e\n \u003cli\u003eVald\u0026eacute;s-Hern\u0026aacute;ndez PA, Sumiyoshi A, Nonaka H, Haga R, Aubert-V\u0026aacute;squez E, Ogawa T, Iturria-Medina Y, Riera JJ, Kawashima R. An in vivo MRI template set for morphometry, tissue segmentation, and fMRI localization in rats. Frontiers in neuroinformatics 2011; 5:26.\u003c/li\u003e\n \u003cli\u003eCortese S, Kelly C, Chabernaud C, Proal E, Di Martino A, Milham MP, Castellanos FX. Toward systems neuroscience of ADHD: a meta-analysis of 55 fMRI studies. American Journal of Psychiatry 2012; 169(10):1038-1055.\u003c/li\u003e\n \u003cli\u003edel Campo N, Fryer TD, Hong YT, Smith R, Brichard L, Acosta-Cabronero J, Chamberlain SR, Tait R, Izquierdo D, Regenthal R. A positron emission tomography study of nigro-striatal dopaminergic mechanisms underlying attention: implications for ADHD and its treatment. Brain 2013; 136(11):3252-3270.\u003c/li\u003e\n \u003cli\u003eSaccaro LF, Schilliger Z, Perroud N, Piguet C. Inflammation, Anxiety, and Stress in Attention-Deficit/Hyperactivity Disorder. Biomedicines 2021; 9(10):1313.\u003c/li\u003e\n \u003cli\u003eFerencova N, Visnovcova Z, Ondrejka I, Hrtanek I, Bujnakova I, Kovacova V, Macejova A, Tonhajzerova I. Peripheral Inflammatory Markers in Autism Spectrum Disorder and Attention Deficit/Hyperactivity Disorder at Adolescent Age. International Journal of Molecular Sciences 2023; 24(14):11710.\u003c/li\u003e\n \u003cli\u003eWong RSY. Psychopathology of attention deficit/hyperactivity disorder: from an inflammatory perspective. The Egyptian Journal of Neurology, Psychiatry and Neurosurgery 2022; 58(1):123.\u003c/li\u003e\n \u003cli\u003eLeffa DT, Torres IL, Rohde LA. A review on the role of inflammation in attention-deficit/hyperactivity disorder. Neuroimmunomodulation 2019; 25(5-6):328-333.\u003c/li\u003e\n \u003cli\u003eCorona JC. Role of Oxidative Stress and Neuroinflammation in Attention-Deficit/Hyperactivity Disorder. Antioxidants (Basel) 2020; 9(11).\u003c/li\u003e\n \u003cli\u003eYokokura M, Takebasashi K, Takao A, Nakaizumi K, Yoshikawa E, Futatsubashi M, Suzuki K, Nakamura K, Yamasue H, Ouchi Y. In vivo imaging of dopamine D1 receptor and activated microglia in attention-deficit/hyperactivity disorder: a positron emission tomography study. Molecular psychiatry 2021; 26(9):4958-4967.\u003c/li\u003e\n \u003cli\u003eYamamoto M, Inada T. Positron emission tomography studies in adult patients with attention-deficit/hyperactivity disorder. Japanese Journal of Radiology 2023; 41(4):382-392.\u003c/li\u003e\n \u003cli\u003eFurlan VA, MacAuslan D, Ha K, Patel N, Adam S, Zanagar B, Venugopal S. Microglial Dyshomeostasis: A Common Substrate in Neurodevelopmental and Neurodegenerative Diseases. Neuroglia 2024; 5(2):119-128.\u003c/li\u003e\n \u003cli\u003eWoodburn SC, Bollinger JL, Wohleb ES. The semantics of microglia activation: neuroinflammation, homeostasis, and stress. Journal of Neuroinflammation 2021; 18(1):258.\u003c/li\u003e\n \u003cli\u003eTsai SR, Hamblin MR. Biological effects and medical applications of infrared radiation. J Photochem Photobiol B 2017; 170:197-207.\u003c/li\u003e\n \u003cli\u003eBush G, Valera EM, Seidman LJ. Functional neuroimaging of attention-deficit/hyperactivity disorder: a review and suggested future directions. Biological psychiatry 2005; 57(11):1273-1284.\u003c/li\u003e\n \u003cli\u003eSeidman LJ, Valera EM, Makris N. Structural brain imaging of attention-deficit/hyperactivity disorder. Biological psychiatry 2005; 57(11):1263-1272.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"lasers-in-medical-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"lims","sideBox":"Learn more about [Lasers in Medical Science](https://link.springer.com/journal/10103)","snPcode":"10103","submissionUrl":"https://submission.springernature.com/new-submission/10103/3","title":"Lasers in Medical Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Photobiomodulation therapy (PBMT), Attention-deficit/hyperactivity disorder (ADHD), Neuroinflammation, Neuronal connectivity","lastPublishedDoi":"10.21203/rs.3.rs-6990648/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6990648/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eSignificance:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eCurrent treatment options for Attention-deficit/hyperactivity disorder (ADHD) primarily involve pharmacological and behavioral interventions, but concerns about side effects, long-term safety, and limited efficacy in certain populations necessitate alternative therapies. This study investigates the therapeutic potential of Photobiomodulation therapy (PBMT) in an ADHD animal model, contributing to the growing field of nonpharmacological interventions. The study aims to evaluate the effects of PBMT on ADHD symptoms using a spontaneously hypertensive rat (SHR) model. Specifically, it examines whether PBMT can reduce impulsivity and hyperactivity by modulating neuroinflammation, preserving neuronal integrity, and enhancing structural brain connectivity.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e\u003c/em\u003e SHRs, a well-established ADHD animal model, were used to assess PBMT’s effects. The experimental design included three groups: a normal control group (WKY), an ADHD model group receiving PBMT (SHR25), and an ADHD model group without PBMT intervention (SHR). PBMT was administered daily for 25 seconds at a wavelength of 808 nm over 21 consecutive days. Behavioral assessments (open field test), diffusion tensor imaging (DTI), and immunohistochemical analysis were conducted to evaluate neuroinflammation, neuronal integrity, and myelination.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003ePBMT significantly reduced impulsivity and hyperactivity in SHRs, as indicated by decreased total track length and average speed in the open field test. DTI analysis revealed improved neural connectivity in ADHD-associated brain regions, particularly the prefrontal cortex, striatum, and hippocampus. Immunohistochemical staining demonstrated a significant reduction in Iba-1 positive cells, indicating decreased neuroinflammation. Additionally, PBMT preserved myelin integrity in ADHD-related brain regions, suggesting neuroprotective effects.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003eThe findings suggest that PBMT is an effective nonpharmacological intervention for ADHD, reducing impulsivity and hyperactivity while preserving neuronal structure and function. By mitigating neuroinflammation and improving neuronal connectivity, PBMT presents a promising therapeutic alternative to conventional ADHD treatments. Further studies are needed to explore its clinical applicability and long-term safety in human populations.\u003c/p\u003e","manuscriptTitle":"Photobiomodulation as a Therapeutic Approach for Attention-Deficit/Hyperactivity Disorder in Model Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-29 12:23:43","doi":"10.21203/rs.3.rs-6990648/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-17T00:19:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-16T19:23:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223086583408504194391811570876535742646","date":"2025-08-14T09:10:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"244487534527735978391503766332296502709","date":"2025-08-08T16:34:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27794216443600343819796487026352797257","date":"2025-08-08T15:43:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-25T11:00:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"193015833132417540380957594230895425966","date":"2025-07-25T06:46:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-24T21:40:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-24T15:23:53+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-08T13:46:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Lasers in Medical Science","date":"2025-06-27T10:15:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"lasers-in-medical-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"lims","sideBox":"Learn more about [Lasers in Medical Science](https://link.springer.com/journal/10103)","snPcode":"10103","submissionUrl":"https://submission.springernature.com/new-submission/10103/3","title":"Lasers in Medical Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3328d596-d3bc-455f-aec1-10da44eff557","owner":[],"postedDate":"July 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-13T15:59:41+00:00","versionOfRecord":{"articleIdentity":"rs-6990648","link":"https://doi.org/10.1007/s10103-025-04653-y","journal":{"identity":"lasers-in-medical-science","isVorOnly":false,"title":"Lasers in Medical Science"},"publishedOn":"2025-10-10 15:57:12","publishedOnDateReadable":"October 10th, 2025"},"versionCreatedAt":"2025-07-29 12:23:43","video":"","vorDoi":"10.1007/s10103-025-04653-y","vorDoiUrl":"https://doi.org/10.1007/s10103-025-04653-y","workflowStages":[]},"version":"v1","identity":"rs-6990648","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6990648","identity":"rs-6990648","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}