Non-invasive modulation of meningeal lymphatics ameliorates ageing and Alzheimer's disease-associated pathology and cognition in mice

The brain was once considered immune-privileged due to the absence of a lymphatic drainage system. However, the discovery of meningeal lymphatic vessels (mLVs) in the dura mater in 2015 revealed an extensive lymphatic network responsible for removing macromolecular waste and inflammatory mediators, transporting immune cells, and coordinating immune responses in the central nervous system (CNS). Subsequent research linked mLVs to aging, Alzheimer's disease (AD), Parkinson's disease (PD), traumatic brain injury (TBI), subarachnoid hemorrhage (SAH), CNS viral infections, and other neurological diseases. The efficiency of mLVs transport significantly impacts disease progression, suggesting that modulating mLVs drainage could be a therapeutic strategy for neurological disorders. AD, a major age-associated neurodegenerative disease, is characterized by abnormal β-amyloid (Aβ) aggregation and neuronal tangles, leading to neuronal dysfunction and cognitive decline. Studies indicate functional degeneration of mLVs with aging and AD progression, potentially contributing to cognitive dysfunction and neural impairment. While invasive therapies like viral-mediated vascular endothelial growth factor C (VEGF-C) treatment can boost mLVs function, improving lymphatic drainage and cognitive ability, these are not feasible for chronic and progressive diseases like AD. Therefore, non-invasive treatment modalities are crucial for AD therapy. The superficial distribution of the mLVs network in the dura mater makes it an ideal target for transcranial neuromodulation therapies. This study investigates the use of near-infrared light to modulate meningeal lymphatic endothelial cell (mLEC) function, impacting mLVs drainage, and subsequently, the pathology and cognitive function of aged and AD mice.

Existing research has established the link between meningeal lymphatic dysfunction and various neurological conditions. Studies have shown impaired meningeal lymphatic drainage in Alzheimer's disease, Parkinson's disease, traumatic brain injury, and subarachnoid hemorrhage. Furthermore, the role of meningeal lymphatics in clearing amyloid beta plaques in Alzheimer's disease has been highlighted. These findings support the notion that enhancing meningeal lymphatic drainage could be a beneficial therapeutic approach for these diseases. However, current methods for enhancing lymphatic drainage are often invasive and not suitable for chronic treatment. This study focuses on developing a non-invasive method for modulating meningeal lymphatic function.

This study used C57BL/6J mice, including aged (15-17 months), young (1.5 months), 5xFAD (6 months), and APP/PS1 (11 months) mice and their respective controls. Aged and AD mice received transcranial near-infrared light treatment (808 nm, 20 mW/cm²) for four weeks (three times per week, 10 minutes per session). Control groups received indoor lighting. Lymphatic drainage was assessed via fluorescence intensity detection of CSF tracers (OVA-A647 or OVA-ICG) in deep cervical lymph nodes (dCLNs) using in vivo imaging. mLV structure was evaluated using immunohistochemistry (LYVE-1 staining). Cognitive function was assessed using open field (OF), novel object localization (NOL), novel object recognition (NOR), and Y-maze tests, and the Morris water maze (MWM) test. AD-associated pathology (Aβ deposition, neuroinflammation, neuronal damage) was assessed using immunostaining (Aβ1-42, Iba1, NeuN, synaptophysin, MAP2). Mitochondrial homeostasis in mLECs was examined using transmission electron microscopy (TEM) and measurements of mitochondrial superoxide generation (MitoSOX) and mitochondrial membrane potential (TMRE). RNA sequencing (RNA-seq) was performed on hippocampal and meningeal tissues and isolated mLECs to analyze gene expression changes. In a subset of experiments, mLVs were ablated using Visudyne photoconversion to determine the role of mLVs in the light treatment effects. Statistical analyses included two-tailed unpaired Student's t-tests, one-way ANOVA, and two-way ANOVA.

Near-infrared light treatment significantly improved cognitive function in both aged and AD mice, as demonstrated by improved performance in behavioral tests (NOL, NOR, Y-maze, MWM). Light treatment reduced Aβ deposition, neuroinflammation (microglia activation), and neuronal damage in the hippocampus and prefrontal cortex of AD mice, mitigating AD-associated pathology. Transmission electron microscopy revealed that near-infrared light treatment restored mitochondrial morphology and improved cellular junctions in mLECs of AD mice, indicating improved mitochondrial function. RNA sequencing analysis identified significant changes in gene expression in both hippocampal and meningeal tissues and isolated mLECs of AD mice following light treatment. Specifically, genes related to oxidative phosphorylation, cell adhesion, and mitochondrial metabolism were upregulated, suggesting improved mitochondrial function and lymphatic drainage. Importantly, the beneficial effects of light treatment on cognition and AD-associated pathology were abolished after mLV ablation, demonstrating that the improvements were dependent on functional mLVs. The study also showed that light treatment enhanced the drainage of CSF tracers into cervical lymph nodes, suggesting an improvement in both meningeal and glymphatic drainage.

This study demonstrates that non-invasive transcranial near-infrared light treatment can effectively improve cognitive function and ameliorate AD-associated pathology in mice by enhancing meningeal lymphatic drainage. The findings highlight the critical role of mLVs in AD pathogenesis and suggest that targeting mLVs through non-invasive photomodulation could be a promising therapeutic approach. The mechanism appears to involve the restoration of mLEC mitochondrial function and cellular junctions, leading to improved lymphatic drainage capacity and clearance of Aβ. The observed changes in gene expression related to oxidative phosphorylation and cell adhesion further support this mechanism. The study's success in a non-invasive approach holds significant promise for translation to clinical settings. However, it's important to consider that other mechanisms, such as regulation of immune cells or direct effects on neuronal activity, may also contribute to the observed effects.

This study presents a novel non-invasive approach to treat neurodegenerative diseases by targeting meningeal lymphatics. Near-infrared light treatment effectively improved cognitive function and reduced AD-associated pathology in mice by restoring mLEC function and enhancing lymphatic drainage. This approach offers a promising therapeutic strategy for AD and other neurological conditions characterized by impaired lymphatic drainage. Further studies are needed to investigate the long-term effects of this treatment, optimize parameters, and explore potential applications in humans.

This study was conducted in mouse models of AD and aging. The results may not be directly generalizable to humans. The long-term effects of near-infrared light treatment are still unknown and require further investigation. The specific photoreceptors and downstream signaling pathways involved in the mechanism of action need further elucidation. The study mainly focused on the effects on the meningeal lymphatic system, while other potential mechanisms, such as direct effects on neuronal activity or immune system modulation, could also be further explored. Finally, the penetration depth of the near-infrared light used in this study was limited to the superficial layers of the brain. Further research is required to optimize the wavelength and intensity of the light to enhance penetration depth.