Photobiomodulation Improves the Inflammatory Response and Intracellular Signaling Proteins Linked To Vascular Function and Cell Survival in the Brain of Aged Rats

Photobiomodulation Improves the Inflammatory Response and Intracellular Signaling Proteins Linked To Vascular Function and Cell Survival in the Brain of Aged 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 Improves the Inflammatory Response and Intracellular Signaling Proteins Linked To Vascular Function and Cell Survival in the Brain of Aged Rats Fabrizio Cardoso, Fernanda Mansur, Bruno Araújo, Francisco Gonzalez-Lima, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-293139/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Photobiomodulation is a non-pharmacological tool widely used to reduce inflammation in many tissues. However, little is known about its effects on the inflammatory response in the aged brain. We conducted the study to examine anti-inflammatory effects of photobiomodulation in aging brains. We used aged rats (20 months old) with control (handled, laser off) or transcranial laser (660 nm wavelength, 100 mW power) treatments for 10 consecutive days and evaluated the level of inflammatory cytokines and chemokines, and the expression and activation of intracellular signaling proteins in the cerebral cortex and the hippocampus. Inflammatory analysis showed that aged rats submitted to transcranial laser treatment had increased levels of IL-1alpha and decreased levels of IL-5 in the cerebral cortex. In the hippocampus, the laser treatment increased the levels of IL-1alpha and decreased levels of IL-5, IL-18 and fractalkine. Regarding the intracellular signaling proteins, a reduction in the ERK and p38 expression and an increase in the STAT3 and ERK activation were observed in the cerebral cortex of aged rats from the laser group. In addition, the laser treatment increased the hippocampal expression of p70S6K, STAT3 and p38 of aged rats. Taken together, our data indicate that transcranial photobiomodulation can improve the inflammatory response and the activation of intracellular signaling proteins linked to vascular function and cell survival in the aged brain. Neurobiology of Disease laser photobiomodulation brain aging inflammation intracellular signaling proteins Figures Figure 1 Figure 2 Figure 3 Figure 4 Highlights (1) Photobiomodulation improves the inflammatory response in the cortex and hippocampus of aged rats (2) Photobiomodulation activates intracellular signaling proteins linked to vascular function and cell survival in the brain of aged rats 1. Introduction Photobiomodulation (PBM), also called low-level laser therapy (Anders et al., 2015 ) has emerged as a non-pharmacological, non-invasive tool capable of stimulating wound healing, reducing pain and inflammation in several diseases (Chung et al., 2012 ). Many human studies have also used transcranial PBM to improve brain functions in several conditions (e.g., Schiffer et al., 2009 ; Muili et al., 2012 ; Muili et al., 2013 ; Barrett and Gonzalez-Lima, 2013 ; Tian et al., 2016 ; Disner et al., 2016 ; Hwang et al., 2016 ; Eells et al., 2016 ; Blanco et al., 2017a ; Blanco et al., 2017b ; Wang et al., 2017 ; Saltmarche et al., 2017 ; Holmes et al., 2019 ). For example, Saltmarche and collaborators (2017) submitted elderly people diagnosed with dementia to PBM and observed an improvement in executive function and a significant improvement in sleep and mood. Many studies using animal models have also shown interesting results of PBM on the brain (e.g., Rojas et al., 2008 ; Rojas and Gonzalez-Lima, 2011 ; El Massri et al., 2017 ; Lu et al., 2017 ). For instance, Lu and collaborators (2017) injected beta amyloid (Aβ) in the hippocampus of rats that were treated with laser PBM for five days. They noted that laser treatment restored spatial memory and object recognition memory. In addition, they observed an increase in the antioxidant capacity of hippocampal CA1 neurons and a decrease of Aβ-induced reactive gliosis and inflammation. Recently, some authors have shown promising results of PBM in the aged brain (Salgado et al., 2015 ; Salehpour et al., 2017 ; Vargas et al., 2017 ; Salehpour et al., 2018 ; O’Donnell et al., 2021 ; Saucedo et al., 2021 ). However, there is little evidence to explain such effects, other than improved mitochondrial respiration and vascular function. It is known that brain aging is characterized by local inflammation with glial cells releasing increasing amount of pro-inflammatory cytokines such as IL-1beta, IL-6 and TNF-alpha (Cribbs et al., 2012 ; Norden and Godbout, 2013 ). This process is directly involved with cellular dysfunctions characteristic of aging and Alzheimer's disease (Stephan et al., 2013 ; Hong et al., 2016 ), which may result in an increase in the activation of signaling pathways linked to inflammation and cellular death such as c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) (O'Donnell et al., 2000 ). Based on the well-documented anti-inflammatory effects of PBM in other tissues (Almeida et al., 2013 ; Haslerud et al., 2017 ; Tomazoni et al., 2017 : Naterstad et al., 2018 ), we evaluated whether a transcranial treatment with a laser diode of 660 nm wavelength and 100 mW power can modulate the inflammatory response and expression and activation of intracellular signaling proteins in the cortex and hippocampus of aged rats. 2. Methods 2.1. Animals Twenty-month-old male Wistar rats (n = 10) were used in this study. The colony room was maintained at 21 ± 2°C with a 12 h light/ dark schedule (light: 7 am until 7 pm), and food and water were provided ad libitum throughout the experimental period. All experimental protocols were approved by the ethics committee of the Universidade de Mogi das Cruzes (UMC) (#003/2020) and all efforts were made to minimize animal suffering in accordance with the proposals of the International Ethical Guideline for Biomedical Research (CIOMS 1985). 2.2. Laser and control protocols The aged rats were randomly distributed into two groups: laser (n = 5) and control (n = 5). The animals of the laser group were manually immobilized and received the treatment with a laser diode of 660 nm wavelength and 100 mW power for 30 s at each of 5 irradiation points on the head, totalizing 15 Joules of Energy, 150 s of irradiation and fluence of 535.7 J/cm 2 , for 10 consecutive days. These laser parameters were chosen based on our previous publications, showing that these parameters had anti-inflammatory effects in other tissues (Table 1 ) (Almeida et al., 2013 ; Haslerud et al., 2017 ; Tomazoni et al., 2017 ; Naterstad et al., 2018 ). The target coordinates on the scalp were: point 1 = AP + 4.20 mm and ML 0.00 mm; point 2 = AP -3.00 mm and ML -6.60 mm; point 3 = AP -3.00 mm and ML + 6.60 mm; point 4 = AP 0.00 mm and ML 0.00 mm; point 5 = AP -5.52 mm and ML 0.00 mm), as in our previous metabolomics study in the rat (Cardoso et al., 2021 ). The animals of the control group were handled the same way, except that the laser was not turned on. Table 1 Laser parameters used in the present study. Parameter (unit) Measurement method or value information source Center wavelength (nm) 660 Operating mode Continuous wave Average radiant power (W) 0.1 Aperture diameter (cm) 0.6 Irradiance at aperture (mW/cm 2 ) 3.33 Beam divergence (degree) Near zero Beam shape Circular Beam spot size (cm 2 ) 0.03 Exposure duration/point (s) 30 Radiant exposure (J/cm 2 ) per session 500.0 Number of points irradiated 5 Delivery mode Contact mode Number and frequency of sessions One session/day for 10 consecutive days Total radiant energy (J) per head 15 2.4. Tissue preparation One hour after the final laser or control session, aged rats from the laser (n = 5) and control (n = 5) groups were euthanized by decapitation and their cerebral cortex and hippocampus were immediately collected and frozen. The whole cerebral cortex and the hippocampus were homogenized in ice-cold RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS) with freshly added protease (Cat# M222–1ml; Lot# 1295C056; Amresco) and phosphatase (Cat# B15001-A and B; Lot# 510011; Biotool) inhibitors. Homogenates were centrifuged at 10,000 x g for 10 minutes at 4 o C and supernatants were collected for cytokine/chemokine quantification. 2.5. Methods for the protein detection and analysis Milliplex® MAP rat cytokine/chemokine magnetic bead panel assay (RECYMAG65K) was used to quantify the levels G-CSF, eotaxin, GM-CSF, IL-1alpha, leptin, MIP-1alpha, IL-4, IL-1beta, IL-2, IL-6, IL-13, IL-10, IL-5, IL-17alpha, IL-18, MCP-1, IP-10, VEGF, fractalkine, MIP-2, TNF-alpha, and RANTES in the brain samples of the studied groups. Milliplex® MAP Kits 48-681MAG and 48-680MAG were used to evaluate the expression and brain activation of signaling proteins Akt, p70S6K, STAT3, STAT5, ERK, JNK, NF-kB and p38. The plates were run on a Luminex ™ Magpix ™ instrument and results were analyzed with the Milliplex Analyst 5.1 Software using a Logistic 5P Weighted regression formula to calculate sample concentrations from the standard curves. 2.6. Statistical analyses Statistical procedures were conducted using the Mann-Whitney U Test that allows comparison of non-parametric data. All analyses were performed using the Statistical Package for the Social Science (SPSS Inc, IBM, version 221.0, Chicago, IL, USA). A statistical difference was considered significant when the P -value was lower than 0.05. All plots were acquired using the Graph Pad Prism (6.0). 3. Results 3.1. Cortical and hippocampal levels of cytokines and chemokines To evaluate whether the PBM had anti-inflammatory effects on the aged brain, we quantified the cortical and hippocampal levels of several chemokines and cytokines in aged rats submitted to 10 consecutive days of laser treatment or control treatment. The detailed results of Mann-Whitney tests are presented in Supplementary Tables 1 and 2. The laser treatment increased the cortical level of IL-1alpha (p = 0.008) and reduced the IL-5 level (p = 0.046) (Fig. 1 ). In the hippocampus, an increase in the IL-1alpha (p = 0.035) level was observed in the laser group. However, the laser treatment was able to reduce the levels of IL5 (p = 0.027), IL-18 (p = 0.049) and fractalkine (p = 0.037) in aged rats (Fig. 2 ). Taken together, these data showed that PBM changed the levels of neuroinflammatory markers in aged rats. 3.2. Cortical and hippocampal expression and activation of signaling proteins We investigated the cortical and hippocampal expression and activation of signaling proteins in aged rats submitted to laser treatment vs. control treatment. The detailed results of Mann-Whitney tests are presented in Supplementary Tables 3 and 4. The laser treatment decreased the cortical expression of ERK (p = 0.028) and p38 (p = 0.009). Interestingly, the laser treatment was able to increase the cortical activation of ERK (p = 0.014) and STAT3 (p = 0.0016) (Fig. 3 ). In the hippocampus, the laser treatment increased the expression of p70S6K (p = 0.016) and STAT5 (p = 0.050), and decreased the expression of p38 (p = 0.028) in aged rats (Fig. 4 ). 4. Discussion The aim of our study was to investigate levels of pro- and anti-inflammatory cytokines and chemokines and the expression and activation of signaling proteins in the brain of aged rats submitted to repeated treatment with a laser diode of 660 nm wavelength and 100 mW power. Our results indicate that transcranial PBM was able to modulate the expression and activation of signaling proteins and the inflammatory profile in the brain of aged rats. 4.1. Anti-inflammatory effects of PBM on the aged brain The laser treatment increased the levels of IL-1alpha and decreased the levels of IL-5 in both the cortex and hippocampus of aged rats. Interestingly, IL-1alpha has been shown to promote angiogenesis and to increase proliferation and migration of endothelial cells (Saini and Bix, 2012 ; Salmeron et al., 2016 ). The IL-1alpha findings are consistent with improving vascular function and oxygenation of the aged brain (Saucedo et al., 2021 ), which may help explain the facilitation of neurocognitive functions shown by transcranial PBM studies in older humans (Saltmarche et al., 2017 ; Vargas et al., 2017 ). In contrast, a reduction of IL-5 may be beneficial to the aged brain because IL-5 induces proliferation and activation of microglia, which is characteristic of inflammatory reactions (Liva and de Vellis, 2001 ). Moreover, PBM promoted an anti-inflammatory effect by reducing the hippocampal levels of IL-18 and fractalkine. IL-18 is a pro-inflammatory cytokine that inhibits cell differentiation and reduces neurogenesis and induces neuronal death in cultured neural progenitors (Liu et al., 2005 ; Zhu et al., 2009 ). Further evidence suggests that IL-18 can activate the p38 signaling pathway (Arend et al., 2008 ). Also, high levels of this cytokine are observed during aging and in neurodegenerative diseases (Bossù et al., 2010 ; Bellaver et al., 2017 ). Fractalkine, in turn, is a chemokine regulated by pro-inflammatory cytokines such as TNFalpha and IL-1beta, which is involved in the communication between neurons and microglia (Desforges et al., 2012 ). High levels of fractalkine were observed in the cortex and hippocampus of a rat model of Alzheimer’s disease (Hanzel et al., 2014 ). In this sense, reducing the levels of both IL-18 and fractalkine by laser treatment may contribute to improving the inflammatory response in the aging brain and in aging-related neurodegenerative diseases. 4.2. Effects of PBM on signaling proteins in the aged brain PBM reduced the expression of ERK and p38 and increased the activation of ERK and STAT3 in the cortex of aged rats. In the hippocampus, PBM increased the expression of p70S6K and STAT5 and decreased the expression of p38. ERK activation is associated with proliferation, differentiation and cellular migration, having an effect opposite to p38, which is activated by cellular stresses such as genotoxic, osmotic, hypoxic, or oxidative stress and it is connected to apoptosis and inflammatory responses (Kim and Choi, 2010 ; Zhang et al., 2016 ). STAT3 modulates the expression of genes responsible for important physiological functions such as cell regulation and apoptosis control (Desrivières et al., 2006 ; Kim et al., 2010 ). STAT5 is linked to neuronal survival (Um and Lodish, 2006 ; Zhang et al., 2007 ). Also, STAT5 is necessary for the neuroprotective and neurotrophic effects of growth hormone on hippocampal neurons (Byts et al., 2008 ). However, STAT3 and STAT5 brain levels are decreased during aging (Bazhanova and Anisimov, 2016 ). Nevertheless, laser treatment increased STAT3 activation and the expression of STAT5 in the cortex of aged rats. These observed changes in intracellular signaling proteins may be linked to the anti-inflammatory effects of PBM in the cortex and hippocampus of aged rats. High levels of neuroinflammatory markers are observed during aging (Lee et al., 2000 ; Godbout et al., 2005 ), indicating a pro- and anti-inflammatory imbalance (Godbout and Johnson, 2009 ). In particular, it is possible that PBM of the neuroinflammatory response is related to increases in the activation of intracellular signaling proteins STAT3 and ERK in the cortical region. In this sense, STAT3 is a transcription factor that interacts with polypeptide receptors in the cell membrane, mediating extracellular signals such as growth factors and cytokines (Levy and Darnell, 2002 ). When activated by tyrosine phosphorylation, STAT3 dimerizes and translocate to the nucleus, thus activating the target genes (You, et al., 2015 ). The activation of STAT3 suppresses the expression of pro-inflammatory mediators, promoting an immune evasion and blocking the production and detection of inflammatory signals by several components of the immune system (Wang et al., 2004 ). ERK can be activated by growth factors, such as the epidermal growth factor (EGF) that is associated with the attenuation of pro-inflammatory mediators (García-Ojalvo et al., 2019 ), activating Ras, which recruits Raf for the membrane. Raf activates Mek, which in turn activates ERK. ERK activation triggers cell proliferation, differentiation and cell migration (Kim and Choi, 2010 ). It is possible that the neuroinflammatory response in the cortex may trigger the activation of the ERK signaling pathways which in turn activates STAT3 (David et al., 1995 ). In this context, it is possible that the increased ERK activation is linked to the anti-inflammatory effect of laser treatment. 4.3. Limitations We did not evaluate functional parameters, such as cognitive functions, since our goal was to investigate whether repeated laser treatment alters the cortical and hippocampal inflammatory and signaling profiles in aged rats. Also, the use of a small number of old rats with one laser dose is also a limitation. We used only one dose due to the difficulty in obtaining aged rats. This dose was used based on our previous studies showing anti-inflammatory effects in other tissues. 4.4. Conclusion Taken together, our data suggest that transcranial PBM improves the inflammatory response and the activation of intracellular signaling proteins linked to vascular function and cell survival in the brain of aged rats. Declarations Funding statement Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; #2017/16443-0). Author contributions Conceived and designed the experiments: FSC, FCBM, and SGS. Performed the experiments: FSC and FCBM. Analyzed the data: FSC, and SGS. Contributed reagents/materials/analysis tools: FSC, BHSA and SGS. Wrote the manuscript: FSC, FGL and SGS. Approved the final version of the manuscript: FSC, FCBM, BHSA, FGL, and SGS. Availability of data and material Not applicable. Compliance with ethical standards All experimental protocols were approved by the ethics committee of the Universidade de Mogi das Cruzes (UMC) (#003/2020). Consent to participate Not applicable. Consent for Publication Not applicable. Conflict of interest The authors declare that they have no conflicts of interest. All authors read and approved the final manuscript. Acknowledgements This study was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; #2017/16443-0). F. Gonzalez-Lima was supported by the Oskar Fischer Project Fund. 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Nature medicine 10(1):48–54 Wang X, Tian F, Reddy DD, Nalawade SS, Barrett DW, Gonzalez-Lima F, Liu H (2017) Up-regulation of cerebral cytochrome-c-oxidase and hemodynamics by transcranial infrared laser stimulation: a broadband near-infrared spectroscopy study. Journal of Cerebral Blood Flow Metabolism 37(12):3789–3802 You L, Wang Z, Li H, Shou J, Jing Z, Xie J, Sui X, Pan H, Han W (2015) The role of STAT3 in autophagy. Autophagy 11(5):729–739 Zhang F, Wang S, Cao G, Gao Y, Chen J (2007) Signal transducers and activators of transcription 5 contributes to erythropoietin-mediated neuroprotection against hippocampal neuronal death after transient global cerebral ischemia. Neurobiol Dis 25(1):45–53 Zhang J, Wang X, Vikash V, Ye Q, Wu D, Liu Y, Dong W (2016) ROS and ROS-mediated cellular signaling. Oxidative medicine and cellular longevity , 2016 Zhu C, Huang Z, Gao J, Zhang Y, Wang X, Karlsson N, Li Q, Lannering B, Bjork-Erikson T, Kuhn HG, Blomgren K (2009) Irradiation to the immature brain attenuates neurogenesis and exacerbates subsequent hypoxic-ischemic brain injury in the adult. Journal of neurochemistry 111(6):1447–1456 Supplementary Files Supplementarymaterial.docx Supplementary material Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 23 Jul, 2021 Reviewers invited by journal 18 Mar, 2021 Editor invited by journal 11 Mar, 2021 Editor assigned by journal 04 Mar, 2021 First submitted to journal 02 Mar, 2021 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. 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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-293139","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":15186583,"identity":"5b39c7bb-f678-471a-9fd9-19b5181e3ebb","order_by":0,"name":"Fabrizio Cardoso","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABIElEQVRIiWNgGAWjYBAC9gYeBoYEBgkID8hm4GdgYIPJsmHTwnMAXYtkAzFaEDwgNjhASAv72WMfHvyxkJNvP/zsw5uKOjnjG8nPHnyoYJDnFzvA9rgCixaevOQZCTwSxgZn0oxnzjlz2NjsRpq54YwzDIYzZyewG57B1GLPkGPMkCAhkbhBgsGYmbftQOK2Gwlm0rxtDAkGtxPYgB7DtIX/DVCLgUTi/Bnsn4Fa6hI3z0j/hl+LBMiWBInEhhs8IFuYgdblELBF4l0yQ8IBkF9yihlBfpE486ZMcsYZCaBfEtsNsTos9zDjjz91wBA7vpkBFGL87enbJD5U2MjzSycfe4hFCxYgkAAiQZHLSJwGYIo5QKTCUTAKRsEoGCkAAC0eX2dGeePWAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-7547-8880","institution":"Universidade de Mogi das Cruzes Nucleo de Ciencias Saude","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Fabrizio","middleName":"","lastName":"Cardoso","suffix":""},{"id":15186584,"identity":"4e26c9e4-d9c1-48b5-a9f5-07166938a9a0","order_by":1,"name":"Fernanda Mansur","email":"","orcid":"","institution":"Hospital israelita Albert Einstein","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fernanda","middleName":"","lastName":"Mansur","suffix":""},{"id":15186585,"identity":"11d85ae1-fd23-4935-8e25-eb255a8e99d9","order_by":2,"name":"Bruno Araújo","email":"","orcid":"","institution":"Centro Nacional de Pesquisa em Energia e Materiais","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bruno","middleName":"","lastName":"Araújo","suffix":""},{"id":15186586,"identity":"a0030587-d218-49c9-83d6-f90723060130","order_by":3,"name":"Francisco Gonzalez-Lima","email":"","orcid":"","institution":"University of Texas at Austin","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"","lastName":"Gonzalez-Lima","suffix":""},{"id":15186587,"identity":"e8f1ecbb-1971-4a4d-b74b-5a139cef715e","order_by":4,"name":"Sérgio Gomes da Silva","email":"","orcid":"","institution":"Universidade de Mogi das Cruzes","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sérgio","middleName":"Gomes da","lastName":"Silva","suffix":""}],"badges":[],"createdAt":"2021-03-03 04:14:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-293139/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-293139/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":6806861,"identity":"975ab392-12cb-43d1-8b58-e8ee2c68c422","added_by":"auto","created_at":"2021-03-10 17:33:45","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":50051,"visible":true,"origin":"","legend":"Cortical levels of IL-1alpha and IL-5 in rats from control (n=5) and laser (n=5) groups. The laser treatment increased IL-1alpha level and reduced IL-5 level in aged rats (*). Data are showed in picogram per microgram of protein (p\u003c0.05; Mann-Whitney test).","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-293139/v1/00f0b18b5779f241525e44ec.jpeg"},{"id":6807302,"identity":"bf29ba74-7a47-4672-b197-3f869ab9fb0d","added_by":"auto","created_at":"2021-03-10 17:36:45","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":108964,"visible":true,"origin":"","legend":"Hippocampal levels of IL-1alpha, IL-5, IL-18 and Fractalkine in rats from control (n=5) and laser (n=4) groups. The laser treatment increased IL-1alpha level and reduced IL-5, IL-18 and fractalkine levels in aged rats (*). Data are showed in picogram per microgram of protein (p\u003c0.05; Mann-Whitney test).","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-293139/v1/1ef65f490497920cb7c9d9ae.jpeg"},{"id":6807300,"identity":"85ecda8b-3757-4c13-b73f-d63f1c87d249","added_by":"auto","created_at":"2021-03-10 17:36:45","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":107825,"visible":true,"origin":"","legend":"Cortical expression of ERK and p38 and activation of STAT3 and ERK in rats from control (n=5) and laser (n=5) groups. The laser treatment reduced the expression of ERK and p38 and increased the activation of STAT3 and ERK in aged rats (*). Data were normalized to the mean fluorescence intensity (MFI) of the control group (p\u003c0.05; Mann-Whitney test).","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-293139/v1/b4705fc1f5f33202377f5963.jpeg"},{"id":6807301,"identity":"5c937997-1ed1-445f-9c34-50db135174b3","added_by":"auto","created_at":"2021-03-10 17:36:45","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":47025,"visible":true,"origin":"","legend":"Hippocampal expression of p70S6K, STAT5 and p38 in rats from control (n=5) and laser (n=5) groups. The laser treatment increased the expression of p70S6K and STAT5 and reduced the expression of p38 in aged rats (*). Data were normalized to the mean fluorescence intensity (MFI) of the control group (p\u003c0.05; Mann-Whitney test).","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-293139/v1/d615737678daa5adcec9e483.jpeg"},{"id":13677842,"identity":"b31a6866-4fbf-41f1-a736-b45a462c0c82","added_by":"auto","created_at":"2021-09-17 11:36:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":523746,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-293139/v1/af976d3a-384f-40be-a0a2-52c663d661a3.pdf"},{"id":6807299,"identity":"3efa84ca-8699-477d-b453-846f38b9f1d4","added_by":"auto","created_at":"2021-03-10 17:36:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":21771,"visible":true,"origin":"","legend":"Supplementary material","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-293139/v1/673142cc76725b1024ed98a4.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePhotobiomodulation Improves the Inflammatory Response and Intracellular Signaling Proteins Linked To Vascular Function and Cell Survival in the Brain of Aged Rats\u003c/p\u003e","fulltext":[{"header":"Highlights","content":"\u003cp\u003e(1) Photobiomodulation improves the inflammatory response in the cortex and hippocampus of aged rats\u003c/p\u003e\n\u003cp\u003e(2) Photobiomodulation activates intracellular signaling proteins linked to vascular function and cell survival in the brain of aged rats\u003c/p\u003e"},{"header":"1. Introduction","content":" \u003cp\u003ePhotobiomodulation (PBM), also called low-level laser therapy (Anders et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) has emerged as a non-pharmacological, non-invasive tool capable of stimulating wound healing, reducing pain and inflammation in several diseases (Chung et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Many human studies have also used transcranial PBM to improve brain functions in several conditions (e.g., Schiffer et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Muili et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Muili et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Barrett and Gonzalez-Lima, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Tian et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Disner et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Hwang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Eells et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Blanco et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017a\u003c/span\u003e; Blanco et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Saltmarche et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Holmes et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For example, Saltmarche and collaborators (2017) submitted elderly people diagnosed with dementia to PBM and observed an improvement in executive function and a significant improvement in sleep and mood. Many studies using animal models have also shown interesting results of PBM on the brain (e.g., Rojas et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Rojas and Gonzalez-Lima, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; El Massri et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Lu et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For instance, Lu and collaborators (2017) injected beta amyloid (Aβ) in the hippocampus of rats that were treated with laser PBM for five days. They noted that laser treatment restored spatial memory and object recognition memory. In addition, they observed an increase in the antioxidant capacity of hippocampal CA1 neurons and a decrease of Aβ-induced reactive gliosis and inflammation.\u003c/p\u003e \u003cp\u003eRecently, some authors have shown promising results of PBM in the aged brain (Salgado et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Salehpour et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Vargas et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Salehpour et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; O\u0026rsquo;Donnell et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Saucedo et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, there is little evidence to explain such effects, other than improved mitochondrial respiration and vascular function. It is known that brain aging is characterized by local inflammation with glial cells releasing increasing amount of pro-inflammatory cytokines such as IL-1beta, IL-6 and TNF-alpha (Cribbs et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Norden and Godbout, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This process is directly involved with cellular dysfunctions characteristic of aging and Alzheimer's disease (Stephan et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hong et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), which may result in an increase in the activation of signaling pathways linked to inflammation and cellular death such as c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) (O'Donnell et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2000\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on the well-documented anti-inflammatory effects of PBM in other tissues (Almeida et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Haslerud et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tomazoni et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e: Naterstad et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), we evaluated whether a transcranial treatment with a laser diode of 660 nm wavelength and 100 mW power can modulate the inflammatory response and expression and activation of intracellular signaling proteins in the cortex and hippocampus of aged rats.\u003c/p\u003e "},{"header":"2. Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Animals\u003c/h2\u003e \u003cp\u003eTwenty-month-old male Wistar rats (n\u0026thinsp;=\u0026thinsp;10) were used in this study. The colony room was maintained at 21\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C with a 12 h light/ dark schedule (light: 7 am until 7 pm), and food and water were provided \u003cem\u003ead libitum\u003c/em\u003e throughout the experimental period. All experimental protocols were approved by the ethics committee of the Universidade de Mogi das Cruzes (UMC) (#003/2020) and all efforts were made to minimize animal suffering in accordance with the proposals of the International Ethical Guideline for Biomedical Research (CIOMS 1985).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Laser and control protocols\u003c/h2\u003e \u003cp\u003eThe aged rats were randomly distributed into two groups: laser (n\u0026thinsp;=\u0026thinsp;5) and control (n\u0026thinsp;=\u0026thinsp;5). The animals of the laser group were manually immobilized and received the treatment with a laser diode of 660 nm wavelength and 100 mW power for 30 s at each of 5 irradiation points on the head, totalizing 15 Joules of Energy, 150 s of irradiation and fluence of 535.7 J/cm\u003csup\u003e2\u003c/sup\u003e, for 10 consecutive days. These laser parameters were chosen based on our previous publications, showing that these parameters had anti-inflammatory effects in other tissues (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Almeida et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Haslerud et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tomazoni et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Naterstad et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The target coordinates on the scalp were: point 1\u0026thinsp;=\u0026thinsp;AP\u0026thinsp;+\u0026thinsp;4.20 mm and ML 0.00 mm; point 2\u0026thinsp;=\u0026thinsp;AP -3.00 mm and ML -6.60 mm; point 3\u0026thinsp;=\u0026thinsp;AP -3.00 mm and ML\u0026thinsp;+\u0026thinsp;6.60 mm; point 4\u0026thinsp;=\u0026thinsp;AP 0.00 mm and ML 0.00 mm; point 5\u0026thinsp;=\u0026thinsp;AP -5.52 mm and ML 0.00 mm), as in our previous metabolomics study in the rat (Cardoso et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The animals of the control group were handled the same way, except that the laser was not turned on.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLaser parameters used in the present study.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter (unit)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeasurement method or value information source\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCenter wavelength (nm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e660\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOperating mode\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eContinuous wave\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage radiant power (W)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAperture diameter (cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIrradiance at aperture (mW/cm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBeam divergence (degree)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNear zero\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBeam shape\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCircular\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBeam spot size (cm\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eExposure duration/point (s)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRadiant exposure (J/cm\u003csup\u003e2\u003c/sup\u003e) per session\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of points irradiated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDelivery mode\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eContact mode\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber and frequency of sessions\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOne session/day for 10 consecutive days\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal radiant energy (J) per head\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Tissue preparation\u003c/h2\u003e \u003cp\u003eOne hour after the final laser or control session, aged rats from the laser (n\u0026thinsp;=\u0026thinsp;5) and control (n\u0026thinsp;=\u0026thinsp;5) groups were euthanized by decapitation and their cerebral cortex and hippocampus were immediately collected and frozen. The whole cerebral cortex and the hippocampus were homogenized in ice-cold RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS) with freshly added protease (Cat# M222\u0026ndash;1ml; Lot# 1295C056; Amresco) and phosphatase (Cat# B15001-A and B; Lot# 510011; Biotool) inhibitors. Homogenates were centrifuged at 10,000 x g for 10 minutes at 4\u003csup\u003eo\u003c/sup\u003eC and supernatants were collected for cytokine/chemokine quantification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Methods for the protein detection and analysis\u003c/h2\u003e \u003cp\u003eMilliplex\u0026reg; MAP rat cytokine/chemokine magnetic bead panel assay (RECYMAG65K) was used to quantify the levels G-CSF, eotaxin, GM-CSF, IL-1alpha, leptin, MIP-1alpha, IL-4, IL-1beta, IL-2, IL-6, IL-13, IL-10, IL-5, IL-17alpha, IL-18, MCP-1, IP-10, VEGF, fractalkine, MIP-2, TNF-alpha, and RANTES in the brain samples of the studied groups. Milliplex\u0026reg; MAP Kits 48-681MAG and 48-680MAG were used to evaluate the expression and brain activation of signaling proteins Akt, p70S6K, STAT3, STAT5, ERK, JNK, NF-kB and p38. The plates were run on a Luminex\u003csup\u003e\u0026trade;\u003c/sup\u003e Magpix\u003csup\u003e\u0026trade;\u003c/sup\u003e instrument and results were analyzed with the Milliplex Analyst 5.1 Software using a Logistic 5P Weighted regression formula to calculate sample concentrations from the standard curves.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analyses\u003c/h2\u003e \u003cp\u003eStatistical procedures were conducted using the Mann-Whitney U Test that allows comparison of non-parametric data. All analyses were performed using the Statistical Package for the Social Science (SPSS Inc, IBM, version 221.0, Chicago, IL, USA). A statistical difference was considered significant when the \u003cem\u003eP\u003c/em\u003e-value was lower than 0.05. All plots were acquired using the Graph Pad Prism (6.0).\u003c/p\u003e \u003c/div\u003e "},{"header":"3. Results","content":" \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Cortical and hippocampal levels of cytokines and chemokines\u003c/h2\u003e \u003cp\u003eTo evaluate whether the PBM had anti-inflammatory effects on the aged brain, we quantified the cortical and hippocampal levels of several chemokines and cytokines in aged rats submitted to 10 consecutive days of laser treatment or control treatment. The detailed results of Mann-Whitney tests are presented in Supplementary Tables\u0026nbsp;1 and 2. The laser treatment increased the cortical level of IL-1alpha (p\u0026thinsp;=\u0026thinsp;0.008) and reduced the IL-5 level (p\u0026thinsp;=\u0026thinsp;0.046) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the hippocampus, an increase in the IL-1alpha (p\u0026thinsp;=\u0026thinsp;0.035) level was observed in the laser group. However, the laser treatment was able to reduce the levels of IL5 (p\u0026thinsp;=\u0026thinsp;0.027), IL-18 (p\u0026thinsp;=\u0026thinsp;0.049) and fractalkine (p\u0026thinsp;=\u0026thinsp;0.037) in aged rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Taken together, these data showed that PBM changed the levels of neuroinflammatory markers in aged rats.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Cortical and hippocampal expression and activation of signaling proteins\u003c/h2\u003e \u003cp\u003eWe investigated the cortical and hippocampal expression and activation of signaling proteins in aged rats submitted to laser treatment vs. control treatment. The detailed results of Mann-Whitney tests are presented in Supplementary Tables\u0026nbsp;3 and 4. The laser treatment decreased the cortical expression of ERK (p\u0026thinsp;=\u0026thinsp;0.028) and p38 (p\u0026thinsp;=\u0026thinsp;0.009). Interestingly, the laser treatment was able to increase the cortical activation of ERK (p\u0026thinsp;=\u0026thinsp;0.014) and STAT3 (p\u0026thinsp;=\u0026thinsp;0.0016) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the hippocampus, the laser treatment increased the expression of p70S6K (p\u0026thinsp;=\u0026thinsp;0.016) and STAT5 (p\u0026thinsp;=\u0026thinsp;0.050), and decreased the expression of p38 (p\u0026thinsp;=\u0026thinsp;0.028) in aged rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e "},{"header":"4. Discussion","content":" \u003cp\u003eThe aim of our study was to investigate levels of pro- and anti-inflammatory cytokines and chemokines and the expression and activation of signaling proteins in the brain of aged rats submitted to repeated treatment with a laser diode of 660 nm wavelength and 100 mW power. Our results indicate that transcranial PBM was able to modulate the expression and activation of signaling proteins and the inflammatory profile in the brain of aged rats.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Anti-inflammatory effects of PBM on the aged brain\u003c/h2\u003e \u003cp\u003eThe laser treatment increased the levels of IL-1alpha and decreased the levels of IL-5 in both the cortex and hippocampus of aged rats. Interestingly, IL-1alpha has been shown to promote angiogenesis and to increase proliferation and migration of endothelial cells (Saini and Bix, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Salmeron et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The IL-1alpha findings are consistent with improving vascular function and oxygenation of the aged brain (Saucedo et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which may help explain the facilitation of neurocognitive functions shown by transcranial PBM studies in older humans (Saltmarche et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Vargas et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In contrast, a reduction of IL-5 may be beneficial to the aged brain because IL-5 induces proliferation and activation of microglia, which is characteristic of inflammatory reactions (Liva and de Vellis, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, PBM promoted an anti-inflammatory effect by reducing the hippocampal levels of IL-18 and fractalkine. IL-18 is a pro-inflammatory cytokine that inhibits cell differentiation and reduces neurogenesis and induces neuronal death in cultured neural progenitors (Liu et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Further evidence suggests that IL-18 can activate the p38 signaling pathway (Arend et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Also, high levels of this cytokine are observed during aging and in neurodegenerative diseases (Boss\u0026ugrave; et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Bellaver et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Fractalkine, in turn, is a chemokine regulated by pro-inflammatory cytokines such as TNFalpha and IL-1beta, which is involved in the communication between neurons and microglia (Desforges et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). High levels of fractalkine were observed in the cortex and hippocampus of a rat model of Alzheimer\u0026rsquo;s disease (Hanzel et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In this sense, reducing the levels of both IL-18 and fractalkine by laser treatment may contribute to improving the inflammatory response in the aging brain and in aging-related neurodegenerative diseases.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Effects of PBM on signaling proteins in the aged brain\u003c/h2\u003e \u003cp\u003ePBM reduced the expression of ERK and p38 and increased the activation of ERK and STAT3 in the cortex of aged rats. In the hippocampus, PBM increased the expression of p70S6K and STAT5 and decreased the expression of p38. ERK activation is associated with proliferation, differentiation and cellular migration, having an effect opposite to p38, which is activated by cellular stresses such as genotoxic, osmotic, hypoxic, or oxidative stress and it is connected to apoptosis and inflammatory responses (Kim and Choi, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSTAT3 modulates the expression of genes responsible for important physiological functions such as cell regulation and apoptosis control (Desrivi\u0026egrave;res et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kim et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). STAT5 is linked to neuronal survival (Um and Lodish, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Also, STAT5 is necessary for the neuroprotective and neurotrophic effects of growth hormone on hippocampal neurons (Byts et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). However, STAT3 and STAT5 brain levels are decreased during aging (Bazhanova and Anisimov, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Nevertheless, laser treatment increased STAT3 activation and the expression of STAT5 in the cortex of aged rats. These observed changes in intracellular signaling proteins may be linked to the anti-inflammatory effects of PBM in the cortex and hippocampus of aged rats.\u003c/p\u003e \u003cp\u003eHigh levels of neuroinflammatory markers are observed during aging (Lee et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Godbout et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), indicating a pro- and anti-inflammatory imbalance (Godbout and Johnson, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In particular, it is possible that PBM of the neuroinflammatory response is related to increases in the activation of intracellular signaling proteins STAT3 and ERK in the cortical region. In this sense, STAT3 is a transcription factor that interacts with polypeptide receptors in the cell membrane, mediating extracellular signals such as growth factors and cytokines (Levy and Darnell, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). When activated by tyrosine phosphorylation, STAT3 dimerizes and translocate to the nucleus, thus activating the target genes (You, et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The activation of STAT3 suppresses the expression of pro-inflammatory mediators, promoting an immune evasion and blocking the production and detection of inflammatory signals by several components of the immune system (Wang et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eERK can be activated by growth factors, such as the epidermal growth factor (EGF) that is associated with the attenuation of pro-inflammatory mediators (Garc\u0026iacute;a-Ojalvo et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), activating Ras, which recruits Raf for the membrane. Raf activates Mek, which in turn activates ERK. ERK activation triggers cell proliferation, differentiation and cell migration (Kim and Choi, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). It is possible that the neuroinflammatory response in the cortex may trigger the activation of the ERK signaling pathways which in turn activates STAT3 (David et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). In this context, it is possible that the increased ERK activation is linked to the anti-inflammatory effect of laser treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Limitations\u003c/h2\u003e \u003cp\u003eWe did not evaluate functional parameters, such as cognitive functions, since our goal was to investigate whether repeated laser treatment alters the cortical and hippocampal inflammatory and signaling profiles in aged rats. Also, the use of a small number of old rats with one laser dose is also a limitation. We used only one dose due to the difficulty in obtaining aged rats. This dose was used based on our previous studies showing anti-inflammatory effects in other tissues.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Conclusion\u003c/h2\u003e \u003cp\u003eTaken together, our data suggest that transcranial PBM improves the inflammatory response and the activation of intracellular signaling proteins linked to vascular function and cell survival in the brain of aged rats.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCoordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES) and Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de S\u0026atilde;o Paulo (FAPESP; #2017/16443-0).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceived and designed the experiments: FSC, FCBM, and SGS. Performed the experiments: FSC and FCBM. Analyzed the data: FSC, and SGS.\u003cbr /\u003e Contributed reagents/materials/analysis tools: FSC, BHSA and SGS.\u003cbr /\u003e Wrote the manuscript: FSC, FGL and SGS. Approved the final version of the manuscript: FSC, FCBM, BHSA, FGL, and SGS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocols were approved by the ethics committee of the Universidade de Mogi das Cruzes (UMC) (#003/2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES) and Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de S\u0026atilde;o Paulo (FAPESP; #2017/16443-0). F. Gonzalez-Lima was supported by the Oskar Fischer Project Fund.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlmeida P, Lopes-Martins R\u0026Aacute;B, Tomazoni SS, Albuquerque‐Pontes GM, Santos LA, Vanin AA, Frigo L, Vieira RP, Albertini R, Carvalho PTC, Leal‐Junior ECP (2013) Low‐Level Laser Therapy and Sodium Diclofenac in Acute Inflammatory Response Induced by Skeletal Muscle Trauma: Effects in Muscle Morphology and m RNA Gene Expression of Inflammatory Markers. 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Journal of neurochemistry 111(6):1447\u0026ndash;1456\u003c/span\u003e\u003c/li\u003e\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":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"laser, photobiomodulation, brain, aging, inflammation, intracellular signaling proteins","lastPublishedDoi":"10.21203/rs.3.rs-293139/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-293139/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhotobiomodulation is a non-pharmacological tool widely used to reduce inflammation in many tissues. However, little is known about its effects on the inflammatory response in the aged brain. We conducted the study to examine anti-inflammatory effects of photobiomodulation in aging brains. We used aged rats (20 months old) with control (handled, laser off) or transcranial laser (660 nm wavelength, 100 mW power) treatments for 10 consecutive days and evaluated the level of inflammatory cytokines and chemokines, and the expression and activation of intracellular signaling proteins in the cerebral cortex and the hippocampus. Inflammatory analysis showed that aged rats submitted to transcranial laser treatment had increased levels of IL-1alpha and decreased levels of IL-5 in the cerebral cortex. In the hippocampus, the laser treatment increased the levels of IL-1alpha and decreased levels of IL-5, IL-18 and fractalkine. Regarding the intracellular signaling proteins, a reduction in the ERK and p38 expression and an increase in the STAT3 and ERK activation were observed in the cerebral cortex of aged rats from the laser group. In addition, the laser treatment increased the hippocampal expression of p70S6K, STAT3 and p38 of aged rats. Taken together, our data indicate that transcranial photobiomodulation can improve the inflammatory response and the activation of intracellular signaling proteins linked to vascular function and cell survival in the aged brain.\u003c/p\u003e","manuscriptTitle":"Photobiomodulation Improves the Inflammatory Response and Intracellular Signaling Proteins Linked To Vascular Function and Cell Survival in the Brain of Aged Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-03-10 17:33:43","doi":"10.21203/rs.3.rs-293139/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-07-23T07:11:51+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-03-19T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Molecular Neurobiology","date":"2021-03-12T00:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-03-05T00:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2021-03-02T09:27:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5279e300-faae-4d55-9463-7c5b51802922","owner":[],"postedDate":"March 10th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":2876040,"name":"Neurobiology of Disease"}],"tags":[],"updatedAt":"2021-10-17T22:32:15+00:00","versionOfRecord":[],"versionCreatedAt":"2021-03-10 17:33:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-293139","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-293139","identity":"rs-293139","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}