Transcranial photobiomodulation induced frequency-specific dual-pathway glial activation for neurovascular protection vs amyloid clearance in Alzheimer’s disease

Transcranial photobiomodulation induced frequency-specific dual-pathway glial activation for neurovascular protection vs amyloid clearance in Alzheimer’s disease | 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 Article Transcranial photobiomodulation induced frequency-specific dual-pathway glial activation for neurovascular protection vs amyloid clearance in Alzheimer’s disease Ting Li, Bowen Zhang, Zemeng Chen, Louzhe Xu, Songqi Yang, Hui Shen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6906760/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Vascular network impairment is a critical driver of Alzheimer’s Disease (AD) progression, yet strategies to effectively regulate vascular pathology remain unclear. Here, we attempted to employ transcranial continuous-wave (CW) and 40Hz near-infrared (NIR) light stimulation to target vascular network in 5xFAD mice. The results showed that NIR light significantly ameliorated cognitive dysfunction via enhancing β-amyloid (Aβ) plaque clearance and providing neurovascular protection. Glial cell activation served as the primary mediator through which NIR light achieved these modulation effects. The colocalization and correlational analyses revealed that CW-NIR light primarily activated astrocyte-vascular synergism to ameliorate vascular dysfunction, thereby conferring synaptic protection. In contrast, 40Hz light primarily activated microglia to increase their aggregation around Aβ plaque, which enhanced Aβ local clearance. These differential mediating pathways suggested that CW and 40Hz exhibited modality-specific therapeutic advantages for distinct AD pathological hallmarks – vascular dysfunction and Aβ plaque deposition, respectively. These findings potentially offer a precision light stimulation strategy targeting different neurodegenerative hallmarks. Physical sciences/Optics and photonics/Other photonics/Biophotonics Physical sciences/Optics and photonics/Lasers, LEDs and light sources Alzheimer’s disease Transcranial photobiomodulation glial cell activation vascular protection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Alzheimer’s disease (AD), as the most common form of dementia worldwide, was mainly characterized by abnormal deposition of β-amyloid (Aβ) plaques and neuronal degeneration 1 – 3 . Numerous studies confirmed that AD was also usually accompanied by cerebral vascular pathology, including the reduction and distortion of small blood vessels, especially capillaries 4 – 6 . Recent disease-modifying therapies mainly targeted clearing Aβ deposition and showed positive clinical outcomes in early-stage AD patients 7 , 8 . However, these monoclonal antibody therapies focused only on reducing Aβ accumulation but failed to ameliorate overall neurodegenerative pathology in the brain. Moreover, anti-Aβ therapies exhibited strong dose-dependency and had the risk of amyloid-related imaging abnormalities (ARIA), including microhemorrhages and cerebral edema 9 , 10 . Therefore, developing novel sustainable therapeutic approaches to complement existing anti-Aβ therapies holds significant value for effective AD intervention. Light stimulation has emerged in recent years as a promising non-invasive physical therapy for neurological diseases with fewer side effects 11 – 13 . Transcranial continuous-wave (CW) light stimulation mitigated brain degeneration and counteracted neuroinflammation by enhancing mitochondrial dynamics and preserving neuronal hemoglobin 14 , 15 in AD mice. Moreover, CW-NIR light also modulated vascular endothelial growth factor (VEGF)-mediated angiogenesis 16 and regulated nitric oxide (NO) release to improve hemodynamics 17 , 18 . In contrast, the current pulsed-wave (PW) light therapy protocol for AD mice was mainly sensory stimulation related to nerve entrainment, such as visual and auditory stimulation, rather than transcranial irradiation 16 , 19 – 22 . Accumulating research has indicated that 40Hz visual flickering stimulation attenuates AD by activating gamma oscillations, triggering a general neuronal protective response that leads to a reduction in Aβ production and an increase in microglial endocytosis to significantly alleviate AD-related pathological changes 23 , 24 . There is still a lack of research on transcranial PW light stimulation in AD mice. Previous studies focusing on traumatic brain injury and stroke indicate that transcranial PW light may yield superior outcomes compared to CW light 25 – 27 . However, the differences in therapeutic mechanisms of these two light stimulations for alleviating neurovascular dysfunction in AD remain unknown. Elucidating these differences is necessary for constructing effective light stimulation for the clinical treatment of AD. Glial cells were strongly implicated in the progression of AD, particularly in vascular dysfunction and synaptic impairment 28 – 31 . As the most abundant glial cells in the brain, astrocytes not only regulate synaptic plasticity but also serve as essential components of both the blood-brain barrier (BBB) and neurovascular unit (NVU) 32 , 33 . The astrocytic atrophy has been found in the early stage of AD 34 , 35 . Smaller cell bodies and less complex processes of astrocytes in AD diminished the synaptic maintenance, thereby contributing to synaptic loss and neurotransmission impairment 36 , 37 . The degeneration of the end-feet structure of astrocytes affected the function of the BBB and the regulation of cerebral blood flow 28 , 29 . Microglia were mainly activated in AD to phagocytose and degrade Aβ aggregates, limiting plaque formation 38 , 39 . Microglia also played an important role in vascular protection, mainly manifested in regulating cerebral blood flow 40 and decreasing cerebral amyloid angiopathy 31 , 41 . Notably, over-activated microglia phagocytose synapses, leading to synaptic loss and also exacerbating neuroinflammation in the late stage of AD 42 . Recent studies have shown that light stimulation alleviated cerebral vessel impairment 16 , 39 . However, the specific mechanisms underlying this vascular protective effect and whether they are related to glial cells remain unclear. In this study, we investigated the effects of CW and 40Hz NIR light stimulation on glial cells and neurovascular system in 5xFAD mice. The results revealed that both types of NIR light stimulation improved cognitive function, while they were associated with different target mechanisms. CW-NIR light stimulation mainly mediated activation of astrocyte-vascular synergism, promoting vascular remodeling and directly supporting synaptic and neuronal repairment. In contrast, 40Hz-NIR light stimulation achieved efficient Aβ clearance and synaptic protection by reprogramming the spatial distribution of microglia (towards Aβ plaque aggregation) to enhance their local clearance, while decreasing the overall inflammatory burden. These findings highlight the important role of glial cell activation in vascular impairment and synaptic protection in AD mice. Results A transcranial near-infrared light (NIR) stimulation device for 5xFAD mice was designed. Before NIR irradiation, the hair of all mice was shaved to make the light source close to the scalp, thereby avoiding the interference of black hair with light penetration. To approximate the experiment conditions at the clinical level, the NIR beam was incident from the prefrontal cortex of 5xFAD mice, without covering eyes and other tissues. During irradiation, 5xFAD mice were restrained in the mouse immobilizer, and the light source was fixed on its movable end (Fig. 1 (B)). The home-built device employed an 810 ± 50 nm light source selected through Monte Carlo simulations, demonstrating superior cerebral tissue penetration at this wavelength (Fig. 1 (E)). Two intervention groups were constructed, including CW 810 nm light and 810 nm light pulsed at 40 Hz (duty cycle: 50%) to compare the therapeutic differences between two photon delivery patterns. NIR light improved cognitive deficits in 5xFAD mice To evaluate the effect of NIR light on cognitive function in 5xFAD mice, we conducted behavioral assessments after one-month stimulation (Fig. 1 (A)). The novel object recognition (NOR) test showed comparable average velocity and total distance between groups (Fig. S1 (A, B)). The discrimination index represented the recognition of a novel object, reflecting short-term memory and learning ability. We found that each mouse of four groups showed more observation time for the novel object than the familiar one, but the discrimination index (DI) had no significant differences between groups (Fig. 1 (G)). Notably, by comparing typical locomotion trajectories of the same mice between training and testing (Fig. 1 (F)), we found that 5xFAD mice in the CW and 40 Hz groups were more inclined to move around the novel object, suggesting that NIR light alleviated the cognitive dysfunction of AD mice to a certain extent, especially the symptoms of fuzzy memory loss. The long-term spatial memory and learning ability were further assessed via a 6-day Morris Water Maze (MWM) test to evaluate (Fig. 1 (D)). Both stimulation groups exhibited the improvement of memory ability, with CW and 40Hz group showing significantly reduced escape latencies than untreated 5xFAD mice on day 3 (5xFAD vs. 5xFAD + CW: 57.98 ± 3.73 vs. 48.32 ± 8.26 s, p < 0.05, 5xFAD vs. 5xFAD + 40Hz: 57.98 ± 3.73 vs. 45.92 ± 1.79 s, p < 0.001, Fig. 1 (I)). On Day 4, only 40Hz group exhibited the advantages of escape latencies (5xFAD vs. 5xFAD + 40Hz: 52.09 ± 4.80 vs. 39.45 ± 9.05 s, p < 0.05). Although the escape time gradually decreased as the training days increased in all groups, the latency and proportion of residence time in southwest (SW) quadrant (with the target platform), and average swimming speed had no dramatic changes (Fig. 1 (J), Fig. S1 (C, D)). During probe trials, 40Hz group navigated paths comparable to WT mice in platform localization, while the path length of CW group was similar to the control 5xFAD group without light treatment (Fig. 1 (H)). The MWM indicators on the test day had no remarkable differences between multiple groups (Fig. S1 (E-H)). These results from behavioral tests indicated that 40 Hz NIR stimulation enhanced spatial memory acquisition in 5xFAD mice, while CW irradiation showed intermediate efficacy. NIR light reduced Aβ deposition in 5xFAD mice To explore the effects of NIR light on amyloid pathology in 5xFAD mice, we detected Aβ 1−42 levels in the cortex and hippocampus via ELISA (Fig. 2 (A)). The results showed that 40Hz NIR light significantly decreased Aβ 1−42 level (5xFAD vs. 5xFAD + 40Hz: 100% ± 74.57% vs. 12.34% ± 9.20%, p < 0.01) in hippocampus, while CW light only exhibited a mild Aβ 1−42 clearance effect (5xFAD vs. 5xFAD + CW: 100% ± 74.57% vs. 39.89% ± 19.19%, p = 0.10) in hippocampus. Both types of light stimulation showed no significant reductions in Aβ 1−42 levels in cortex. To further investigate Aβ burden of specific brain regions, we employed immunofluorescence with Aβ-specific antibody D54D2 to semi-quantitatively assess Aβ deposition in hippocampus and cortex. hippocampus region was differentiated into distinct functional regions, including DG, CA1, CA2, and CA3. Given the fuzzy boundary between CA2 and CA3, we combined the two regions into a single statistical module, designated as CA23. The results showed that Aβ plaques were mostly deposited in the DG region compared to CA1 and CA23 (Fig. 2 (B)). Unexpectedly, these two stimulation protocols precisely were the most effective for DG in three subregions, including the reduction of the Aβ plaque number (CW: 47.56%, 40Hz: 41.12%, Fig. 2 (C)) and Aβ area proportion (CW: 59.69%, 40Hz: 37.52%, Fig. 2 (D)). Moreover, 40Hz NIR irradiation significantly reduced the number of Aβ plaques in CA1 (55.99%, Fig. 2 (C)), while CW light failed to decrease any Aβ burden of CA1. CA23 demonstrated the least response to the therapeutic intervention of CW and 40Hz light, with no significant differences in almost all indicators (Fig. 2 (C-E)). Notably, compared to 40Hz light, CW irradiation more effectively reduced the average size of Aβ in CA1 (181.67%) and CA23 (117.77%), which has not been reported in previous studies (Fig. 2 (E)). Immunostaining results of cortex (Fig. 2 (F, J)) revealed that CW and 40 Hz irradiation both significantly decreased the number of Aβ plaques in 5xFAD mice (CW: 27.28%, 40Hz: 24.94%, Fig. 2 (G)), compared to the no-irradiation group. The average size of Aβ plaques and the total Aβ plaques area in each ROI were also remarkably decreased in the 40Hz group than the control group (Average size: 12.40%, Total area proportion: 32.15%, Fig. 2 (H, I)). In contrast, CW group showed a similar average size of Aβ plaques and a significantly lower total area of Aβ plaques in comparison to the control group (Average size: 7.55%, Total area proportion: 28.80%). NIR light improved vascular impairment in 5xFAD mice To assess whether NIR light improved the cognitive function in 5xFAD mice via reducing vascular dysfunction, we marked and reconstructed the vascular network of the whole brain (Fig. 3 (A)). The overall qualitative comparison revealed that compared to the control group, both CW and 40Hz NIR light stimulation increased the density of small blood vessels, while exhibiting no effect on large vessels. Moreover, the local vessel images were extracted and reconstructed to analyze the vessel density, vessel length, and vessel mean diameter in different brain regions, including CA1, CA23, DG, and cortex (Fig. 3 (B), Fig. S2(A)). The vessel density was defined as the ratio of vascular volume to the total volume in the region of interest (ROI), which reflected the blood flow and was associated with oxygen supply 6 , 43 . The vessel length represented the sum of all vessel segment lengths within the ROI, which directly correlated with the complexity of vascular network and morphological changes in vasculature. The vessel mean diameter referred to the average diameter of all vessel segments within the ROI, which served as an indirect indicator of blood perfusion area. The quantitative results based on fMOST revealed that NIR light stimulation significantly improved vascular impairment of hippocampus and cortex in 5xFAD mice. CW-NIR and 40Hz-NIR groups both exhibited the higher vessel density (CW: 474.07%, 446.60%, 149.92%, and 304.31%; 40Hz: 141.81%, 117.00%, 63.92%, and 74.29% for CA1, CA23, DG, and cortex, Fig. 3 (C)) and vessel mean diameter (CW: 83.54%, 114.40%, 105.82%, and 100.71%; 40Hz: 21.63%, 33.81%, 28.67%, and 28.88% for CA1, CA23, DG, and cortex, Fig. S2(B)) in each subregion, compared with the control group. In contrast, vascular length exhibited significant regional heterogeneity, potentially attributable to the inclusion of major vessels within the ROIs (Fig. 3 (D)). Specifically, 40Hz-NIR light stimulation significantly increased the vascular length in CA1 (33.19%) and cortex (15.77%), slightly enhanced it in CA23 (22.84%) and DG (11.83%). While CW light stimulation increased vascular length in CA1 (64.44%), it significantly reduced vascular length in DG (-24.44%). We also found that both CW and 40Hz light stimulation decreased vascular straightness in CA1 (CW: -2.37%, 40Hz: -1.60%, Fig. S2(C)). NIR light promoted glial cell response in 5xFAD mice Numerous studies revealed that glial cells responded to light stimulation and played a vital role in mediating its biological effects 23 , 39 , 44 . To further investigate the effects of CW and 40Hz NIR light stimulation on glial cells, we performed immunostaining for the astrocyte marker (GFAP and S100B) and microglia marker (Iba1) in hippocampus and cortex (Fig. 4 (A, D)). We observed that CW and 40Hz NIR light both significantly increased GFAP + astrocytes number (CW: 84.62%, 40Hz: 58.82%, Fig. 4 (B)) and their area fraction (CW: 73.05%, 40Hz: 58.95%, Fig. S3(A)) in cortex. Notably, CW irradiation also enhanced astrocyte populations in hippocampus subfields, with GFAP + cell number increasing by 78.70% and area fraction by 90.35% in the DG (Fig. 4 (B), Fig. S3(A)), alongside a 33.60% increase in GFAP + cell size of CA23 region (Fig. S3(B)). S100B + astrocytes exhibited even more pronounced expansion in CA23, with cell number and area fraction respectively expanding by 160.47% and 245.53% (p < 0.05 vs.5xFAD, Fig. 4 (C), Fig. S3(C)). However, NIR light stimulation had no significant effects on the average size of S100B + astrocytes across all brain regions (Fig. S3(D)). In AD, microglia typically undergo activation with somatic hypertrophy to phagocytose Aβ plaques, often accumulating around these deposits. The quantitative analysis of Iba1 + microglia revealed that 40Hz-NIR light stimulation selectively reduced total microglia number in the DG subregion (46.00%, Fig. 4 (E)), whereas CW light showed no significant effects on microglial morphology (Fig. S3(E-F)). However, given the close spatial association between microglial distribution and Aβ plaques, we quantified two parameters to control for potential confounding effects from the intergroup differences of Aβ deposition: the area fraction of microglia within plaques, and the average number of microglia around plaques. Intriguingly, 40Hz-NIR light stimulation instead exhibited higher microglial density tightly surrounding Aβ plaques in DG subregion, compared to the 5xFAD group (Fig. 4 (F)). In CA1 region of hippocampus, 40Hz stimulation doubled peri-plaque microglial numbers (2-fold increase, p < 0.05, Fig. 4 (G)), while CW irradiation induced a 2.5-fold elevation of microglial clustering around plaques in CA23 (p < 0.01). These results suggested that NIR light stimulation robustly activated both astrocyte and microglia, consistent with prior light stimulation studies. However, we further revealed that CW and 40Hz NIR light stimulation exhibited complementary glial cell selectivity, mainly manifested as astrocytic activation dominated under CW irradiation, while 40Hz specifically potentiated microglial Aβ clearance. NIR light induced connections between glial cells and blood vessels in 5xFAD mice Accumulating evidence implicated astrocytes and microglia in cerebrovascular network regulation 29 , 30 , 32 , 40 . Thus, to determine whether NIR-mediated vascular improvements involved glial activation, we conducted co-immunostaining of vascular markers (CD31 or Lectin) and glial markers (GFAP and Iba1). The quantitative colocalization analysis was performed to evaluate the connection between glial cells and vessels. We observed that two-dimensional CD31 (Fig. 5 (C)) and Lectin (Fig. 5 (H)) labeling confirmed CW-induced vascular density improvement, consistent with fMOST three-dimensional quantification results. The colocalization analysis revealed that CW stimulation significantly enhanced the spatial coupling between GFAP + astrocytes and CD31 + vessels in both CA1 (co-localization coefficient: 0.08 ± 0.05 vs 5xFAD 0.04 ± 0.02, p < 0.05, Fig. 5 (A, D)) and cortex regions (co-localization coefficient: 0.10 ± 0.04 vs 5xFAD 0.07 ± 0.04, p < 0.05, Fig. 5 (B, D)), but not in CA23 and DG (Fig. S4(A, B)). The increased proportion of CD31 co-localized structures (Fig. S4(D)) without proportional GFAP colocalization changes (Fig. S4(E)) suggested that CW stimulation-induced astrocytic increase facilitated more process extension toward vascular endothelia (Increased co-localized area of GFAP and CD31, Fig. S4(C)). Moreover, the positive correlation (r = 0.34, p = 0.086, Fig. S6(A, B)) between GFAP + astrocyte number and the co-localization coefficient of GFAP and CD31 further corroborated this observation. We also found that this co-localization coefficient of GFAP and CD31 was positively correlated with CD31 + vessel density (r = 0.45, p = 0.006, Fig. 5 (E)). Together, these data indicated that the vascular protective effects of CW stimulation were possibly mediated through enhanced astrocyte-vascular interactions. In contrast, no significant effects of NIR light stimulation were exhibited in the connection of microglia and vessels (Fig. 5 (F, G), Fig. S4(F, G)), although the co-localization level of Iba1 and Lectin showed a slight increase in CA1 region of 40Hz-NIR group compared to CW-NIR group (Fig. 5 (I), Fig. S4(H-J)). However, we observed that the co-localization coefficient between Iba1 and Lectin showed a negative correlation with Lectin + vessel density (r = -0.56, p = 0.005, Fig. 5 (J)), primarily due to NIR-induced vascular density augmentation without concomitant increase of microglia. Notably, this co-localization coefficient also positively correlated with GFAP and CD31 coupling (r = 0.45, p = 0.015, Fig. S6(E)), which may involve synergistic effects secondary to increased vascular density. NIR light improved synaptic impairment via activating glial cell–vascular coupling in 5xFAD mice In AD, Aβ-driven neuronal loss and synaptic dysfunction underlie cognitive decline. To evaluate the neuroprotective effect of NIR light stimulation, immunofluorescence analyses were performed with NeuN + neurons and synaptophysin+ (Syn) synapses. Due to dense hippocampal neuronal packing, we measured neuronal band widths (50 µm intervals) as a proxy for cell numbers. We found that both CW and 40Hz NIR light significantly alleviated NeuN + cell loss in CA23 (CW: 10.57%, 40Hz: 20.37%, Fig. 6 (A, C))) and increased cortical neuron density (CW: 22.95%, 40Hz: 17.35%, Fig. 6 (A, D)), with no effects in CA1 and DG. Synaptophysin, as a membrane protein localized on synaptic vesicles, participated in synaptic transmission and maintained synaptic plasticity 45 . We observed that both CW and 40Hz light stimulation significantly increased the number of synapses in the cortex (CW: 98.17%, 40Hz: 114.17%, Fig. 6 (B, E)). In contrast, a slight improvement in synaptic density was detected in hippocampus subregions (Fig. 6 (E), Fig. S5(A, B)). To further investigate whether the synapse protective effects of NIR light stimulation were mediated by the aforementioned glial cells activation, we analyzed the correlation between synaptic markers and glial cell related indicators (Fig. S6(A)). The results showed that the synapse number had both positive correlation with CD31 + vessel density (r = 0.38, p = 0.034, Fig. 6 (F)) and GFAP-CD31 co-localization coefficient (r = 0.36, p = 0.047, Fig. 6 (G)), which suggested that enhanced astrocyte-vasculature coupling not only ameliorated vascular damage, but also achieved synaptic protection through improving blood supply. Conversely, synaptic quantitative metrics showed no significant correlation with microglial or their vascular co-localization coefficients (Fig. 6 (H), Fig. S6(A)). Thus, we inferred that the protective effects of NIR stimulation on synapses were primarily mediated through astrocytes, with no direct involvement of microglia. Furthermore, although previous studies showed that light stimulation triggered the release of NO 46 , no significant improvement of nNOS was found in CW and 40Hz groups (Fig. S5(C)). Discussion In summary, we systematically evaluated the effects of CW and 40Hz NIR light stimulation on neurovascular network in 5xFAD mice. The results demonstrated that both modalities improved cognitive deficits, while they engaged distinct glial-mediated modulation pathways. CW-NIR preferentially enhanced astrocyte-vascular interactions, improving structural impairment of cerebral vasculature and providing indirect synaptic protection. In contrast, 40Hz-NIR induced spatial reconfiguration of microglia, driving their targeted migration to Aβ plaque-enriched zones. This microglial repositioning enhanced local Aβ clearance and reduced plaque burden, which collectively protected nervous system. Our findings elucidated the critical role of glial modulation in neurovascular protection in AD, and proposed that NIR stimulation modalities were selectively targeted to specific AD pathologies, astrocyte-mediated vascular-synaptic dysfunction or microglial amyloid clearance. By establishing this modality-dependent mechanistic specificity, our study advances precision light stimulation tailored to the multi-pathological progression of AD. Recent advances in light stimulation for AD have delineated two principal paradigms 47 , including frequency-entrained neural modulation and mitochondrial-targeted transcranial photobiomodulation (tPBM). Iaccarino et al. demonstrated gamma-frequency visual stimulation reduces AD pathology through neural oscillation entrainment 23 , while Miao et al. found that tPBM with CW light enhanced meningeal lymphatic Aβ clearance via chromophore activation of mitochondrial 45 . Our work integrated these two modalities and explored the effects of gamma-frequency (40Hz) tPBM in AD mice. While tPBM traditionally relies on photon-chromophore interactions (e.g., CCO activation) 14 , 48 and exhibits biphasic dose-response 49 – 51 , our data suggested 40Hz tPBM exhibited positive effects on improving cognitive function similar to those of CW light, despite delivering half the total photons (50% duty cycle). tPBM based on PW light has not been extensively investigated in degenerative neurological diseases, but PW was preliminarily attempted in other application scenarios of light stimulation with controversial results, such as traumatic brain injury (TBI), wound healing 52 . Takahiro et al. found that 10 Hz laser irradiation induced more effective neuroprotection compared to CW and 100 Hz light in TBI mice 26 . In contrast, CW light was superior to PW irradiation in promoting wound healing 53 . In this study, we identified tPBM with PW light also as an effective strategy against AD-induced neurovascular dysfunction, proposing a promising light stimulation modality for neurodegenerative diseases. However, diverging from the potent vascular protective effects of CW light, PW stimulation exerted its cognitive benefits primarily through enhancing microglia-dependent Aβ clearance. One potential explanation for these discrepancies is that light stimulation at non-neural tissues remains mostly dependent on the biological effects of photons, whereas PW-based tPBM additionally stimulates other biological activities (such as neural entrainment), which was not possible with CW. Previous studies have established that AD is predominantly accompanied by cerebrovascular dysfunction, characterized by diminished perfusion area and impaired oxygen supply 54 – 56 . This hypoperfusion state fails to sustain normal neural activity, ultimately precipitating neurodegenerative pathology. Given this potential therapeutic target in AD, many researchers have investigated the vascular-protection effects of light stimulation, focusing on its capacity to induce angiogenesis 16 and restore cerebral hemodynamics 57 . However, the effects of light stimulation on the vascular network remain underexplored. Our study employed fMOST to achieve whole-brain cerebrovascular architecture visualization in post-stimulation 5xFAD mice. Our findings revealed that both CW and 40Hz NIR light stimulation significantly ameliorated AD-induced vessel diameter impairment, which provided a structural foundation for cerebral perfusion improvement. Moreover, consistent with prior MOST vessel imaging from Zhang et al 6 , we observed significant heterogeneity in vascular complexity across different brain regions in 6-month-old 5xFAD mice. These differences were primarily manifested in vascular complexity, with both DG and cortex regions exhibiting significantly higher vessel length compared to CA1 and CA23 across all experimental groups. Therefore, we suggested that NIR light stimulation may mitigate neuronal injury through augmenting cerebrovascular perfusion, and thereby enhance cognitive function. However, precise mechanistic pathways through which NIR light stimulation modulates vascular architecture and synapses remain incompletely elucidated. The existing evidence supported that astrocytes were the anatomical and functional link between vasculature and nervous system 58 . They interfaced with blood vessels, forming the BBB and neurogliovascular unit to regulate cerebral blood flow and energy supply. 40Hz auditory-induced gamma entrainment has been confirmed to robustly activate astrocytes and elicit vascular responses in 5xFAD mice 39 . Our results revealed that NIR light stimulation also exhibited similar effects and further confirmed that NIR light enhanced structural connectivity between astrocytes and vasculature, which established a critical foundation for protecting or forming additional BBB and NVU structural modules. Astrocytes also regulate synaptic function by controlling neurotransmitter clearance (such as glutamate, GABA, and others), and thereby maintaining synaptic transmission and neuroprotection 59 . Our findings demonstrated that CW-NIR light stimulation triggered significant astrocyte activation while concurrently exerting synapse-protective effects. Moreover, the connection proportion of astrocyte and vasculature exhibited a positive correlation with synapse density (r = 0.36) in 5xFAD mice. Thus, we posit astrocytes as the pivotal cellular mediators through which CW-NIR light stimulation exerts its neurovascular protective effects. In contrast, 40Hz stimulation demonstrated significantly greater efficacy in activating microglia, consistent with transcranial and visual light stimulation outcomes across other studies. Notably, although microglial activation facilitated Aβ clearance, the excessive activation concurrently provoked detrimental neuroinflammatory cascades 42 . We found that transcranial 40Hz NIR light stimulation specifically promoted microglia clustering around Aβ plaques, without inducing global microgliosis. This targeted recruitment effectively mitigated the side effects of neuroinflammatory. It should be acknowledged that this study still has some limitations. Herein, we propose astrocytes and microglia as putative mediator cells for the modulation effect of NIR light stimulation on neurovascular. However, these findings are mainly phenotype-based, and the specific signaling pathways involved require further genomics-based results. Secondly, although we observed that CW and 40Hz light preferentially engaged different glial populations (astrocytes vs. microglia) to enhance cognition, the mechanistic underpinnings of this selective activation remain elusive. By comparing prior research on PW stimulation and CW tPBM, we infer that this difference may be associated with gating frequency-dependence ion channels, which necessitated further validation through electrophysiological assays and in vitro verification. To summarize, our results revealed that both CW and 40Hz transcranial NIR light stimulation enhanced cognitive performance and ameliorated neurovascular impairment, which was mediated through glial cell activation. Specifically, CW light primarily activated astrocytes, augmenting their anatomical connection with vasculature, thereby improving vascular impairment and conferring synaptic protection. In contrast, 40Hz light primarily modulated the spatial distribution of microglia, promoting them clustering around Aβ plaques, which enhanced Aβ clearance and thereby protected neurovascular. Collectively, our findings highlighted the distinct advantages of CW and 40Hz light in cerebrovascular network improvement and amyloid plaque clearance respectively, providing a theoretical foundation for precision light stimulation tailored to AD subtypes with different pathological signatures. Materials and methods Animals and experimental design Adult (five-month-old) male and female 5xFAD mice with an average body weight of 24–28 g and age-matched littermate wild-type (WT) C57BL/6J mice were utilized in this study 60 . All mice were in the C57BL/6J background and obtained from Yangzhou Youdu Biotechnology Co. Ltd (Yangzhou, China). WT mice all received sham light treatment (n = 5). All 5xFAD mice were divided into the following three groups: (1) 5xFAD mice with sham NIR light treatment (5xFAD, n = 5), (2) 5xFAD mice with CW light treatment (5xFAD + CW, n = 5), and (3) 5xFAD mice with 40 Hz light treatment (5xFAD + 40Hz, n = 5). All mice were housed at 23 ± 3 ℃ at constant humidity (40% ~ 70%), with a light/dark cycle of 12 / 12 h and a light phase of 8:00 a.m. to 8:00 p.m. Up to five mice of the same sex and group were fed in each cage, with standard water and food. All interventions in this study complied with the Guide for the Care and Use of Laboratory Animals. The experiment was reviewed and approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences (approval number: IRM/1-IACUC-240510-01). Our light stimulation lasted for 10 min per day and 37 consecutive days (Fig. 1 (A)). In the last 7 days of treatment, we sequentially employed 1-day NOR tests and 6-day MWM tests to evaluate spatial memory and learning ability in mice. After these cognitive function tests, we humanely executed mice and collected brain tissues for subsequent pathological analysis. Transcranial NIR light stimulation The stimulation device is composed of a control circuit and several LED light sources connected in series. Before TLS, all hair on the mice's heads was gently shaved using depilatory cream. Thus, the light source can be closely aligned with the skin of the mouse's head (Fig. 1 (B)). The irradiation wavelength of the LED was 810 ± 50 nm, and the optical power density was 25 mW/cm 2 in both CW and 40 Hz (duty cycle 50%) groups. The previous study showed that 25% of around 810 nm light penetrated the mouse skull with a hairless scalp. Thus, for the CW group, about 6.25 mW/cm 2 reached cerebral tissue, while for the PW group, about 3.125 mW/cm 2 . The control and intervention groups differed only in the absence or presence of NIR light exposure, otherwise, the experimental conditions were identical. Behavioral tests Novel Object Recognition (NOR) test The NOR test, which relies on a rodent's natural preference to explore novelty without additional external reinforcement, has been used to examine cognitive function, especially recognition and memory 61 , 62 . During the habituation session, mice were placed in the test rooms. After 24 h, two identical non-toxic cylinders were placed in opposite and symmetrical corners of the 40 cm \(\:\times\:\) 40 cm \(\:\times\:\) 50 cm box (Fig. 1 (C)), then each mouse was released into the arena and allowed to explore freely for 10 min while a camera above the arena recorded its movements. After a 1 h break, one of the previously explored objects was replaced by a novel cuboid, which is consistent in the same height and volume with the familiar one. The mice were returned to explore the arena for another 10 min. The trajectory, time spent exploring each object and total distance traveled were recorded using Tracking Master-V4.0-YM (Shanghai Fanbi Intelligent Technology Co., Shanghai, China). The discrimination index (DI = (T novel – T familiar ) / (T novel + T familiar ), where T novel and T familiar indicate the exploration time during testing for the novel and familiar objects, respectively), average velocity, and total distance traveled were calculated and analyzed. Morris Water Maze (MWM) test The MWM test is a widely used method for assessing spatial learning and memory and related forms of abilities 63 , 64 . All mice were trained in a 1.5 m open field water maze. The pool was filled to a depth of 30 cm with water maintained at 25 ± 1 ℃. The escape platform was located 2 ~ 3 cm below the surface of the water (Fig. 1 (D)). White edible pigment was added to the water to make the platform invisible during tests. The pool was divided into four quadrants. All mice underwent the learning phase four times daily for four consecutive days. Mice were randomly introduced into different quadrants and allowed to search for the hidden platform for 60 s. The mice were left on the hidden platform for 15 s to memorize the spatial cues if the mice found it on their own or time was over. A test phase was conducted 24 h later with a hidden platform removed from the tank. The percentage of total time the mice spent searching for the platform from the opposite side of the quadrant where the original platform was located, and the number of times they crossed the original platform area within 60 s were recorded. Latency and swimming speed were analyzed. Throughout the experiment, the swimming path of the mice was recorded by Tracking Master-V4.0-MWM (Shanghai Fanbi Intelligent Technology Co., Shanghai, China), which was placed approximately 2.5 m vertically above the center of the circular water trough. Brain tissue preparation After the behavioral tests, to eliminate the interference of blood in the immunofluorescence staining experiments, the mouse heart was perfused with cold phosphate-buffered saline (PBS) under deep anesthesia. Then, paraformaldehyde (PFA) was used to fix the brain tissue. Brains were removed and separated into the two hemispheres, which were fixed in 4% PFA or snap frozen in liquid nitrogen and stored at − 80 ℃ until processing. ELISA Prior to the testing, mice brains were collected according to brain regions, the hippocampus and cortex. The mouse amyloid beta peptide 1–42(Aβ 1−42 ) ELISA kit (No. CSB-E10787m, CUSABIO) was employed for assessing the expression profiles of Aβ. According to the manufacturer’s protocol, 50 mg of brain tissue was added to 450 µL PBS. After grinding thoroughly and centrifuging, the supernatants of the brain tissue were used to measure the absorbance of the resulting solution at 450 nm for each well by a microplate reader. Immunofluorescence The paraffin-embedded mouse brain was sectioned coronally at a thickness of 4 µm on a pathology sectioning machine. Paraffin sections were sequentially placed in xylene for two times, 20 min each, then the anhydrous ethanol, anhydrous ethanol, 85% alcohol, and 75% alcohol, each for 5 min, and washed with distilled water. The sections were placed in citrate antigen retrieval solution (pH 6.0) in a microwave oven for antigen repair, with medium-high and high heat for 5 min each. After cooling, the slides were immersed in PBS (pH 7.4) with shaking and washed 3 times for 5 min each to perform antigen retrieval. The slides were soaked in PBS with shaking and washing for 3 times for 5 min each. Then the 5% goat serum was added to cover the tissue evenly, and the sections were sealed for 30 minutes at room temperature to perform the serum seal. The primary antibody were β-Amyloid (D54D2) (1:500, 8243T, Cell Signaling Technology), NeuN (1:200, 66836-1-IG, Proteintech), CD31 (1:2000, AB182981, Abcam), GFAP (1:500, 16825-1-AP, Proteintech), S100B (1:500, OB-PPGP015-02, Oasis biofarm), nNOS (1:500, OB-PGP070-02, Oasis biofarm), Synaptophysin (1:500, OB-PRB115-02, Oasis biofarm), Iba1 (1:500, OB-PGP049-02, Oasis biofarm), and Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight® 594 (1:200, DL-1177-1, Vector Laboratories). Sections were immersed in PBS with shaking and washing for 3 times, 5 min each. Thereafter, sections were incubated with Goat Anti-Rabbit IgG H&L (1:1000, ab6717, Abcam), Goat anti-Guinea pig IgG, AF488 (1:1000, G-GP488, Oasis biofarm), GoraLite Plus 594-Goat anti-Rabbit IgG H&L (1:1000, RGAR004, Proteintech). The sections were soaked in PBS with 0.1 mg/mL DAPI 3 times, 5 min each, with shaking and washing. The sections were sealed with an anti-fluorescence quenching sealer and imaged by confocal laser scanning microscopy (AX / AX R with NSPARC, Nikon) and a panoramic scanner (Panoramic Scan, 3DHISTECH) with a 20 objective. Images were analyzed using ImageJ software (National Institutes of Health, USA) and Imaris 9.9.0 (Bitplane). For each immunofluorescence image, we divided the mouse brain into four regions based on the DAPI image: cortex, hippocampus CA1, hippocampus CA2/3, and hippocampus DG. Fluorescence micro-optical sectioning tomography (fMOST) After perfusing with cold PBS and PFA, the mouse hearts were then perfused with a mixture of 9 mg Fluorescein 5-isothiocyanate (ND420177, Bomeibio), 3 g gelatin, and 30 mL 0.01 M PBS. Before the mouse brains were excised, the mouse carcasses were immersed in cold water for 30 min. The LR White resin (AGR1280A, Agar Scientific) was employed to embed the mouse brain. The samples were rinsed with a graded ethanol series (50%, 75%, 100%, and 100% treatments, each incubated for 2 h), and finally in 100% ethanol for 12 h. The next day, the samples were infiltrated with a graded LR White series (50%, 75%, and 100% treatments, each for 2 h), then in 100% LR White for 48 h. Gelatin capsules were used as an embedding mold to polymerize the tissue block. The LR White was allowed to polymerize at 48°C for 24 hours. The fMOST system was used to image the mouse brain layer by layer, with a resolution of 2*0.35*0.35um. The BioMapping 7000 system (Wuhan OE-Bio Co., Ltd.) consisted of a 40× Olympus microscope objective, 488nm and 561 nm lasers, and a TDI-CCD camera, which was used for detecting images from the fMOST system. The Imaris program (10.1.0) was used to reconstruct the cerebrovascular network. Statistical analysis All statistical analyses were performed using IBM SPSS Statistics 27.0 and GraphPad Prism 9.5 for Windows software. Quantitative results were expressed as mean ± standard error (SEM) or mean ± standard deviation (SD). One-way or two-way analysis of variance (ANOVAs) with the least significant difference (LSD) or Tukey’s post hoc tests were applied to make the statistical comparisons between groups of behavioral tests and immunofluorescence. Significance levels were set at p < 0.05 for all analyses. Declarations Conflict of interest. We have no conflicts of interest to disclose. Author contributions. Conceptualization, B.Z., Z.C., and T.L.; methodology, B.Z., Z.C., and L.X.; validation, B.Z., Z.C., S. Y. and T.L.; formal analysis, B.Z., Z.C.; investigation, B.Z., Z.C. and T.L.; resources, B.Z., Z.C.; data curation, B.Z., Z.C.; writing—original draft preparation, B.Z., Z.C.; writing, review and editing, B.Z., Z.C., T.L., and L.X.; supervision, T.L.; project administration, T.L.; funding acquisition, T.L. All authors have read and agreed to the published version of the manuscript. Acknowledgements. This research was funded by Chinese Academy of Medical Science health innovation project (grant nos. 2021-I2M-1-042 and 2021-I2M-1-058). Data availability statements. Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request. References Scheltens P, De Strooper B, Kivipelto M, Holstege H, Chételat G, Teunissen CE et al. Alzheimer’s disease. The Lancet 2021; 397 : 1577–1590. Benilova I, Karran E, De Strooper B. The toxic Aβ oligomer and Alzheimer’s disease: an emperor in need of clothes. Nat Neurosci 2012; 15 : 349–357. Reitz C, Brayne C, Mayeux R. Epidemiology of Alzheimer disease. Nat Rev Neurol 2011; 7 : 137–152. Nortley R, Korte N, Izquierdo P, Hirunpattarasilp C, Mishra A, Jaunmuktane Z et al. Amyloid β oligomers constrict human capillaries in Alzheimer’s disease via signaling to pericytes. Science 2019; 365 : eaav9518. Nielsen RB, Egefjord L, Angleys H, Mouridsen K, Gejl M, Møller A et al. Capillary dysfunction is associated with symptom severity and neurodegeneration in Alzheimer’s disease. Alzheimer’s & Dementia 2017; 13 : 1143–1153. Zhang X, Yin X, Zhang J, Li A, Gong H, Luo Q et al. High-resolution mapping of brain vasculature and its impairment in the hippocampus of Alzheimer’s disease mice. National Science Review 2019; 6 : 1223–1238. Swanson CJ, Zhang Y, Dhadda S, Wang J, Kaplow J, Lai RYK et al. A randomized, double-blind, phase 2b proof-of-concept clinical trial in early Alzheimer’s disease with lecanemab, an anti-Aβ protofibril antibody. Alz Res Therapy 2021; 13 : 80. Van Dyck CH, Swanson CJ, Aisen P, Bateman RJ, Chen C, Gee M et al. Lecanemab in Early Alzheimer’s Disease. N Engl J Med 2023; 388 : 9–21. Hampel H, Elhage A, Cho M, Apostolova LG, Nicoll JAR, Atri A. Amyloid-related imaging abnormalities (ARIA): radiological, biological and clinical characteristics. Brain 2023; 146 : 4414–4424. Filippi M, Cecchetti G, Spinelli EG, Vezzulli P, Falini A, Agosta F. Amyloid-Related Imaging Abnormalities and β-Amyloid–Targeting Antibodies: A Systematic Review. JAMA Neurol 2022; 79 : 291. Hamblin MR, Salehpour F. Photobiomodulation of the Brain: Shining Light on Alzheimer’s and Other Neuropathological Diseases. JAD 2021; 83 : 1395–1397. Enengl J, Hamblin MR, Dungel P. Photobiomodulation for Alzheimer’s Disease: Translating Basic Research to Clinical Application. JAD 2020; 75 : 1073–1082. Huang Z, Hamblin MR, Zhang Q. Photobiomodulation in experimental models of Alzheimer’s disease: state-of-the-art and translational perspectives. Alz Res Therapy 2024; 16 : 114. Yang L, Wu C, Parker E, Li Y, Dong Y, Tucker L et al. Non-invasive photobiomodulation treatment in an Alzheimer Disease-like transgenic rat model. Theranostics 2022; 12 : 2205–2231. Yang L, Youngblood H, Wu C, Zhang Q. Mitochondria as a target for neuroprotection: role of methylene blue and photobiomodulation. Transl Neurodegener 2020; 9 : 19. Tao L, Liu Q, Zhang F, Fu Y, Zhu X, Weng X et al. Microglia modulation with 1070-nm light attenuates Aβ burden and cognitive impairment in Alzheimer’s disease mouse model. Light Sci Appl 2021; 10 : 179. Keszler A, Lindemer B, Weihrauch D, Jones D, Hogg N, Lohr NL. Red/near infrared light stimulates release of an endothelium dependent vasodilator and rescues vascular dysfunction in a diabetes model. Free Radical Biology and Medicine 2017; 113 : 157–164. Feng Y, Huang Z, Ma X, Zong X, Tesic V, Ding B et al. Photobiomodulation Inhibits Ischemia-Induced Brain Endothelial Senescence via Endothelial Nitric Oxide Synthase. Antioxidants 2024; 13 : 633. Park M, Hoang GM, Nguyen T, Lee E, Jung HJ, Choe Y et al. Effects of transcranial ultrasound stimulation pulsed at 40 Hz on Aβ plaques and brain rhythms in 5×FAD mice. Transl Neurodegener 2021; 10 : 48. Zheng L, Yu M, Lin R, Wang Y, Zhuo Z, Cheng N et al. Rhythmic light flicker rescues hippocampal low gamma and protects ischemic neurons by enhancing presynaptic plasticity. Nat Commun 2020; 11 : 3012. Singer AC, Martorell AJ, Douglas JM, Abdurrob F, Attokaren MK, Tipton J et al. Noninvasive 40-Hz light flicker to recruit microglia and reduce amyloid beta load. Nat Protoc 2018; 13 : 1850–1868. Shen Q, Wu X, Zhang Z, Zhang D, Yang S, Xing D. Gamma frequency light flicker regulates amyloid precursor protein trafficking for reducing β‐amyloid load in Alzheimer’s disease model. Aging Cell 2022; 21 . doi:10.1111/acel.13573. Iaccarino HF, Singer AC, Martorell AJ, Rudenko A, Gao F, Gillingham TZ et al. Gamma frequency entrainment attenuates amyloid load and modifies microglia. Nature 2016; 540 : 230–235. Martorell AJ, Paulson AL, Suk H-J, Abdurrob F, Drummond GT, Guan W et al. Multi-sensory Gamma Stimulation Ameliorates Alzheimer’s-Associated Pathology and Improves Cognition. Cell 2019; 177 : 256-271.e22. Hamblin MR. Photobiomodulation for traumatic brain injury and stroke. J Neuro Res 2018; 96 : 731–743. Ando T, Xuan W, Xu T, Dai T, Sharma SK, Kharkwal GB et al. Comparison of Therapeutic Effects between Pulsed and Continuous Wave 810-nm Wavelength Laser Irradiation for Traumatic Brain Injury in Mice. PLoS ONE 2011; 6 : e26212. Lapchak PA, Salgado KF, Chao CH, Zivin JA. Transcranial near-infrared light therapy improves motor function following embolic strokes in rabbits: an extended therapeutic window study using continuous and pulse frequency delivery modes. Neuroscience 2007; 148 : 907–914. Verkhratsky A, Olabarria M, Noristani HN, Yeh C-Y, Rodriguez JJ. Astrocytes in Alzheimer’s Disease. Neurotherapeutics 2010; 7 : 399–412. Kimbrough IF, Robel S, Roberson ED, Sontheimer H. Vascular amyloidosis impairs the gliovascular unit in a mouse model of Alzheimer’s disease. Brain 2015; 138 : 3716–3733. Dudvarski Stankovic N, Teodorczyk M, Ploen R, Zipp F, Schmidt MHH. Microglia–blood vessel interactions: a double-edged sword in brain pathologies. Acta Neuropathol 2016; 131 : 347–363. Kiani Shabestari S, Morabito S, Danhash EP, McQuade A, Sanchez JR, Miyoshi E et al. Absence of microglia promotes diverse pathologies and early lethality in Alzheimer’s disease mice. Cell Reports 2022; 39 : 110961. Williamson MR, Fuertes CJA, Dunn AK, Drew MR, Jones TA. Reactive astrocytes facilitate vascular repair and remodeling after stroke. Cell Reports 2021; 35 : 109048. Schumacher S, Tahiri H, Ezan P, Rouach N, Witschas K, Leybaert L. Inhibiting astrocyte connexin‐43 hemichannels blocks radiation‐induced vesicular VEGF‐A release and blood–brain barrier dysfunction. Glia 2024; 72 : 34–50. Yeh C-Y, Vadhwana B, Verkhratsky A, Rodríguez JJ. Early Astrocytic Atrophy in the Entorhinal Cortex of a Triple Transgenic Animal Model of Alzheimer’s Disease. ASN Neuro 2011; 3 : AN20110025. Olabarria M, Noristani HN, Verkhratsky A, Rodríguez JJ. Concomitant astroglial atrophy and astrogliosis in a triple transgenic animal model of Alzheimer’s disease. Glia 2010; 58 : 831–838. Ota Y, Zanetti AT, Hallock RM. The Role of Astrocytes in the Regulation of Synaptic Plasticity and Memory Formation. Neural Plasticity 2013; 2013 : 1–11. Popov A, Brazhe A, Denisov P, Sutyagina O, Li L, Lazareva N et al. Astrocyte dystrophy in ageing brain parallels impaired synaptic plasticity. Aging Cell 2021; 20 : e13334. Uchihara T, Akiyama H, Kondo H, Ikeda K. Activated Microglial Cells Are Colocalized With Perivascular Deposits of Amyloid-β Protein in Alzheimer’s Disease Brain. Stroke 1997; 28 : 1948–1950. Martorell AJ, Paulson AL, Suk H-J, Abdurrob F, Drummond GT, Guan W et al. Multi-sensory Gamma Stimulation Ameliorates Alzheimer’s-Associated Pathology and Improves Cognition. Cell 2019; 177 : 256-271.e22. Császár E, Lénárt N, Cserép C, Környei Z, Fekete R, Pósfai B et al. Microglia modulate blood flow, neurovascular coupling, and hypoperfusion via purinergic actions. Journal of Experimental Medicine 2022; 219 : e20211071. Van Den Brink H, Voigt S, Kozberg M, Van Etten ES. The role of neuroinflammation in cerebral amyloid angiopathy. eBioMedicine 2024; 110 : 105466. Hansen DV, Hanson JE, Sheng M. Microglia in Alzheimer’s disease. Journal of Cell Biology 2018; 217 : 459–472. Wu Y, Ke J, Ye S, Shan L-L, Xu S, Guo S-F et al. 3D Visualization of Whole Brain Vessels and Quantification of Vascular Pathology in a Chronic Hypoperfusion Model Causing White Matter Damage. Transl Stroke Res 2023. doi:10.1007/s12975-023-01157-1. Chen H, Li N, Liu N, Zhu H, Ma C, Ye Y et al. Photobiomodulation modulates mitochondrial energy metabolism and ameliorates neurological damage in an APP/PS1 mousmodel of Alzheimer’s disease. Alz Res Therapy 2025; 17 : 72. Wang M, Yan C, Li X, Yang T, Wu S, Liu Q et al. Non-invasive modulation of meningeal lymphatics ameliorates ageing and Alzheimer’s disease-associated pathology and cognition in mice. Nat Commun 2024; 15 : 1453. Hamblin M. Photobiomodulation for Alzheimer’s Disease: Has the Light Dawned? Photonics 2019; 6 : 77. Xu H, Luo Z, Zhang R, Golovynska I, Huang Y, Samanta S et al. Exploring the effect of photobiomodulation and gamma visual stimulation induced by 808 nm and visible LED in Alzheimer’s disease mouse model. Journal of Photochemistry and Photobiology B: Biology 2024; 250 : 112816. Lin H, Li D, Zhu J, Liu S, Li J, Yu T et al. Transcranial photobiomodulation for brain diseases: review of animal and human studies including mechanisms and emerging trends. Neurophoton 2024; 11 . doi:10.1117/1.NPh.11.1.010601. Stepanov YV, Golovynska I, Zhang R, Golovynskyi S, Stepanova LI, Gorbach O et al. Near-infrared light reduces β-amyloid-stimulated microglial toxicity and enhances survival of neurons: mechanisms of light therapy for Alzheimer’s disease. Alz Res Therapy 2022; 14 : 84. Zhang R, Zhou T, Liu L, Ohulchanskyy TY, Qu J. Dose–effect relationships for PBM in the treatment of Alzheimer’s disease. J Phys D: Appl Phys 2021; 54 : 353001. Spera V, Sitnikova T, Ward MJ, Farzam P, Hughes J, Gazecki S et al. Pilot Study on Dose-Dependent Effects of Transcranial Photobiomodulation on Brain Electrical Oscillations: A Potential Therapeutic Target in Alzheimer’s Disease. JAD 2021; 83 : 1481–1498. Hashmi JT, Huang Y, Sharma SK, Kurup DB, De Taboada L, Carroll JD et al. Effect of pulsing in low‐level light therapy. Lasers Surg Med 2010; 42 : 450–466. Al-Watban FAH, Zhang XY. The Comparison of Effects between Pulsed and CW Lasers on Wound Healing. Journal of Clinical Laser Medicine & Surgery 2004; 22 : 15–18. Winchester LM, Powell J, Lovestone S, Nevado-Holgado AJ. Red blood cell indices and anaemia as causative factors for cognitive function deficits and for Alzheimer’s disease. Genome Med 2018; 10 : 51. Meyer EP, Ulmann-Schuler A, Staufenbiel M, Krucker T. Altered morphology and 3D architecture of brain vasculature in a mouse model for Alzheimer’s disease. Proc Natl Acad Sci USA 2008; 105 : 3587–3592. Kimbrough IF, Robel S, Roberson ED, Sontheimer H. Vascular amyloidosis impairs the gliovascular unit in a mouse model of Alzheimer’s disease. Brain 2015; 138 : 3716–3733. Saucedo CL, Courtois EC, Wade ZS, Kelley MN, Kheradbin N, Barrett DW et al. Transcranial laser stimulation: Mitochondrial and cerebrovascular effects in younger and older healthy adults. Brain Stimulation 2021; 14 : 440–449. Preman P, Alfonso-Triguero M, Alberdi E, Verkhratsky A, Arranz AM. Astrocytes in Alzheimer’s Disease: Pathological Significance and Molecular Pathways. Cells 2021; 10 : 540. Andersen JV, Schousboe A, Verkhratsky A. Astrocyte energy and neurotransmitter metabolism in Alzheimer’s disease: Integration of the glutamate/GABA-glutamine cycle. Progress in Neurobiology 2022; 217 : 102331. Oakley H, Cole SL, Logan S, Maus E, Shao P, Craft J et al. Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: potential factors in amyloid plaque formation. J Neurosci 2006; 26 : 10129–10140. Leger M, Quiedeville A, Bouet V, Haelewyn B, Boulouard M, Schumann-Bard P et al. Object recognition test in mice. Nat Protoc 2013; 8 : 2531–2537. Zhang R, Xue G, Wang S, Zhang L, Shi C, Xie X. Novel object recognition as a facile behavior test for evaluating drug effects in AβPP/PS1 Alzheimer’s disease mouse model. J Alzheimers Dis 2012; 31 : 801–812. Vorhees CV, Williams MT. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc 2006; 1 : 848–858. Nehra G, Promsan S, Yubolphan R, Chumboatong W, Vivithanaporn P, Maloney BJ et al. Cognitive decline, Aβ pathology, and blood-brain barrier function in aged 5xFAD mice. Fluids Barriers CNS 2024; 21 : 29. Additional Declarations There is no conflict of interest Supplementary Files SupplementaryInformation20250614.docx Supplementary Information Graphicalabstract.png Graphical Abstract Cite Share Download PDF Status: Posted Version 1 posted 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-6906760","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":472347571,"identity":"d0d1a176-cf1e-4a51-8999-c8d12b4f3e65","order_by":0,"name":"Ting Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAx0lEQVRIiWNgGAWjYBACPmYGhgMMDDYJxGthY2YGaUkjRQsDM4g6TIoWdv6Dhwt+nc/TbW9g/PAzp46Bf3YDYYcdntl3u9jszAFmyd5thxkk7hwgQgtvz+3EbTcS2Bh4tx1gMJAg4EiolnOJ2+4/YGP8u62OSC08Pw4AbQGyebcxE6XF4DBvQzLQL4nN0rLbDvNI3CCghZ//4OPPPH/s8syOHz748e22Ojn+GQS0gAFjG5hsAJE8RKgHgT9EqhsFo2AUjIKRCQB/3z+vkQr1jQAAAABJRU5ErkJggg==","orcid":"","institution":"Institute of Biomedical Engineering, Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":true,"prefix":"","firstName":"Ting","middleName":"","lastName":"Li","suffix":""},{"id":472347572,"identity":"b558299b-dd12-4d6e-ae82-a47f83953529","order_by":1,"name":"Bowen Zhang","email":"","orcid":"","institution":"Institute of Biomedical Engineering, Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Bowen","middleName":"","lastName":"Zhang","suffix":""},{"id":472347573,"identity":"3323419b-6af3-47be-baf9-49b855af500d","order_by":2,"name":"Zemeng Chen","email":"","orcid":"","institution":"Institute of Biomedical Engineering, Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Zemeng","middleName":"","lastName":"Chen","suffix":""},{"id":472347574,"identity":"506c274a-d3a7-423f-afe8-0aae3a74d119","order_by":3,"name":"Louzhe Xu","email":"","orcid":"https://orcid.org/0009-0005-7122-4647","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Louzhe","middleName":"","lastName":"Xu","suffix":""},{"id":472347575,"identity":"b3be17c8-7362-4d9b-b237-9ab52bddf6b9","order_by":4,"name":"Songqi Yang","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences and Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Songqi","middleName":"","lastName":"Yang","suffix":""},{"id":472347576,"identity":"f7f56a10-ca30-4011-89c7-f92348b29c6a","order_by":5,"name":"Hui Shen","email":"","orcid":"","institution":"Tianjin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Shen","suffix":""},{"id":472347577,"identity":"14e2b9fc-60a2-465a-979b-d7221f32fc09","order_by":6,"name":"Xunbin Wei","email":"","orcid":"","institution":"Peking University","correspondingAuthor":false,"prefix":"","firstName":"Xunbin","middleName":"","lastName":"Wei","suffix":""}],"badges":[],"createdAt":"2025-06-16 15:00:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6906760/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6906760/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84963530,"identity":"d2d20350-3e2b-42ec-86fb-6903ed7993f7","added_by":"auto","created_at":"2025-06-19 09:25:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":743059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNear-infrared light stimulation ameliorated cognitive dysfunction in 5xFAD mice.\u003c/strong\u003e (A) Scheme of the experimental design. (B) Schematic representation of a mouse undergoing transcranial light stimulation. The orange-colored, unilaterally open, hollow cylinder is the body of a mouse immobilizer, the blue cylinder with a conical hollow inside is the movable end of the mouse immobilizer, the red dot represents LED light source affixed to the inside of the mouse immobilizer, and the green screw was used to secure the movable end of the mouse immobilizer. (C, D) Schematic diagram of the NOR (C) and MWM (D) experimental site. (E) Photon fluence distribution from Monte Carlo simulation. (F) Typical path of mice in NOR during training (upper) and testing (lower). (G) The discrimination index during the NOR test. (H) Typical swimming path of mice on day 4. (I, J) The latency (I) and swimming speed (J) in the four groups during the 5-day MWM test. Data in (F, G) are analyzed by one-way ANOVA with Turkey’s post-hoc tests of multiple groups. Data in (I, J) are presented as mean ± SEM and analyzed by two-way ANOVA with LSD post-hoc tests of multiple groups. p-value: *p \u0026lt; 0.05, **p\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig.1Behavior.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6906760/v1/e349bb8ee31f91d18de15537.jpg"},{"id":84963531,"identity":"02131b21-d5ca-4fd0-a030-f0c6dc222e48","added_by":"auto","created_at":"2025-06-19 09:25:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5060849,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNear-infrared light stimulation reduced Aβ deposition in 5xFAD mice.\u003c/strong\u003e (A) Relative Aβ\u003csub\u003e1-42 \u003c/sub\u003elevels in hippocampus and cortex. (B) Representative images of brain sections in hippocampus stained with anti-Aβ (D54D2, green) and DAPI (blue) in 5xFAD mice. Scale bar, 500 μm. (C-E) The count (C), area ratio (D), and average size (E) of Aβ in each hippocampus subregion, including CA1, CA23, and DG. (F) Representative images of brain sections in cortex stained with anti-Aβ (D54D2, green) and DAPI (blue) in 5xFAD mice. Scale bar, 200um. (G-I) The count (G), area ratio (H), and average size (I) of Aβ in cortex. (J) Aβ deposition in the coronal section of the hemibrain. Scale bar, 1000 μm. Data in (A, C-E, G-I) are presented as mean ± SD and analyzed by one-way or two-way ANOVA with Turkey’s post-hoc tests of multiple groups. p-value: *p \u0026lt; 0.05, *p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.2D54D2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6906760/v1/3af9e809aaab42cfbc80a2d5.jpg"},{"id":84965003,"identity":"646d25ae-e744-45e4-bdb3-b94fb4fc6fa9","added_by":"auto","created_at":"2025-06-19 09:41:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6583282,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNear-infrared light stimulation improved vascular network impairment in 5xFAD mice.\u003c/strong\u003e (A) Whole-brain vascular network visualized by fMOST imaging. Scale bar, 1000 μm. (B) Representative 3D reconstruction images of vascular network in three subregions of hippocampus, including CA1, CA23, and DG. The pseudo-color in reconstructed images represents the vascular mean diameter. Scale bar, 500um. (C, D) The vascular density (C) and total vascular length (D) in hippocampus and cortex. Data in (C, D) are presented as mean ± SD and analyzed by two-way ANOVA with Turkey’s post-hoc tests of multiple groups. p-value: *p \u0026lt; 0.05, *p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.3fmostvesselfig.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6906760/v1/856bde0f806dc66f39016a4a.jpg"},{"id":84963534,"identity":"681dcf58-3c5a-4920-9133-6af6912aae6e","added_by":"auto","created_at":"2025-06-19 09:25:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7256996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNear-infrared light stimulation induced glial cell response in 5xFAD mice.\u003c/strong\u003e (A) Immunofluorescence with anti-GFAP (purple) and anti-S100B (green) antibodies in the hippocampus and cortex of 5xFAD mice. (B-C) Number of GFAP+ (B) and S100B+ (C) astrocytes per ROI in hippocampus and cortex. (D) Immunofluorescence with anti-Aβ (D54D2, red) and anti-Iba1 (green) antibodies in the hippocampus and cortex of 5xFAD mice. (E) Number of Iba+ microglia per ROI in hippocampus and cortex. (F) Percentage of Aβ amyloid that are also Iba1+ microglia in hippocampus and cortex. (G) Average number of Iba+ microglia close to Aβ amyloid per ROI in hippocampus and cortex. Data in (B, C, E-G) are presented as mean ± SD and analyzed by two-way ANOVA with Turkey’s post-hoc tests of multiple groups. p-value: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001. Scale bar, 100 μm.\u003c/p\u003e","description":"","filename":"Fig.4Gilaresults.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6906760/v1/71bdbf7267fca0a2fd636429.jpg"},{"id":84963537,"identity":"027ca897-5a2b-4066-8aff-cb0d0ee59dc5","added_by":"auto","created_at":"2025-06-19 09:25:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5773800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNear-infrared light stimulation induced connections between glial cells and blood vessels.\u003c/strong\u003e (A, B) Immunofluorescence with anti-CD31 (red) and anti-GFAP (green) antibodies in the CA1 (A) and cortex (B) of 5xFAD mice. (C) CD31+ vessel density in hippocampus and cortex. (D) Co-localization coefficient of GFAP and CD31 in hippocampus and cortex. (E) The Pearson’s correlation analysis between CD31+ vessel density and the co-localization coefficient of GFAP+ astrocytes and CD31+ vessel. (F, G) Immunofluorescence with Lectin (red) and anti-Iba1 (green) antibodies in the CA1 (F) and cortex (G) of 5xFAD mice. (H) Lectin+ vessel density in hippocampus and cortex. (I) Co-localization coefficient of Iba1 and Lectin in hippocampus and cortex. (J) The Pearson’s correlation analysis between Lectin+ vessel density and the co-localization coefficient of Iba1+ microglia and Lectin+ vessel. Data in (C-D, H-I) are presented as mean ± SD and analyzed by two-way ANOVA with Turkey’s post-hoc test of multiple groups. p-value: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.5Gliavesselcorrresults.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6906760/v1/a7097c3954b451683f0c4851.jpg"},{"id":84964121,"identity":"f678a1ee-69af-48ce-aff2-c6b1f94505f3","added_by":"auto","created_at":"2025-06-19 09:33:43","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6012138,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNear-infrared light stimulation mitigated neurodegenerative pathology in 5xFAD mice\u003c/strong\u003e. (A) The representative images were immunofluorescence with anti-NeuN (yellow) and DAPI (blue) in hippocampus and cortex of 5xFAD mice. (B) The representative images were immunofluorescence with anti-Syn (red) and DAPI (blue) in hippocampus and cortex of 5xFAD mice. Scale bar, 50 μm. (C) The neuron bandwidth in hippocampus. (D) The neuron number in cortex. (E) The synapse number in hippocampus and cortex. (F) The Pearson’s correlation analysis between synapse number and vessel density. (G) The Pearson’s correlation analysis between synapse number and the co-localization coefficient of GFAP+ astrocytes and CD31+ vessels. (H) The Pearson’s correlation analysis between synapse number and the co-localization coefficient of Iba1+ microglia and Lectin+ vessel. Data in (C-E) are presented as mean ± SD and analyzed by one-way or two-way ANOVA with Turkey’s post-hoc test of multiple groups. p-value: *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig.6NeuNcombined.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6906760/v1/b0c22857efb2025f2660107d.jpg"},{"id":87157358,"identity":"386d1d91-74ac-4f63-9a03-bf1518ce94c3","added_by":"auto","created_at":"2025-07-21 03:28:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":32576212,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6906760/v1/807d031e-2e7c-4f3a-9ed8-cb52c40ddce1.pdf"},{"id":84963539,"identity":"1cec4ae9-358b-450a-86d4-28286804a505","added_by":"auto","created_at":"2025-06-19 09:25:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8638584,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformation20250614.docx","url":"https://assets-eu.researchsquare.com/files/rs-6906760/v1/91171ca023519e033fc3fa5a.docx"},{"id":84963535,"identity":"1ab2656d-608f-406d-8c53-0a5488ad4c78","added_by":"auto","created_at":"2025-06-19 09:25:43","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":294199,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-6906760/v1/29b96e72f471f9b04c548569.png"}],"financialInterests":"There is no conflict of interest","formattedTitle":"Transcranial photobiomodulation induced frequency-specific dual-pathway glial activation for neurovascular protection vs amyloid clearance in Alzheimer’s disease","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlzheimer\u0026rsquo;s disease (AD), as the most common form of dementia worldwide, was mainly characterized by abnormal deposition of β-amyloid (Aβ) plaques and neuronal degeneration \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Numerous studies confirmed that AD was also usually accompanied by cerebral vascular pathology, including the reduction and distortion of small blood vessels, especially capillaries \u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Recent disease-modifying therapies mainly targeted clearing Aβ deposition and showed positive clinical outcomes in early-stage AD patients \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, these monoclonal antibody therapies focused only on reducing Aβ accumulation but failed to ameliorate overall neurodegenerative pathology in the brain. Moreover, anti-Aβ therapies exhibited strong dose-dependency and had the risk of amyloid-related imaging abnormalities (ARIA), including microhemorrhages and cerebral edema \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Therefore, developing novel sustainable therapeutic approaches to complement existing anti-Aβ therapies holds significant value for effective AD intervention.\u003c/p\u003e \u003cp\u003eLight stimulation has emerged in recent years as a promising non-invasive physical therapy for neurological diseases with fewer side effects \u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Transcranial continuous-wave (CW) light stimulation mitigated brain degeneration and counteracted neuroinflammation by enhancing mitochondrial dynamics and preserving neuronal hemoglobin \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e in AD mice. Moreover, CW-NIR light also modulated vascular endothelial growth factor (VEGF)-mediated angiogenesis \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and regulated nitric oxide (NO) release to improve hemodynamics \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. In contrast, the current pulsed-wave (PW) light therapy protocol for AD mice was mainly sensory stimulation related to nerve entrainment, such as visual and auditory stimulation, rather than transcranial irradiation \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Accumulating research has indicated that 40Hz visual flickering stimulation attenuates AD by activating gamma oscillations, triggering a general neuronal protective response that leads to a reduction in Aβ production and an increase in microglial endocytosis to significantly alleviate AD-related pathological changes \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. There is still a lack of research on transcranial PW light stimulation in AD mice. Previous studies focusing on traumatic brain injury and stroke indicate that transcranial PW light may yield superior outcomes compared to CW light \u003csup\u003e\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. However, the differences in therapeutic mechanisms of these two light stimulations for alleviating neurovascular dysfunction in AD remain unknown. Elucidating these differences is necessary for constructing effective light stimulation for the clinical treatment of AD.\u003c/p\u003e \u003cp\u003eGlial cells were strongly implicated in the progression of AD, particularly in vascular dysfunction and synaptic impairment \u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. As the most abundant glial cells in the brain, astrocytes not only regulate synaptic plasticity but also serve as essential components of both the blood-brain barrier (BBB) and neurovascular unit (NVU) \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The astrocytic atrophy has been found in the early stage of AD \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Smaller cell bodies and less complex processes of astrocytes in AD diminished the synaptic maintenance, thereby contributing to synaptic loss and neurotransmission impairment \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. The degeneration of the end-feet structure of astrocytes affected the function of the BBB and the regulation of cerebral blood flow \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Microglia were mainly activated in AD to phagocytose and degrade Aβ aggregates, limiting plaque formation \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Microglia also played an important role in vascular protection, mainly manifested in regulating cerebral blood flow \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e and decreasing cerebral amyloid angiopathy \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Notably, over-activated microglia phagocytose synapses, leading to synaptic loss and also exacerbating neuroinflammation in the late stage of AD \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Recent studies have shown that light stimulation alleviated cerebral vessel impairment \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. However, the specific mechanisms underlying this vascular protective effect and whether they are related to glial cells remain unclear.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the effects of CW and 40Hz NIR light stimulation on glial cells and neurovascular system in 5xFAD mice. The results revealed that both types of NIR light stimulation improved cognitive function, while they were associated with different target mechanisms. CW-NIR light stimulation mainly mediated activation of astrocyte-vascular synergism, promoting vascular remodeling and directly supporting synaptic and neuronal repairment. In contrast, 40Hz-NIR light stimulation achieved efficient Aβ clearance and synaptic protection by reprogramming the spatial distribution of microglia (towards Aβ plaque aggregation) to enhance their local clearance, while decreasing the overall inflammatory burden. These findings highlight the important role of glial cell activation in vascular impairment and synaptic protection in AD mice.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA transcranial near-infrared light (NIR) stimulation device for 5xFAD mice was designed. Before NIR irradiation, the hair of all mice was shaved to make the light source close to the scalp, thereby avoiding the interference of black hair with light penetration. To approximate the experiment conditions at the clinical level, the NIR beam was incident from the prefrontal cortex of 5xFAD mice, without covering eyes and other tissues. During irradiation, 5xFAD mice were restrained in the mouse immobilizer, and the light source was fixed on its movable end (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(B)). The home-built device employed an 810\u0026thinsp;\u0026plusmn;\u0026thinsp;50 nm light source selected through Monte Carlo simulations, demonstrating superior cerebral tissue penetration at this wavelength (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(E)). Two intervention groups were constructed, including CW 810 nm light and 810 nm light pulsed at 40 Hz (duty cycle: 50%) to compare the therapeutic differences between two photon delivery patterns.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eNIR light improved cognitive deficits in 5xFAD mice\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of NIR light on cognitive function in 5xFAD mice, we conducted behavioral assessments after one-month stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(A)). The novel object recognition (NOR) test showed comparable average velocity and total distance between groups (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e(A, B)). The discrimination index represented the recognition of a novel object, reflecting short-term memory and learning ability. We found that each mouse of four groups showed more observation time for the novel object than the familiar one, but the discrimination index (DI) had no significant differences between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(G)). Notably, by comparing typical locomotion trajectories of the same mice between training and testing (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(F)), we found that 5xFAD mice in the CW and 40 Hz groups were more inclined to move around the novel object, suggesting that NIR light alleviated the cognitive dysfunction of AD mice to a certain extent, especially the symptoms of fuzzy memory loss.\u003c/p\u003e \u003cp\u003eThe long-term spatial memory and learning ability were further assessed via a 6-day Morris Water Maze (MWM) test to evaluate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(D)). Both stimulation groups exhibited the improvement of memory ability, with CW and 40Hz group showing significantly reduced escape latencies than untreated 5xFAD mice on day 3 (5xFAD vs. 5xFAD\u0026thinsp;+\u0026thinsp;CW: 57.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.73 vs. 48.32\u0026thinsp;\u0026plusmn;\u0026thinsp;8.26 s, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, 5xFAD vs. 5xFAD\u0026thinsp;+\u0026thinsp;40Hz: 57.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.73 vs. 45.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79 s, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(I)). On Day 4, only 40Hz group exhibited the advantages of escape latencies (5xFAD vs. 5xFAD\u0026thinsp;+\u0026thinsp;40Hz: 52.09\u0026thinsp;\u0026plusmn;\u0026thinsp;4.80 vs. 39.45\u0026thinsp;\u0026plusmn;\u0026thinsp;9.05 s, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Although the escape time gradually decreased as the training days increased in all groups, the latency and proportion of residence time in southwest (SW) quadrant (with the target platform), and average swimming speed had no dramatic changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (J), Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (C, D)). During probe trials, 40Hz group navigated paths comparable to WT mice in platform localization, while the path length of CW group was similar to the control 5xFAD group without light treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(H)). The MWM indicators on the test day had no remarkable differences between multiple groups (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (E-H)). These results from behavioral tests indicated that 40 Hz NIR stimulation enhanced spatial memory acquisition in 5xFAD mice, while CW irradiation showed intermediate efficacy.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNIR light reduced Aβ deposition in 5xFAD mice\u003c/h3\u003e\n\u003cp\u003eTo explore the effects of NIR light on amyloid pathology in 5xFAD mice, we detected Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e levels in the cortex and hippocampus via ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(A)). The results showed that 40Hz NIR light significantly decreased Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e level (5xFAD vs. 5xFAD\u0026thinsp;+\u0026thinsp;40Hz: 100% \u0026plusmn; 74.57% vs. 12.34% \u0026plusmn; 9.20%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in hippocampus, while CW light only exhibited a mild Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e clearance effect (5xFAD vs. 5xFAD\u0026thinsp;+\u0026thinsp;CW: 100% \u0026plusmn; 74.57% vs. 39.89% \u0026plusmn; 19.19%, p\u0026thinsp;=\u0026thinsp;0.10) in hippocampus. Both types of light stimulation showed no significant reductions in Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e levels in cortex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate Aβ burden of specific brain regions, we employed immunofluorescence with Aβ-specific antibody D54D2 to semi-quantitatively assess Aβ deposition in hippocampus and cortex. hippocampus region was differentiated into distinct functional regions, including DG, CA1, CA2, and CA3. Given the fuzzy boundary between CA2 and CA3, we combined the two regions into a single statistical module, designated as CA23. The results showed that Aβ plaques were mostly deposited in the DG region compared to CA1 and CA23 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (B)). Unexpectedly, these two stimulation protocols precisely were the most effective for DG in three subregions, including the reduction of the Aβ plaque number (CW: 47.56%, 40Hz: 41.12%, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (C)) and Aβ area proportion (CW: 59.69%, 40Hz: 37.52%, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (D)). Moreover, 40Hz NIR irradiation significantly reduced the number of Aβ plaques in CA1 (55.99%, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e(C)), while CW light failed to decrease any Aβ burden of CA1. CA23 demonstrated the least response to the therapeutic intervention of CW and 40Hz light, with no significant differences in almost all indicators (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (C-E)). Notably, compared to 40Hz light, CW irradiation more effectively reduced the average size of Aβ in CA1 (181.67%) and CA23 (117.77%), which has not been reported in previous studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (E)). Immunostaining results of cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (F, J)) revealed that CW and 40 Hz irradiation both significantly decreased the number of Aβ plaques in 5xFAD mice (CW: 27.28%, 40Hz: 24.94%, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (G)), compared to the no-irradiation group. The average size of Aβ plaques and the total Aβ plaques area in each ROI were also remarkably decreased in the 40Hz group than the control group (Average size: 12.40%, Total area proportion: 32.15%, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e (H, I)). In contrast, CW group showed a similar average size of Aβ plaques and a significantly lower total area of Aβ plaques in comparison to the control group (Average size: 7.55%, Total area proportion: 28.80%).\u003c/p\u003e\n\u003ch3\u003eNIR light improved vascular impairment in 5xFAD mice\u003c/h3\u003e\n\u003cp\u003eTo assess whether NIR light improved the cognitive function in 5xFAD mice via reducing vascular dysfunction, we marked and reconstructed the vascular network of the whole brain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(A)). The overall qualitative comparison revealed that compared to the control group, both CW and 40Hz NIR light stimulation increased the density of small blood vessels, while exhibiting no effect on large vessels. Moreover, the local vessel images were extracted and reconstructed to analyze the vessel density, vessel length, and vessel mean diameter in different brain regions, including CA1, CA23, DG, and cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(B), Fig. S2(A)). The vessel density was defined as the ratio of vascular volume to the total volume in the region of interest (ROI), which reflected the blood flow and was associated with oxygen supply \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. The vessel length represented the sum of all vessel segment lengths within the ROI, which directly correlated with the complexity of vascular network and morphological changes in vasculature. The vessel mean diameter referred to the average diameter of all vessel segments within the ROI, which served as an indirect indicator of blood perfusion area.\u003c/p\u003e \u003cp\u003eThe quantitative results based on fMOST revealed that NIR light stimulation significantly improved vascular impairment of hippocampus and cortex in 5xFAD mice. CW-NIR and 40Hz-NIR groups both exhibited the higher vessel density (CW: 474.07%, 446.60%, 149.92%, and 304.31%; 40Hz: 141.81%, 117.00%, 63.92%, and 74.29% for CA1, CA23, DG, and cortex, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(C)) and vessel mean diameter (CW: 83.54%, 114.40%, 105.82%, and 100.71%; 40Hz: 21.63%, 33.81%, 28.67%, and 28.88% for CA1, CA23, DG, and cortex, Fig. S2(B)) in each subregion, compared with the control group. In contrast, vascular length exhibited significant regional heterogeneity, potentially attributable to the inclusion of major vessels within the ROIs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(D)). Specifically, 40Hz-NIR light stimulation significantly increased the vascular length in CA1 (33.19%) and cortex (15.77%), slightly enhanced it in CA23 (22.84%) and DG (11.83%). While CW light stimulation increased vascular length in CA1 (64.44%), it significantly reduced vascular length in DG (-24.44%). We also found that both CW and 40Hz light stimulation decreased vascular straightness in CA1 (CW: -2.37%, 40Hz: -1.60%, Fig. S2(C)).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eNIR light promoted glial cell response in 5xFAD mice\u003c/h3\u003e\n\u003cp\u003eNumerous studies revealed that glial cells responded to light stimulation and played a vital role in mediating its biological effects \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. To further investigate the effects of CW and 40Hz NIR light stimulation on glial cells, we performed immunostaining for the astrocyte marker (GFAP and S100B) and microglia marker (Iba1) in hippocampus and cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(A, D)). We observed that CW and 40Hz NIR light both significantly increased GFAP\u0026thinsp;+\u0026thinsp;astrocytes number (CW: 84.62%, 40Hz: 58.82%, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(B)) and their area fraction (CW: 73.05%, 40Hz: 58.95%, Fig. S3(A)) in cortex. Notably, CW irradiation also enhanced astrocyte populations in hippocampus subfields, with GFAP\u0026thinsp;+\u0026thinsp;cell number increasing by 78.70% and area fraction by 90.35% in the DG (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(B), Fig. S3(A)), alongside a 33.60% increase in GFAP\u0026thinsp;+\u0026thinsp;cell size of CA23 region (Fig. S3(B)). S100B\u0026thinsp;+\u0026thinsp;astrocytes exhibited even more pronounced expansion in CA23, with cell number and area fraction respectively expanding by 160.47% and 245.53% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs.5xFAD, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(C), Fig. S3(C)). However, NIR light stimulation had no significant effects on the average size of S100B\u0026thinsp;+\u0026thinsp;astrocytes across all brain regions (Fig. S3(D)).\u003c/p\u003e \u003cp\u003eIn AD, microglia typically undergo activation with somatic hypertrophy to phagocytose Aβ plaques, often accumulating around these deposits. The quantitative analysis of Iba1\u0026thinsp;+\u0026thinsp;microglia revealed that 40Hz-NIR light stimulation selectively reduced total microglia number in the DG subregion (46.00%, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(E)), whereas CW light showed no significant effects on microglial morphology (Fig. S3(E-F)). However, given the close spatial association between microglial distribution and Aβ plaques, we quantified two parameters to control for potential confounding effects from the intergroup differences of Aβ deposition: the area fraction of microglia within plaques, and the average number of microglia around plaques. Intriguingly, 40Hz-NIR light stimulation instead exhibited higher microglial density tightly surrounding Aβ plaques in DG subregion, compared to the 5xFAD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(F)). In CA1 region of hippocampus, 40Hz stimulation doubled peri-plaque microglial numbers (2-fold increase, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e(G)), while CW irradiation induced a 2.5-fold elevation of microglial clustering around plaques in CA23 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). These results suggested that NIR light stimulation robustly activated both astrocyte and microglia, consistent with prior light stimulation studies. However, we further revealed that CW and 40Hz NIR light stimulation exhibited complementary glial cell selectivity, mainly manifested as astrocytic activation dominated under CW irradiation, while 40Hz specifically potentiated microglial Aβ clearance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eNIR light induced connections between glial cells and blood vessels in 5xFAD mice\u003c/h3\u003e\n\u003cp\u003eAccumulating evidence implicated astrocytes and microglia in cerebrovascular network regulation \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Thus, to determine whether NIR-mediated vascular improvements involved glial activation, we conducted co-immunostaining of vascular markers (CD31 or Lectin) and glial markers (GFAP and Iba1). The quantitative colocalization analysis was performed to evaluate the connection between glial cells and vessels. We observed that two-dimensional CD31 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(C)) and Lectin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(H)) labeling confirmed CW-induced vascular density improvement, consistent with fMOST three-dimensional quantification results.\u003c/p\u003e \u003cp\u003eThe colocalization analysis revealed that CW stimulation significantly enhanced the spatial coupling between GFAP\u0026thinsp;+\u0026thinsp;astrocytes and CD31\u0026thinsp;+\u0026thinsp;vessels in both CA1 (co-localization coefficient: 0.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 vs 5xFAD 0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(A, D)) and cortex regions (co-localization coefficient: 0.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 vs 5xFAD 0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(B, D)), but not in CA23 and DG (Fig. S4(A, B)). The increased proportion of CD31 co-localized structures (Fig. S4(D)) without proportional GFAP colocalization changes (Fig. S4(E)) suggested that CW stimulation-induced astrocytic increase facilitated more process extension toward vascular endothelia (Increased co-localized area of GFAP and CD31, Fig. S4(C)). Moreover, the positive correlation (r\u0026thinsp;=\u0026thinsp;0.34, p\u0026thinsp;=\u0026thinsp;0.086, Fig. S6(A, B)) between GFAP\u0026thinsp;+\u0026thinsp;astrocyte number and the co-localization coefficient of GFAP and CD31 further corroborated this observation. We also found that this co-localization coefficient of GFAP and CD31 was positively correlated with CD31\u0026thinsp;+\u0026thinsp;vessel density (r\u0026thinsp;=\u0026thinsp;0.45, p\u0026thinsp;=\u0026thinsp;0.006, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(E)). Together, these data indicated that the vascular protective effects of CW stimulation were possibly mediated through enhanced astrocyte-vascular interactions.\u003c/p\u003e \u003cp\u003eIn contrast, no significant effects of NIR light stimulation were exhibited in the connection of microglia and vessels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(F, G), Fig. S4(F, G)), although the co-localization level of Iba1 and Lectin showed a slight increase in CA1 region of 40Hz-NIR group compared to CW-NIR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(I), Fig. S4(H-J)). However, we observed that the co-localization coefficient between Iba1 and Lectin showed a negative correlation with Lectin\u0026thinsp;+\u0026thinsp;vessel density (r = -0.56, p\u0026thinsp;=\u0026thinsp;0.005, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e(J)), primarily due to NIR-induced vascular density augmentation without concomitant increase of microglia. Notably, this co-localization coefficient also positively correlated with GFAP and CD31 coupling (r\u0026thinsp;=\u0026thinsp;0.45, p\u0026thinsp;=\u0026thinsp;0.015, Fig. S6(E)), which may involve synergistic effects secondary to increased vascular density.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNIR light improved synaptic impairment via activating glial cell\u0026ndash;vascular coupling in 5xFAD mice\u003c/h2\u003e \u003cp\u003eIn AD, Aβ-driven neuronal loss and synaptic dysfunction underlie cognitive decline. To evaluate the neuroprotective effect of NIR light stimulation, immunofluorescence analyses were performed with NeuN\u0026thinsp;+\u0026thinsp;neurons and synaptophysin+ (Syn) synapses. Due to dense hippocampal neuronal packing, we measured neuronal band widths (50 \u0026micro;m intervals) as a proxy for cell numbers. We found that both CW and 40Hz NIR light significantly alleviated NeuN\u0026thinsp;+\u0026thinsp;cell loss in CA23 (CW: 10.57%, 40Hz: 20.37%, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (A, C))) and increased cortical neuron density (CW: 22.95%, 40Hz: 17.35%, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e (A, D)), with no effects in CA1 and DG.\u003c/p\u003e \u003cp\u003eSynaptophysin, as a membrane protein localized on synaptic vesicles, participated in synaptic transmission and maintained synaptic plasticity \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. We observed that both CW and 40Hz light stimulation significantly increased the number of synapses in the cortex (CW: 98.17%, 40Hz: 114.17%, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(B, E)). In contrast, a slight improvement in synaptic density was detected in hippocampus subregions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(E), Fig. S5(A, B)). To further investigate whether the synapse protective effects of NIR light stimulation were mediated by the aforementioned glial cells activation, we analyzed the correlation between synaptic markers and glial cell related indicators (Fig. S6(A)). The results showed that the synapse number had both positive correlation with CD31\u0026thinsp;+\u0026thinsp;vessel density (r\u0026thinsp;=\u0026thinsp;0.38, p\u0026thinsp;=\u0026thinsp;0.034, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(F)) and GFAP-CD31 co-localization coefficient (r\u0026thinsp;=\u0026thinsp;0.36, p\u0026thinsp;=\u0026thinsp;0.047, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(G)), which suggested that enhanced astrocyte-vasculature coupling not only ameliorated vascular damage, but also achieved synaptic protection through improving blood supply. Conversely, synaptic quantitative metrics showed no significant correlation with microglial or their vascular co-localization coefficients (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e(H), Fig. S6(A)). Thus, we inferred that the protective effects of NIR stimulation on synapses were primarily mediated through astrocytes, with no direct involvement of microglia. Furthermore, although previous studies showed that light stimulation triggered the release of NO \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, no significant improvement of nNOS was found in CW and 40Hz groups (Fig. S5(C)).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn summary, we systematically evaluated the effects of CW and 40Hz NIR light stimulation on neurovascular network in 5xFAD mice. The results demonstrated that both modalities improved cognitive deficits, while they engaged distinct glial-mediated modulation pathways. CW-NIR preferentially enhanced astrocyte-vascular interactions, improving structural impairment of cerebral vasculature and providing indirect synaptic protection. In contrast, 40Hz-NIR induced spatial reconfiguration of microglia, driving their targeted migration to Aβ plaque-enriched zones. This microglial repositioning enhanced local Aβ clearance and reduced plaque burden, which collectively protected nervous system. Our findings elucidated the critical role of glial modulation in neurovascular protection in AD, and proposed that NIR stimulation modalities were selectively targeted to specific AD pathologies, astrocyte-mediated vascular-synaptic dysfunction or microglial amyloid clearance. By establishing this modality-dependent mechanistic specificity, our study advances precision light stimulation tailored to the multi-pathological progression of AD.\u003c/p\u003e \u003cp\u003eRecent advances in light stimulation for AD have delineated two principal paradigms \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, including frequency-entrained neural modulation and mitochondrial-targeted transcranial photobiomodulation (tPBM). Iaccarino et al. demonstrated gamma-frequency visual stimulation reduces AD pathology through neural oscillation entrainment \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, while Miao et al. found that tPBM with CW light enhanced meningeal lymphatic Aβ clearance via chromophore activation of mitochondrial \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Our work integrated these two modalities and explored the effects of gamma-frequency (40Hz) tPBM in AD mice. While tPBM traditionally relies on photon-chromophore interactions (e.g., CCO activation) \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e and exhibits biphasic dose-response \u003csup\u003e\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, our data suggested 40Hz tPBM exhibited positive effects on improving cognitive function similar to those of CW light, despite delivering half the total photons (50% duty cycle). tPBM based on PW light has not been extensively investigated in degenerative neurological diseases, but PW was preliminarily attempted in other application scenarios of light stimulation with controversial results, such as traumatic brain injury (TBI), wound healing \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Takahiro et al. found that 10 Hz laser irradiation induced more effective neuroprotection compared to CW and 100 Hz light in TBI mice \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In contrast, CW light was superior to PW irradiation in promoting wound healing \u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In this study, we identified tPBM with PW light also as an effective strategy against AD-induced neurovascular dysfunction, proposing a promising light stimulation modality for neurodegenerative diseases. However, diverging from the potent vascular protective effects of CW light, PW stimulation exerted its cognitive benefits primarily through enhancing microglia-dependent Aβ clearance. One potential explanation for these discrepancies is that light stimulation at non-neural tissues remains mostly dependent on the biological effects of photons, whereas PW-based tPBM additionally stimulates other biological activities (such as neural entrainment), which was not possible with CW.\u003c/p\u003e \u003cp\u003ePrevious studies have established that AD is predominantly accompanied by cerebrovascular dysfunction, characterized by diminished perfusion area and impaired oxygen supply \u003csup\u003e\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. This hypoperfusion state fails to sustain normal neural activity, ultimately precipitating neurodegenerative pathology. Given this potential therapeutic target in AD, many researchers have investigated the vascular-protection effects of light stimulation, focusing on its capacity to induce angiogenesis \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e and restore cerebral hemodynamics \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. However, the effects of light stimulation on the vascular network remain underexplored. Our study employed fMOST to achieve whole-brain cerebrovascular architecture visualization in post-stimulation 5xFAD mice. Our findings revealed that both CW and 40Hz NIR light stimulation significantly ameliorated AD-induced vessel diameter impairment, which provided a structural foundation for cerebral perfusion improvement. Moreover, consistent with prior MOST vessel imaging from Zhang et al \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, we observed significant heterogeneity in vascular complexity across different brain regions in 6-month-old 5xFAD mice. These differences were primarily manifested in vascular complexity, with both DG and cortex regions exhibiting significantly higher vessel length compared to CA1 and CA23 across all experimental groups. Therefore, we suggested that NIR light stimulation may mitigate neuronal injury through augmenting cerebrovascular perfusion, and thereby enhance cognitive function.\u003c/p\u003e \u003cp\u003eHowever, precise mechanistic pathways through which NIR light stimulation modulates vascular architecture and synapses remain incompletely elucidated. The existing evidence supported that astrocytes were the anatomical and functional link between vasculature and nervous system \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. They interfaced with blood vessels, forming the BBB and neurogliovascular unit to regulate cerebral blood flow and energy supply. 40Hz auditory-induced gamma entrainment has been confirmed to robustly activate astrocytes and elicit vascular responses in 5xFAD mice \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Our results revealed that NIR light stimulation also exhibited similar effects and further confirmed that NIR light enhanced structural connectivity between astrocytes and vasculature, which established a critical foundation for protecting or forming additional BBB and NVU structural modules. Astrocytes also regulate synaptic function by controlling neurotransmitter clearance (such as glutamate, GABA, and others), and thereby maintaining synaptic transmission and neuroprotection \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Our findings demonstrated that CW-NIR light stimulation triggered significant astrocyte activation while concurrently exerting synapse-protective effects. Moreover, the connection proportion of astrocyte and vasculature exhibited a positive correlation with synapse density (r\u0026thinsp;=\u0026thinsp;0.36) in 5xFAD mice. Thus, we posit astrocytes as the pivotal cellular mediators through which CW-NIR light stimulation exerts its neurovascular protective effects. In contrast, 40Hz stimulation demonstrated significantly greater efficacy in activating microglia, consistent with transcranial and visual light stimulation outcomes across other studies. Notably, although microglial activation facilitated Aβ clearance, the excessive activation concurrently provoked detrimental neuroinflammatory cascades \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. We found that transcranial 40Hz NIR light stimulation specifically promoted microglia clustering around Aβ plaques, without inducing global microgliosis. This targeted recruitment effectively mitigated the side effects of neuroinflammatory.\u003c/p\u003e \u003cp\u003eIt should be acknowledged that this study still has some limitations. Herein, we propose astrocytes and microglia as putative mediator cells for the modulation effect of NIR light stimulation on neurovascular. However, these findings are mainly phenotype-based, and the specific signaling pathways involved require further genomics-based results. Secondly, although we observed that CW and 40Hz light preferentially engaged different glial populations (astrocytes vs. microglia) to enhance cognition, the mechanistic underpinnings of this selective activation remain elusive. By comparing prior research on PW stimulation and CW tPBM, we infer that this difference may be associated with gating frequency-dependence ion channels, which necessitated further validation through electrophysiological assays and in vitro verification.\u003c/p\u003e \u003cp\u003eTo summarize, our results revealed that both CW and 40Hz transcranial NIR light stimulation enhanced cognitive performance and ameliorated neurovascular impairment, which was mediated through glial cell activation. Specifically, CW light primarily activated astrocytes, augmenting their anatomical connection with vasculature, thereby improving vascular impairment and conferring synaptic protection. In contrast, 40Hz light primarily modulated the spatial distribution of microglia, promoting them clustering around Aβ plaques, which enhanced Aβ clearance and thereby protected neurovascular. Collectively, our findings highlighted the distinct advantages of CW and 40Hz light in cerebrovascular network improvement and amyloid plaque clearance respectively, providing a theoretical foundation for precision light stimulation tailored to AD subtypes with different pathological signatures.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and experimental design\u003c/h2\u003e \u003cp\u003eAdult (five-month-old) male and female 5xFAD mice with an average body weight of 24\u0026ndash;28 g and age-matched littermate wild-type (WT) C57BL/6J mice were utilized in this study \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. All mice were in the C57BL/6J background and obtained from Yangzhou Youdu Biotechnology Co. Ltd (Yangzhou, China). WT mice all received sham light treatment (n\u0026thinsp;=\u0026thinsp;5). All 5xFAD mice were divided into the following three groups: (1) 5xFAD mice with sham NIR light treatment (5xFAD, n\u0026thinsp;=\u0026thinsp;5), (2) 5xFAD mice with CW light treatment (5xFAD\u0026thinsp;+\u0026thinsp;CW, n\u0026thinsp;=\u0026thinsp;5), and (3) 5xFAD mice with 40 Hz light treatment (5xFAD\u0026thinsp;+\u0026thinsp;40Hz, n\u0026thinsp;=\u0026thinsp;5). All mice were housed at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;3 ℃ at constant humidity (40% ~ 70%), with a light/dark cycle of 12 / 12 h and a light phase of 8:00 a.m. to 8:00 p.m. Up to five mice of the same sex and group were fed in each cage, with standard water and food. All interventions in this study complied with the Guide for the Care and Use of Laboratory Animals. The experiment was reviewed and approved by the Institutional Animal Care and Use Committee of the Chinese Academy of Medical Sciences (approval number: IRM/1-IACUC-240510-01). Our light stimulation lasted for 10 min per day and 37 consecutive days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e(A)). In the last 7 days of treatment, we sequentially employed 1-day NOR tests and 6-day MWM tests to evaluate spatial memory and learning ability in mice. After these cognitive function tests, we humanely executed mice and collected brain tissues for subsequent pathological analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTranscranial NIR light stimulation\u003c/h2\u003e \u003cp\u003eThe stimulation device is composed of a control circuit and several LED light sources connected in series. Before TLS, all hair on the mice's heads was gently shaved using depilatory cream. Thus, the light source can be closely aligned with the skin of the mouse's head (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (B)). The irradiation wavelength of the LED was 810\u0026thinsp;\u0026plusmn;\u0026thinsp;50 nm, and the optical power density was 25 mW/cm\u003csup\u003e2\u003c/sup\u003e in both CW and 40 Hz (duty cycle 50%) groups. The previous study showed that 25% of around 810 nm light penetrated the mouse skull with a hairless scalp. Thus, for the CW group, about 6.25 mW/cm\u003csup\u003e2\u003c/sup\u003e reached cerebral tissue, while for the PW group, about 3.125 mW/cm\u003csup\u003e2\u003c/sup\u003e. The control and intervention groups differed only in the absence or presence of NIR light exposure, otherwise, the experimental conditions were identical.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral tests\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eNovel Object Recognition (NOR) test\u003c/h2\u003e \u003cp\u003eThe NOR test, which relies on a rodent's natural preference to explore novelty without additional external reinforcement, has been used to examine cognitive function, especially recognition and memory \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. During the habituation session, mice were placed in the test rooms. After 24 h, two identical non-toxic cylinders were placed in opposite and symmetrical corners of the 40 cm \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e 40 cm \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e 50 cm box (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (C)), then each mouse was released into the arena and allowed to explore freely for 10 min while a camera above the arena recorded its movements. After a 1 h break, one of the previously explored objects was replaced by a novel cuboid, which is consistent in the same height and volume with the familiar one. The mice were returned to explore the arena for another 10 min. The trajectory, time spent exploring each object and total distance traveled were recorded using Tracking Master-V4.0-YM (Shanghai Fanbi Intelligent Technology Co., Shanghai, China). The discrimination index (DI = (T\u003csub\u003enovel\u003c/sub\u003e \u0026ndash; T\u003csub\u003efamiliar\u003c/sub\u003e) / (T\u003csub\u003enovel\u003c/sub\u003e + T\u003csub\u003efamiliar\u003c/sub\u003e), where T\u003csub\u003enovel\u003c/sub\u003e and T\u003csub\u003efamiliar\u003c/sub\u003e indicate the exploration time during testing for the novel and familiar objects, respectively), average velocity, and total distance traveled were calculated and analyzed.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMorris Water Maze (MWM) test\u003c/h2\u003e \u003cp\u003eThe MWM test is a widely used method for assessing spatial learning and memory and related forms of abilities \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. All mice were trained in a 1.5 m open field water maze. The pool was filled to a depth of 30 cm with water maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1 ℃. The escape platform was located 2\u0026thinsp;~\u0026thinsp;3 cm below the surface of the water (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (D)). White edible pigment was added to the water to make the platform invisible during tests. The pool was divided into four quadrants. All mice underwent the learning phase four times daily for four consecutive days. Mice were randomly introduced into different quadrants and allowed to search for the hidden platform for 60 s. The mice were left on the hidden platform for 15 s to memorize the spatial cues if the mice found it on their own or time was over. A test phase was conducted 24 h later with a hidden platform removed from the tank. The percentage of total time the mice spent searching for the platform from the opposite side of the quadrant where the original platform was located, and the number of times they crossed the original platform area within 60 s were recorded. Latency and swimming speed were analyzed. Throughout the experiment, the swimming path of the mice was recorded by Tracking Master-V4.0-MWM (Shanghai Fanbi Intelligent Technology Co., Shanghai, China), which was placed approximately 2.5 m vertically above the center of the circular water trough.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBrain tissue preparation\u003c/h2\u003e \u003cp\u003eAfter the behavioral tests, to eliminate the interference of blood in the immunofluorescence staining experiments, the mouse heart was perfused with cold phosphate-buffered saline (PBS) under deep anesthesia. Then, paraformaldehyde (PFA) was used to fix the brain tissue. Brains were removed and separated into the two hemispheres, which were fixed in 4% PFA or snap frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80 ℃ until processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003ePrior to the testing, mice brains were collected according to brain regions, the hippocampus and cortex. The mouse amyloid beta peptide 1\u0026ndash;42(Aβ\u003csub\u003e1\u0026minus;42\u003c/sub\u003e) ELISA kit (No. CSB-E10787m, CUSABIO) was employed for assessing the expression profiles of Aβ. According to the manufacturer\u0026rsquo;s protocol, 50 mg of brain tissue was added to 450 \u0026micro;L PBS. After grinding thoroughly and centrifuging, the supernatants of the brain tissue were used to measure the absorbance of the resulting solution at 450 nm for each well by a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eThe paraffin-embedded mouse brain was sectioned coronally at a thickness of 4 \u0026micro;m on a pathology sectioning machine. Paraffin sections were sequentially placed in xylene for two times, 20 min each, then the anhydrous ethanol, anhydrous ethanol, 85% alcohol, and 75% alcohol, each for 5 min, and washed with distilled water. The sections were placed in citrate antigen retrieval solution (pH 6.0) in a microwave oven for antigen repair, with medium-high and high heat for 5 min each. After cooling, the slides were immersed in PBS (pH 7.4) with shaking and washed 3 times for 5 min each to perform antigen retrieval. The slides were soaked in PBS with shaking and washing for 3 times for 5 min each. Then the 5% goat serum was added to cover the tissue evenly, and the sections were sealed for 30 minutes at room temperature to perform the serum seal. The primary antibody were β-Amyloid (D54D2) (1:500, 8243T, Cell Signaling Technology), NeuN (1:200, 66836-1-IG, Proteintech), CD31 (1:2000, AB182981, Abcam), GFAP (1:500, 16825-1-AP, Proteintech), S100B (1:500, OB-PPGP015-02, Oasis biofarm), nNOS (1:500, OB-PGP070-02, Oasis biofarm), Synaptophysin (1:500, OB-PRB115-02, Oasis biofarm), Iba1 (1:500, OB-PGP049-02, Oasis biofarm), and Lycopersicon Esculentum (Tomato) Lectin (LEL, TL), DyLight\u0026reg; 594 (1:200, DL-1177-1, Vector Laboratories). Sections were immersed in PBS with shaking and washing for 3 times, 5 min each. Thereafter, sections were incubated with Goat Anti-Rabbit IgG H\u0026amp;L (1:1000, ab6717, Abcam), Goat anti-Guinea pig IgG, AF488 (1:1000, G-GP488, Oasis biofarm), GoraLite Plus 594-Goat anti-Rabbit IgG H\u0026amp;L (1:1000, RGAR004, Proteintech). The sections were soaked in PBS with 0.1 mg/mL DAPI 3 times, 5 min each, with shaking and washing. The sections were sealed with an anti-fluorescence quenching sealer and imaged by confocal laser scanning microscopy (AX / AX R with NSPARC, Nikon) and a panoramic scanner (Panoramic Scan, 3DHISTECH) with a 20 objective. Images were analyzed using ImageJ software (National Institutes of Health, USA) and Imaris 9.9.0 (Bitplane). For each immunofluorescence image, we divided the mouse brain into four regions based on the DAPI image: cortex, hippocampus CA1, hippocampus CA2/3, and hippocampus DG.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eFluorescence micro-optical sectioning tomography (fMOST)\u003c/h2\u003e \u003cp\u003eAfter perfusing with cold PBS and PFA, the mouse hearts were then perfused with a mixture of 9 mg Fluorescein 5-isothiocyanate (ND420177, Bomeibio), 3 g gelatin, and 30 mL 0.01 M PBS. Before the mouse brains were excised, the mouse carcasses were immersed in cold water for 30 min. The LR White resin (AGR1280A, Agar Scientific) was employed to embed the mouse brain. The samples were rinsed with a graded ethanol series (50%, 75%, 100%, and 100% treatments, each incubated for 2 h), and finally in 100% ethanol for 12 h. The next day, the samples were infiltrated with a graded LR White series (50%, 75%, and 100% treatments, each for 2 h), then in 100% LR White for 48 h. Gelatin capsules were used as an embedding mold to polymerize the tissue block. The LR White was allowed to polymerize at 48\u0026deg;C for 24 hours. The fMOST system was used to image the mouse brain layer by layer, with a resolution of 2*0.35*0.35um. The BioMapping 7000 system (Wuhan OE-Bio Co., Ltd.) consisted of a 40\u0026times; Olympus microscope objective, 488nm and 561 nm lasers, and a TDI-CCD camera, which was used for detecting images from the fMOST system. The Imaris program (10.1.0) was used to reconstruct the cerebrovascular network.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using IBM SPSS Statistics 27.0 and GraphPad Prism 9.5 for Windows software. Quantitative results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SEM) or mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). One-way or two-way analysis of variance (ANOVAs) with the least significant difference (LSD) or Tukey\u0026rsquo;s post hoc tests were applied to make the statistical comparisons between groups of behavioral tests and immunofluorescence. Significance levels were set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all analyses.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest.\u003c/h2\u003e \u003cp\u003eWe have no conflicts of interest to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor contributions.\u003c/h2\u003e \u003cp\u003eConceptualization, B.Z., Z.C., and T.L.; methodology, B.Z., Z.C., and L.X.; validation, B.Z., Z.C., S. Y. and T.L.; formal analysis, B.Z., Z.C.; investigation, B.Z., Z.C. and T.L.; resources, B.Z., Z.C.; data curation, B.Z., Z.C.; writing\u0026mdash;original draft preparation, B.Z., Z.C.; writing, review and editing, B.Z., Z.C., T.L., and L.X.; supervision, T.L.; project administration, T.L.; funding acquisition, T.L. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements.\u003c/h2\u003e \u003cp\u003eThis research was funded by Chinese Academy of Medical Science health innovation project (grant nos. 2021-I2M-1-042 and 2021-I2M-1-058).\u003c/p\u003e\u003ch2\u003eData availability statements.\u003c/h2\u003e \u003cp\u003eData underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eScheltens P, De Strooper B, Kivipelto M, Holstege H, Ch\u0026eacute;telat G, Teunissen CE \u003cem\u003eet al.\u003c/em\u003e Alzheimer\u0026rsquo;s disease. \u003cem\u003eThe Lancet\u003c/em\u003e 2021; \u003cstrong\u003e397\u003c/strong\u003e: 1577\u0026ndash;1590.\u003c/li\u003e\n\u003cli\u003eBenilova I, Karran E, De Strooper B. The toxic A\u0026beta; oligomer and Alzheimer\u0026rsquo;s disease: an emperor in need of clothes. \u003cem\u003eNat Neurosci\u003c/em\u003e 2012; \u003cstrong\u003e15\u003c/strong\u003e: 349\u0026ndash;357.\u003c/li\u003e\n\u003cli\u003eReitz C, Brayne C, Mayeux R. Epidemiology of Alzheimer disease. \u003cem\u003eNat Rev Neurol\u003c/em\u003e 2011; \u003cstrong\u003e7\u003c/strong\u003e: 137\u0026ndash;152.\u003c/li\u003e\n\u003cli\u003eNortley R, Korte N, Izquierdo P, Hirunpattarasilp C, Mishra A, Jaunmuktane Z \u003cem\u003eet al.\u003c/em\u003e Amyloid \u0026beta; oligomers constrict human capillaries in Alzheimer\u0026rsquo;s disease via signaling to pericytes. \u003cem\u003eScience\u003c/em\u003e 2019; \u003cstrong\u003e365\u003c/strong\u003e: eaav9518.\u003c/li\u003e\n\u003cli\u003eNielsen RB, Egefjord L, Angleys H, Mouridsen K, Gejl M, M\u0026oslash;ller A \u003cem\u003eet al.\u003c/em\u003e Capillary dysfunction is associated with symptom severity and neurodegeneration in Alzheimer\u0026rsquo;s disease. \u003cem\u003eAlzheimer\u0026rsquo;s \u0026amp;amp; Dementia\u003c/em\u003e 2017; \u003cstrong\u003e13\u003c/strong\u003e: 1143\u0026ndash;1153.\u003c/li\u003e\n\u003cli\u003eZhang X, Yin X, Zhang J, Li A, Gong H, Luo Q \u003cem\u003eet al.\u003c/em\u003e High-resolution mapping of brain vasculature and its impairment in the hippocampus of Alzheimer\u0026rsquo;s disease mice. \u003cem\u003eNational Science Review\u003c/em\u003e 2019; \u003cstrong\u003e6\u003c/strong\u003e: 1223\u0026ndash;1238.\u003c/li\u003e\n\u003cli\u003eSwanson CJ, Zhang Y, Dhadda S, Wang J, Kaplow J, Lai RYK \u003cem\u003eet al.\u003c/em\u003e A randomized, double-blind, phase 2b proof-of-concept clinical trial in early Alzheimer\u0026rsquo;s disease with lecanemab, an anti-A\u0026beta; protofibril antibody. \u003cem\u003eAlz Res Therapy\u003c/em\u003e 2021; \u003cstrong\u003e13\u003c/strong\u003e: 80.\u003c/li\u003e\n\u003cli\u003eVan Dyck CH, Swanson CJ, Aisen P, Bateman RJ, Chen C, Gee M \u003cem\u003eet al.\u003c/em\u003e Lecanemab in Early Alzheimer\u0026rsquo;s Disease. \u003cem\u003eN Engl J Med\u003c/em\u003e 2023; \u003cstrong\u003e388\u003c/strong\u003e: 9\u0026ndash;21.\u003c/li\u003e\n\u003cli\u003eHampel H, Elhage A, Cho M, Apostolova LG, Nicoll JAR, Atri A. Amyloid-related imaging abnormalities (ARIA): radiological, biological and clinical characteristics. \u003cem\u003eBrain\u003c/em\u003e 2023; \u003cstrong\u003e146\u003c/strong\u003e: 4414\u0026ndash;4424.\u003c/li\u003e\n\u003cli\u003eFilippi M, Cecchetti G, Spinelli EG, Vezzulli P, Falini A, Agosta F. Amyloid-Related Imaging Abnormalities and \u0026beta;-Amyloid\u0026ndash;Targeting Antibodies: A Systematic Review. \u003cem\u003eJAMA Neurol\u003c/em\u003e 2022; \u003cstrong\u003e79\u003c/strong\u003e: 291.\u003c/li\u003e\n\u003cli\u003eHamblin MR, Salehpour F. Photobiomodulation of the Brain: Shining Light on Alzheimer\u0026rsquo;s and Other Neuropathological Diseases. \u003cem\u003eJAD\u003c/em\u003e 2021; \u003cstrong\u003e83\u003c/strong\u003e: 1395\u0026ndash;1397.\u003c/li\u003e\n\u003cli\u003eEnengl J, Hamblin MR, Dungel P. Photobiomodulation for Alzheimer\u0026rsquo;s Disease: Translating Basic Research to Clinical Application. \u003cem\u003eJAD\u003c/em\u003e 2020; \u003cstrong\u003e75\u003c/strong\u003e: 1073\u0026ndash;1082.\u003c/li\u003e\n\u003cli\u003eHuang Z, Hamblin MR, Zhang Q. Photobiomodulation in experimental models of Alzheimer\u0026rsquo;s disease: state-of-the-art and translational perspectives. \u003cem\u003eAlz Res Therapy\u003c/em\u003e 2024; \u003cstrong\u003e16\u003c/strong\u003e: 114.\u003c/li\u003e\n\u003cli\u003eYang L, Wu C, Parker E, Li Y, Dong Y, Tucker L \u003cem\u003eet al.\u003c/em\u003e Non-invasive photobiomodulation treatment in an Alzheimer Disease-like transgenic rat model. \u003cem\u003eTheranostics\u003c/em\u003e 2022; \u003cstrong\u003e12\u003c/strong\u003e: 2205\u0026ndash;2231.\u003c/li\u003e\n\u003cli\u003eYang L, Youngblood H, Wu C, Zhang Q. Mitochondria as a target for neuroprotection: role of methylene blue and photobiomodulation. \u003cem\u003eTransl Neurodegener\u003c/em\u003e 2020; \u003cstrong\u003e9\u003c/strong\u003e: 19.\u003c/li\u003e\n\u003cli\u003eTao L, Liu Q, Zhang F, Fu Y, Zhu X, Weng X \u003cem\u003eet al.\u003c/em\u003e Microglia modulation with 1070-nm light attenuates A\u0026beta; burden and cognitive impairment in Alzheimer\u0026rsquo;s disease mouse model. \u003cem\u003eLight Sci Appl\u003c/em\u003e 2021; \u003cstrong\u003e10\u003c/strong\u003e: 179.\u003c/li\u003e\n\u003cli\u003eKeszler A, Lindemer B, Weihrauch D, Jones D, Hogg N, Lohr NL. Red/near infrared light stimulates release of an endothelium dependent vasodilator and rescues vascular dysfunction in a diabetes model. \u003cem\u003eFree Radical Biology and Medicine\u003c/em\u003e 2017; \u003cstrong\u003e113\u003c/strong\u003e: 157\u0026ndash;164.\u003c/li\u003e\n\u003cli\u003eFeng Y, Huang Z, Ma X, Zong X, Tesic V, Ding B \u003cem\u003eet al.\u003c/em\u003e Photobiomodulation Inhibits Ischemia-Induced Brain Endothelial Senescence via Endothelial Nitric Oxide Synthase. \u003cem\u003eAntioxidants\u003c/em\u003e 2024; \u003cstrong\u003e13\u003c/strong\u003e: 633.\u003c/li\u003e\n\u003cli\u003ePark M, Hoang GM, Nguyen T, Lee E, Jung HJ, Choe Y \u003cem\u003eet al.\u003c/em\u003e Effects of transcranial ultrasound stimulation pulsed at 40 Hz on A\u0026beta; plaques and brain rhythms in 5\u0026times;FAD mice. \u003cem\u003eTransl Neurodegener\u003c/em\u003e 2021; \u003cstrong\u003e10\u003c/strong\u003e: 48.\u003c/li\u003e\n\u003cli\u003eZheng L, Yu M, Lin R, Wang Y, Zhuo Z, Cheng N \u003cem\u003eet al.\u003c/em\u003e Rhythmic light flicker rescues hippocampal low gamma and protects ischemic neurons by enhancing presynaptic plasticity. \u003cem\u003eNat Commun\u003c/em\u003e 2020; \u003cstrong\u003e11\u003c/strong\u003e: 3012.\u003c/li\u003e\n\u003cli\u003eSinger AC, Martorell AJ, Douglas JM, Abdurrob F, Attokaren MK, Tipton J \u003cem\u003eet al.\u003c/em\u003e Noninvasive 40-Hz light flicker to recruit microglia and reduce amyloid beta load. \u003cem\u003eNat Protoc\u003c/em\u003e 2018; \u003cstrong\u003e13\u003c/strong\u003e: 1850\u0026ndash;1868.\u003c/li\u003e\n\u003cli\u003eShen Q, Wu X, Zhang Z, Zhang D, Yang S, Xing D. Gamma frequency light flicker regulates amyloid precursor protein trafficking for reducing \u0026beta;‐amyloid load in Alzheimer\u0026rsquo;s disease model. \u003cem\u003eAging Cell\u003c/em\u003e 2022; \u003cstrong\u003e21\u003c/strong\u003e. doi:10.1111/acel.13573.\u003c/li\u003e\n\u003cli\u003eIaccarino HF, Singer AC, Martorell AJ, Rudenko A, Gao F, Gillingham TZ \u003cem\u003eet al.\u003c/em\u003e Gamma frequency entrainment attenuates amyloid load and modifies microglia. \u003cem\u003eNature\u003c/em\u003e 2016; \u003cstrong\u003e540\u003c/strong\u003e: 230\u0026ndash;235.\u003c/li\u003e\n\u003cli\u003eMartorell AJ, Paulson AL, Suk H-J, Abdurrob F, Drummond GT, Guan W \u003cem\u003eet al.\u003c/em\u003e Multi-sensory Gamma Stimulation Ameliorates Alzheimer\u0026rsquo;s-Associated Pathology and Improves Cognition. \u003cem\u003eCell\u003c/em\u003e 2019; \u003cstrong\u003e177\u003c/strong\u003e: 256-271.e22.\u003c/li\u003e\n\u003cli\u003eHamblin MR. Photobiomodulation for traumatic brain injury and stroke. \u003cem\u003eJ Neuro Res\u003c/em\u003e 2018; \u003cstrong\u003e96\u003c/strong\u003e: 731\u0026ndash;743.\u003c/li\u003e\n\u003cli\u003eAndo T, Xuan W, Xu T, Dai T, Sharma SK, Kharkwal GB \u003cem\u003eet al.\u003c/em\u003e Comparison of Therapeutic Effects between Pulsed and Continuous Wave 810-nm Wavelength Laser Irradiation for Traumatic Brain Injury in Mice. \u003cem\u003ePLoS ONE\u003c/em\u003e 2011; \u003cstrong\u003e6\u003c/strong\u003e: e26212.\u003c/li\u003e\n\u003cli\u003eLapchak PA, Salgado KF, Chao CH, Zivin JA. Transcranial near-infrared light therapy improves motor function following embolic strokes in rabbits: an extended therapeutic window study using continuous and pulse frequency delivery modes. \u003cem\u003eNeuroscience\u003c/em\u003e 2007; \u003cstrong\u003e148\u003c/strong\u003e: 907\u0026ndash;914.\u003c/li\u003e\n\u003cli\u003eVerkhratsky A, Olabarria M, Noristani HN, Yeh C-Y, Rodriguez JJ. Astrocytes in Alzheimer\u0026rsquo;s Disease. \u003cem\u003eNeurotherapeutics\u003c/em\u003e 2010; \u003cstrong\u003e7\u003c/strong\u003e: 399\u0026ndash;412.\u003c/li\u003e\n\u003cli\u003eKimbrough IF, Robel S, Roberson ED, Sontheimer H. Vascular amyloidosis impairs the gliovascular unit in a mouse model of Alzheimer\u0026rsquo;s disease. \u003cem\u003eBrain\u003c/em\u003e 2015; \u003cstrong\u003e138\u003c/strong\u003e: 3716\u0026ndash;3733.\u003c/li\u003e\n\u003cli\u003eDudvarski Stankovic N, Teodorczyk M, Ploen R, Zipp F, Schmidt MHH. Microglia\u0026ndash;blood vessel interactions: a double-edged sword in brain pathologies. \u003cem\u003eActa Neuropathol\u003c/em\u003e 2016; \u003cstrong\u003e131\u003c/strong\u003e: 347\u0026ndash;363.\u003c/li\u003e\n\u003cli\u003eKiani Shabestari S, Morabito S, Danhash EP, McQuade A, Sanchez JR, Miyoshi E \u003cem\u003eet al.\u003c/em\u003e Absence of microglia promotes diverse pathologies and early lethality in Alzheimer\u0026rsquo;s disease mice. \u003cem\u003eCell Reports\u003c/em\u003e 2022; \u003cstrong\u003e39\u003c/strong\u003e: 110961.\u003c/li\u003e\n\u003cli\u003eWilliamson MR, Fuertes CJA, Dunn AK, Drew MR, Jones TA. Reactive astrocytes facilitate vascular repair and remodeling after stroke. \u003cem\u003eCell Reports\u003c/em\u003e 2021; \u003cstrong\u003e35\u003c/strong\u003e: 109048.\u003c/li\u003e\n\u003cli\u003eSchumacher S, Tahiri H, Ezan P, Rouach N, Witschas K, Leybaert L. Inhibiting astrocyte connexin‐43 hemichannels blocks radiation‐induced vesicular VEGF‐A release and blood\u0026ndash;brain barrier dysfunction. \u003cem\u003eGlia\u003c/em\u003e 2024; \u003cstrong\u003e72\u003c/strong\u003e: 34\u0026ndash;50.\u003c/li\u003e\n\u003cli\u003eYeh C-Y, Vadhwana B, Verkhratsky A, Rodr\u0026iacute;guez JJ. Early Astrocytic Atrophy in the Entorhinal Cortex of a Triple Transgenic Animal Model of Alzheimer\u0026rsquo;s Disease. \u003cem\u003eASN Neuro\u003c/em\u003e 2011; \u003cstrong\u003e3\u003c/strong\u003e: AN20110025.\u003c/li\u003e\n\u003cli\u003eOlabarria M, Noristani HN, Verkhratsky A, Rodr\u0026iacute;guez JJ. Concomitant astroglial atrophy and astrogliosis in a triple transgenic animal model of Alzheimer\u0026rsquo;s disease. \u003cem\u003eGlia\u003c/em\u003e 2010; \u003cstrong\u003e58\u003c/strong\u003e: 831\u0026ndash;838.\u003c/li\u003e\n\u003cli\u003eOta Y, Zanetti AT, Hallock RM. The Role of Astrocytes in the Regulation of Synaptic Plasticity and Memory Formation. \u003cem\u003eNeural Plasticity\u003c/em\u003e 2013; \u003cstrong\u003e2013\u003c/strong\u003e: 1\u0026ndash;11.\u003c/li\u003e\n\u003cli\u003ePopov A, Brazhe A, Denisov P, Sutyagina O, Li L, Lazareva N \u003cem\u003eet al.\u003c/em\u003e Astrocyte dystrophy in ageing brain parallels impaired synaptic plasticity. \u003cem\u003eAging Cell\u003c/em\u003e 2021; \u003cstrong\u003e20\u003c/strong\u003e: e13334.\u003c/li\u003e\n\u003cli\u003eUchihara T, Akiyama H, Kondo H, Ikeda K. Activated Microglial Cells Are Colocalized With Perivascular Deposits of Amyloid-\u0026beta; Protein in Alzheimer\u0026rsquo;s Disease Brain. \u003cem\u003eStroke\u003c/em\u003e 1997; \u003cstrong\u003e28\u003c/strong\u003e: 1948\u0026ndash;1950.\u003c/li\u003e\n\u003cli\u003eMartorell AJ, Paulson AL, Suk H-J, Abdurrob F, Drummond GT, Guan W \u003cem\u003eet al.\u003c/em\u003e Multi-sensory Gamma Stimulation Ameliorates Alzheimer\u0026rsquo;s-Associated Pathology and Improves Cognition. \u003cem\u003eCell\u003c/em\u003e 2019; \u003cstrong\u003e177\u003c/strong\u003e: 256-271.e22.\u003c/li\u003e\n\u003cli\u003eCs\u0026aacute;sz\u0026aacute;r E, L\u0026eacute;n\u0026aacute;rt N, Cser\u0026eacute;p C, K\u0026ouml;rnyei Z, Fekete R, P\u0026oacute;sfai B \u003cem\u003eet al.\u003c/em\u003e Microglia modulate blood flow, neurovascular coupling, and hypoperfusion via purinergic actions. \u003cem\u003eJournal of Experimental Medicine\u003c/em\u003e 2022; \u003cstrong\u003e219\u003c/strong\u003e: e20211071.\u003c/li\u003e\n\u003cli\u003eVan Den Brink H, Voigt S, Kozberg M, Van Etten ES. The role of neuroinflammation in cerebral amyloid angiopathy. \u003cem\u003eeBioMedicine\u003c/em\u003e 2024; \u003cstrong\u003e110\u003c/strong\u003e: 105466.\u003c/li\u003e\n\u003cli\u003eHansen DV, Hanson JE, Sheng M. Microglia in Alzheimer\u0026rsquo;s disease. \u003cem\u003eJournal of Cell Biology\u003c/em\u003e 2018; \u003cstrong\u003e217\u003c/strong\u003e: 459\u0026ndash;472.\u003c/li\u003e\n\u003cli\u003eWu Y, Ke J, Ye S, Shan L-L, Xu S, Guo S-F \u003cem\u003eet al.\u003c/em\u003e 3D Visualization of Whole Brain Vessels and Quantification of Vascular Pathology in a Chronic Hypoperfusion Model Causing White Matter Damage. \u003cem\u003eTransl Stroke Res\u003c/em\u003e 2023. doi:10.1007/s12975-023-01157-1.\u003c/li\u003e\n\u003cli\u003eChen H, Li N, Liu N, Zhu H, Ma C, Ye Y \u003cem\u003eet al.\u003c/em\u003e Photobiomodulation modulates mitochondrial energy metabolism and ameliorates neurological damage in an APP/PS1 mousmodel of Alzheimer\u0026rsquo;s disease. \u003cem\u003eAlz Res Therapy\u003c/em\u003e 2025; \u003cstrong\u003e17\u003c/strong\u003e: 72.\u003c/li\u003e\n\u003cli\u003eWang M, Yan C, Li X, Yang T, Wu S, Liu Q \u003cem\u003eet al.\u003c/em\u003e Non-invasive modulation of meningeal lymphatics ameliorates ageing and Alzheimer\u0026rsquo;s disease-associated pathology and cognition in mice. \u003cem\u003eNat Commun\u003c/em\u003e 2024; \u003cstrong\u003e15\u003c/strong\u003e: 1453.\u003c/li\u003e\n\u003cli\u003eHamblin M. Photobiomodulation for Alzheimer\u0026rsquo;s Disease: Has the Light Dawned? \u003cem\u003ePhotonics\u003c/em\u003e 2019; \u003cstrong\u003e6\u003c/strong\u003e: 77.\u003c/li\u003e\n\u003cli\u003eXu H, Luo Z, Zhang R, Golovynska I, Huang Y, Samanta S \u003cem\u003eet al.\u003c/em\u003e Exploring the effect of photobiomodulation and gamma visual stimulation induced by 808 nm and visible LED in Alzheimer\u0026rsquo;s disease mouse model. \u003cem\u003eJournal of Photochemistry and Photobiology B: Biology\u003c/em\u003e 2024; \u003cstrong\u003e250\u003c/strong\u003e: 112816.\u003c/li\u003e\n\u003cli\u003eLin H, Li D, Zhu J, Liu S, Li J, Yu T \u003cem\u003eet al.\u003c/em\u003e Transcranial photobiomodulation for brain diseases: review of animal and human studies including mechanisms and emerging trends. \u003cem\u003eNeurophoton\u003c/em\u003e 2024; \u003cstrong\u003e11\u003c/strong\u003e. doi:10.1117/1.NPh.11.1.010601.\u003c/li\u003e\n\u003cli\u003eStepanov YV, Golovynska I, Zhang R, Golovynskyi S, Stepanova LI, Gorbach O \u003cem\u003eet al.\u003c/em\u003e Near-infrared light reduces \u0026beta;-amyloid-stimulated microglial toxicity and enhances survival of neurons: mechanisms of light therapy for Alzheimer\u0026rsquo;s disease. \u003cem\u003eAlz Res Therapy\u003c/em\u003e 2022; \u003cstrong\u003e14\u003c/strong\u003e: 84.\u003c/li\u003e\n\u003cli\u003eZhang R, Zhou T, Liu L, Ohulchanskyy TY, Qu J. Dose\u0026ndash;effect relationships for PBM in the treatment of Alzheimer\u0026rsquo;s disease. \u003cem\u003eJ Phys D: Appl Phys\u003c/em\u003e 2021; \u003cstrong\u003e54\u003c/strong\u003e: 353001.\u003c/li\u003e\n\u003cli\u003eSpera V, Sitnikova T, Ward MJ, Farzam P, Hughes J, Gazecki S \u003cem\u003eet al.\u003c/em\u003e Pilot Study on Dose-Dependent Effects of Transcranial Photobiomodulation on Brain Electrical Oscillations: A Potential Therapeutic Target in Alzheimer\u0026rsquo;s Disease. \u003cem\u003eJAD\u003c/em\u003e 2021; \u003cstrong\u003e83\u003c/strong\u003e: 1481\u0026ndash;1498.\u003c/li\u003e\n\u003cli\u003eHashmi JT, Huang Y, Sharma SK, Kurup DB, De Taboada L, Carroll JD \u003cem\u003eet al.\u003c/em\u003e Effect of pulsing in low‐level light therapy. \u003cem\u003eLasers Surg Med\u003c/em\u003e 2010; \u003cstrong\u003e42\u003c/strong\u003e: 450\u0026ndash;466.\u003c/li\u003e\n\u003cli\u003eAl-Watban FAH, Zhang XY. The Comparison of Effects between Pulsed and CW Lasers on Wound Healing. \u003cem\u003eJournal of Clinical Laser Medicine \u0026amp; Surgery\u003c/em\u003e 2004; \u003cstrong\u003e22\u003c/strong\u003e: 15\u0026ndash;18.\u003c/li\u003e\n\u003cli\u003eWinchester LM, Powell J, Lovestone S, Nevado-Holgado AJ. Red blood cell indices and anaemia as causative factors for cognitive function deficits and for Alzheimer\u0026rsquo;s disease. \u003cem\u003eGenome Med\u003c/em\u003e 2018; \u003cstrong\u003e10\u003c/strong\u003e: 51.\u003c/li\u003e\n\u003cli\u003eMeyer EP, Ulmann-Schuler A, Staufenbiel M, Krucker T. Altered morphology and 3D architecture of brain vasculature in a mouse model for Alzheimer\u0026rsquo;s disease. \u003cem\u003eProc Natl Acad Sci USA\u003c/em\u003e 2008; \u003cstrong\u003e105\u003c/strong\u003e: 3587\u0026ndash;3592.\u003c/li\u003e\n\u003cli\u003eKimbrough IF, Robel S, Roberson ED, Sontheimer H. Vascular amyloidosis impairs the gliovascular unit in a mouse model of Alzheimer\u0026rsquo;s disease. \u003cem\u003eBrain\u003c/em\u003e 2015; \u003cstrong\u003e138\u003c/strong\u003e: 3716\u0026ndash;3733.\u003c/li\u003e\n\u003cli\u003eSaucedo CL, Courtois EC, Wade ZS, Kelley MN, Kheradbin N, Barrett DW \u003cem\u003eet al.\u003c/em\u003e Transcranial laser stimulation: Mitochondrial and cerebrovascular effects in younger and older healthy adults. \u003cem\u003eBrain Stimulation\u003c/em\u003e 2021; \u003cstrong\u003e14\u003c/strong\u003e: 440\u0026ndash;449.\u003c/li\u003e\n\u003cli\u003ePreman P, Alfonso-Triguero M, Alberdi E, Verkhratsky A, Arranz AM. Astrocytes in Alzheimer\u0026rsquo;s Disease: Pathological Significance and Molecular Pathways. \u003cem\u003eCells\u003c/em\u003e 2021; \u003cstrong\u003e10\u003c/strong\u003e: 540.\u003c/li\u003e\n\u003cli\u003eAndersen JV, Schousboe A, Verkhratsky A. Astrocyte energy and neurotransmitter metabolism in Alzheimer\u0026rsquo;s disease: Integration of the glutamate/GABA-glutamine cycle. \u003cem\u003eProgress in Neurobiology\u003c/em\u003e 2022; \u003cstrong\u003e217\u003c/strong\u003e: 102331.\u003c/li\u003e\n\u003cli\u003eOakley H, Cole SL, Logan S, Maus E, Shao P, Craft J \u003cem\u003eet al.\u003c/em\u003e Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer\u0026rsquo;s disease mutations: potential factors in amyloid plaque formation. \u003cem\u003eJ Neurosci\u003c/em\u003e 2006; \u003cstrong\u003e26\u003c/strong\u003e: 10129\u0026ndash;10140.\u003c/li\u003e\n\u003cli\u003eLeger M, Quiedeville A, Bouet V, Haelewyn B, Boulouard M, Schumann-Bard P \u003cem\u003eet al.\u003c/em\u003e Object recognition test in mice. \u003cem\u003eNat Protoc\u003c/em\u003e 2013; \u003cstrong\u003e8\u003c/strong\u003e: 2531\u0026ndash;2537.\u003c/li\u003e\n\u003cli\u003eZhang R, Xue G, Wang S, Zhang L, Shi C, Xie X. Novel object recognition as a facile behavior test for evaluating drug effects in A\u0026beta;PP/PS1 Alzheimer\u0026rsquo;s disease mouse model. \u003cem\u003eJ Alzheimers Dis\u003c/em\u003e 2012; \u003cstrong\u003e31\u003c/strong\u003e: 801\u0026ndash;812.\u003c/li\u003e\n\u003cli\u003eVorhees CV, Williams MT. Morris water maze: procedures for assessing spatial and related forms of learning and memory. \u003cem\u003eNat Protoc\u003c/em\u003e 2006; \u003cstrong\u003e1\u003c/strong\u003e: 848\u0026ndash;858.\u003c/li\u003e\n\u003cli\u003eNehra G, Promsan S, Yubolphan R, Chumboatong W, Vivithanaporn P, Maloney BJ \u003cem\u003eet al.\u003c/em\u003e Cognitive decline, A\u0026beta; pathology, and blood-brain barrier function in aged 5xFAD mice. \u003cem\u003eFluids Barriers CNS\u003c/em\u003e 2024; \u003cstrong\u003e21\u003c/strong\u003e: 29.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer’s disease, Transcranial photobiomodulation, glial cell activation, vascular protection","lastPublishedDoi":"10.21203/rs.3.rs-6906760/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6906760/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eVascular network impairment is a critical driver of Alzheimer’s Disease (AD) progression, yet strategies to effectively regulate vascular pathology remain unclear. Here, we attempted to employ transcranial continuous-wave (CW) and 40Hz near-infrared (NIR) light stimulation to target vascular network in 5xFAD mice. The results showed that NIR light significantly ameliorated cognitive dysfunction via enhancing β-amyloid (Aβ) plaque clearance and providing neurovascular protection. Glial cell activation served as the primary mediator through which NIR light achieved these modulation effects. The colocalization and correlational analyses revealed that CW-NIR light primarily activated astrocyte-vascular synergism to ameliorate vascular dysfunction, thereby conferring synaptic protection. In contrast, 40Hz light primarily activated microglia to increase their aggregation around Aβ plaque, which enhanced Aβ local clearance. These differential mediating pathways suggested that CW and 40Hz exhibited modality-specific therapeutic advantages for distinct AD pathological hallmarks – vascular dysfunction and Aβ plaque deposition, respectively. These findings potentially offer a precision light stimulation strategy targeting different neurodegenerative hallmarks.\u003c/p\u003e","manuscriptTitle":"Transcranial photobiomodulation induced frequency-specific dual-pathway glial activation for neurovascular protection vs amyloid clearance in Alzheimer’s disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-19 09:25:38","doi":"10.21203/rs.3.rs-6906760/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fb2b57ce-b99c-4245-acd4-22e9df72e9ca","owner":[],"postedDate":"June 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50159964,"name":"Physical sciences/Optics and photonics/Other photonics/Biophotonics"},{"id":50159965,"name":"Physical sciences/Optics and photonics/Lasers, LEDs and light sources"}],"tags":[],"updatedAt":"2025-07-21T03:20:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-19 09:25:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6906760","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6906760","identity":"rs-6906760","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}