Photobiomodulation Under Electroencephalographic Controls of Sleep for Stimulation of Lymphatic Removal of Toxins from Mouse Brain
Summary
This study presents the non-invasive and portable technology of transcranial photobiomodulation under electroencephalographic control for stimulation lymphatic removal of toxins (e.g., soluble amyloid beta) from the brain of aged and non-anesthetized BALB/c male mice during natural deep sleep.
Abstract
The meningeal lymphatic vessels (MLVs) play an important role in the removal of toxins from the brain. The development of innovative technologies for the stimulation of MLV functions is a promising direction in the progress of the treatment of various brain diseases associated with MLV abnormalities, including Alzheimer's and Parkinson's diseases, brain tumors, traumatic brain injuries, and intracranial hemorrhages. Sleep is a natural state when the brain's drainage processes are most active. Therefore, stimulation of the brain's drainage and MLVs during sleep may have the most pronounced therapeutic effects. However, such commercial technologies do not currently exist.
This study presents a new portable technology of transcranial photobiomodulation (tPBM) under electroencephalographic (EEG) control of sleep designed to photo-stimulate removal of toxins (e.g., soluble amyloid beta (Aβ)) from the brain of aged BALB/c mice with the ability to compare the therapeutic effectiveness of different optical resources. The technology can be used in the natural condition of a home cage without anesthesia, maintaining the motor activity of mice. These data open up new prospects for developing non-invasive and clinically promising photo-technologies for the correction of age-related changes in the MLV functions and brain's drainage processes and for effectively cleansing brain tissues from metabolites and toxins. This technology is intended both for preclinical studies of the functions of the sleeping brain and for developing clinically relevant treatments for sleep-related brain diseases.
Introduction
Meningeal lymphatic vessels (MLVs) play an important role in the removal of toxins and metabolites from brain tissues1,2,3. Damage of MLVs in various brain diseases, including tumors, traumatic brain injuries, hemorrhages, and neurodegenerative processes, is accompanied by a decrease in the MLV functions leading to the progression of these pathologies1,2,3,4,5,6. Therefore, the development of methods for the stimulation of MLVs opens new horizons in the emergence of effective technologies for the treatment of brain diseases. Recently, non-invasive technology for effective transcranial photobiomodulation (tPBM) has been proposed to stimulate MLVs and remove toxins such as blood and Aβ from the brain5,7,8,9,10,11,12. It is interesting to note that deep sleep is a natural factor for the activation of lymphatic drainage processes in the brain13,14. Based on this fact, it is logical to assume that the tPBM of MLVs during sleep may have more effective therapeutic effects than during wakefulness9,11,12,15. However, there are currently no commercial technologies for tPBM during sleep16. In addition, animal experiments to study the therapeutic effects of tPBM are performed under anesthesia, which is required to accurately deliver light to the brain. However, anesthesia significantly affects the brain's drainage, which reduces the quality of research results17.
Aβ is a metabolic product of normal neural activity18. As it was established in cultured rat cortical neurons, Aβ is released from them at high rates into the extracellular space (2-4 molecules/neuron/s for Aβ)19. There is evidence that the dissolved form of Aβ, located in the extracellular and perivascular spaces, is most toxic to neurons and synapses20. The soluble Aβ is rapidly cleared from the human brain during 1-2.5 h21. MLVs are the tunnels for removal of the soluble Aβ from the brain1,7 that declines with age, leading to the accumulation of Aβ in the aged brain1,22. There is evidence that extracellular abnormalities of Aβ levels in the brain correlate with cognitive performance in aging and are associated with the development of Alzheimer's disease (AD)23,24. Therefore, aged and old rodents are considered alternatives to transgenic models for the study of amyloidosis, including AD25,26.
This study presents an original and portable tPBM technology under electroencephalographic (EEG) control of deep or non-rapid eye movement (NREM) sleep in non-anesthetized male BALB/c mice of different ages to stimulate lymphatic clearance of Aβ from the brain into the peripheral lymphatic system (the deep cervical lymph nodes, dcLNs).
Protocol
Representative Results
Discussion
MLVs are an important target for the development of innovative technologies for modulation of the brain's drainage and removal of cellular debris and wastes from the brain, especially in aged subjects whose MLV function declines1,22. In a homeostatic state, deep sleep is associated with the natural activation of brain tissue cleansing13,14. Therefore, it is obvious to expect that stimulation of MLVs d…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was supported by a grant from the Russian Science Foundation (No. 23-75-30001).
Materials
| 0.1% Tween20 | Helicon, Russia | SB-G2009-100ML | |
| Catheter | Scientific Commodities Inc., USA | PE-10, 0.28 mm ID × 0.61 mm OD | |
| CO2 chamber | Binder, Germany | CB-S 170 | |
| Confocal microscop | Nikon, Japan | A1R MP | |
| Dental acrylic | Zermack, Poland-Russia | Villacryl S, V130V4Z05 | |
| Drill | Foredom, Russia | SR W-0016 | |
| Dumont forceps | Stoelting, USA | 52100-07 | |
| Evans Blue dye | Sigma-Aldrich, St. Louis, MO, USA | 206334 | |
| Hamilton | Hamilton Bonaduz AG, Switzerland | 29 G needle | |
| Ibuprofen | Sintez OJSC, Russia | N/A | Analgesic drug |
| Insulin needle | INSUPEN, Italy | 31 G, 0.25 mm x 6 mm | |
| Micro forceps | Stoelting, USA | 52102-02P | |
| Microcentrifuge | Gyrozen, South Korea | GZ-1312 | |
| Microinjector | Stoelting, USA | 53311 | |
| Non-sharp tweezer | Stoelting, USA | 52108-83P | |
| PINNACLE system | Pinnacle Technology, USA | 8400-K3-SL | System for recording EEG (2 channels) and EMG (1 channel) of mice |
| Shaving machine | Braun | Series 3310s | |
| Single and multi-channel pipettes | Eppendorf, Austria | Epp 3120 000.020, Epp 3122 000.019 | |
| Sodium chloride | Kraspharma, Russia | N/A | |
| Soldering station | AOYUE, China | N/A | |
| Stereotaxic frame | Stoelting, USA | 51500 | |
| Straight dissecting scissors | Stoelting, USA | 52132-10P | |
| Tetracycline | JSC Tatkhimfarmpreparaty, Russia | N/A | Eye ointment |
| Tweezer | Stoelting, USA | 52100-03 | |
| Ultrasonic cell disrupter | Biobase, China | USD-500 | |
| Wound retractor | Stoelting, USA | 52125 | |
| Xylanit | Nita-Farm, Russia | N/A | Muscle relaxant |
| Zoletil 100 | Virbac Sante Animale, France | N/A | General anesthesia |
References
- Da Mesquita, S., et al. Functional aspects of meningeal lymphatics in ageing and Alzheimer’s disease. Nature. 560 (7717), 185-191 (2018).
- Chen, J., et al. Meningeal lymphatics clear erythrocytes that arise from subarachnoid hemorrhage. Nat Commun. 11, 3159 (2020).
- Zou, W., et al. Blocking meningeal lymphatic drainage aggravates Parkinson’s disease-like pathology in mice overexpressing mutated α-synuclein. Transl Neurodegener. 8, 7 (2019).
- Hu, X., et al. Meningeal lymphatic vessels regulate brain tumor drainage and immunity. Cell Res. 30 (3), 229-243 (2020).
- Dong-Yu, L., et al. Photostimulation of brain lymphatics in male newborn and adult rodents for therapy of intraventricular hemorrhage. Nat Comm. 14 (1), 6104 (2023).
- Bolte, A., et al. Meningeal lymphatic dysfunction exacerbates traumatic brain injury pathogenesis. Nat Commun. 11 (1), 4524 (2020).
- Semyachkina-Glushkovskaya, O., et al. Mechanisms of phototherapy of Alzheimer’s disease during sleep and wakefulness: the role of the meningeal lymphatics. Front Optoelectron. 16, 22 (2023).
- Dongyu, L., et al. Photostimulation of lymphatic clearance of β- amyloid from mouse brain: new strategy for the therapy of Alzheimer’s disease. Front Optoelectron. 16, 45 (2023).
- Semyachkina-Glushkovskaya, O., et al. Mechanisms of phototherapy of Alzheimer’s disease during sleep and wakefulness: the role of the meningeal lymphatics. Front Optoelectron. 16, 22 (2023).
- Semyachkina-Glushkovskaya, O., et al. Intranasal delivery of liposomes to glioblastoma by photostimulation of the lymphatic system. Pharmaceutics. 15 (1), 36 (2023).
- Semyachkina-Glushkovskaya, O., et al. Night photostimulation of clearance of beta-amyloid from mouse brain: New strategies in preventing Alzheimer’s disease. Cells. 10 (12), 3289 (2021).
- Semyachkina-Glushkovskaya, O., et al. Technology of the photobiostimulation of the brain’s drainage system during sleep for improvement of learning and memory in male mice. Biomed Opt Express. 15 (1), 44-58 (2024).
- Fultz, N., et al. Coupled electrophysiological, hemodynamic, and cerebrospinal fluid oscillations in human sleep. Science. 366 (6465), 628-631 (2019).
- Xie, L., et al. Sleep drives metabolite clearance from the adult brain. Science. 342 (6156), 373-377 (2013).
- Semyachkina-Glushkovskaya, O., et al. Phototherapy of Alzheimer’s disease: Photostimulation of brain lymphatics during sleep: A systematic review. Int J Mol Sci. 24 (13), 10946 (2023).
- Semyachkina-Glushkovskaya, O., et al. Brain waste removal system and sleep: Photobiomodulation as an innovative strategy for night therapy of brain diseases. Int J Mol Sci. 24 (4), 3221 (2023).
- Hablitz, L. M., et al. Increased glymphatic influx is correlated with high EEG delta power and low heart rate in mice under anesthesia. Sci Adv. 5 (2), eaav5447 (2019).
- Fukumoto, H., et al. Primary cultures of neuronal and non-neuronal rat brain cells secrete similar proportions of amyloid beta peptides ending at A beta40 and A beta42. Neuroreport. 10 (14), 2965-2969 (1999).
- Moghekar, A., et al. Large quantities of Abeta peptide are constitutively released during amyloid precursor protein metabolism in vivo and in vitro. J Biol Chem. 286 (16), 15989-15997 (2011).
- Wells, C., Brennan, S., Keon, M., Ooi, L. The role of amyloid oligomers in neurodegenerative pathologies. Int J Biol Macromol. 181, 582-604 (2021).
- Savage, M., et al. Turnover of amyloid beta-protein in mouse brain and acute reduction of its level by phorbol ester. J Neurosci. 18 (5), 1743-1752 (1998).
- Ahn, J., et al. Meningeal lymphatic vessels at the skull base drain cerebrospinal fluid. Nature. 572 (7767), 62-66 (2019).
- Stevens, D., et al. Regional amyloid correlates of cognitive performance in ageing and mild cognitive impairment. Brain Commun. 4 (1), fcac016 (2022).
- Ma, C., Hong, F., Yang, S. Amyloidosis in Alzheimer’s disease: Pathogeny, etiology, and related therapeutic directions. Molecules. 27 (4), 1210 (2022).
- Kobro-Flatmoen, A., Hormann, T., Gouras, G. Intracellular amyloid-β in the normal rat brain and human subjects and its relevance for Alzheimer’s disease. J Alzheimers Dis. 95 (2), 719-733 (2023).
- Ahlemeyer, B., Halupczok, S., Rodenberg-Frank, E., Valerius, K., Baumgart-Vogt, E. Endogenous murine amyloid-β peptide assembles into aggregates in the aged C57BL/6J mouse suggesting these animals as a model to study pathogenesis of amyloid-β plaque formation. J Alzheimers Dis. 61 (4), 1425-1450 (2018).
- Zhinchenko, E., et al. Pilot study of transcranial photobiomodulation of lymphatic clearance of beta-amyloid from the mouse brain: Breakthrough strategies for nonpharmacologic therapy of Alzheimer’s disease. Biomed Opt Express. 10 (8), 4003-4017 (2019).
- Semyachkina-Glushkovskaya, O., et al. Transcranial photobiomodulation of clearance of beta-amyloid from the mouse brain: Effects on the meningeal lymphatic drainage and blood oxygen saturation of the brain. Adv Exp Med Biol. 1269, 57-61 (2021).
- Semyachkina-Glushkovskaya, O., et al. Photobiomodulation of lymphatic drainage and clearance: Perspective strategy for augmentation of meningeal lymphatic functions. Biomed Opt Express. 11 (2), 725-734 (2020).
- Zhinchenko, E., et al. Photostimulation of extravasation of beta-amyloid through the model of blood-brain barrier. Electronics. 9 (6), 1056 (2020).
- Semyachkina-Glushkovskaya, O., et al. Photostimulation of cerebral and peripheral lymphatic functions. Transl Biophotonics. 2 (1-2), e201900036 (2020).
- Semyachkina-Glushkovskaya, O., et al. Photomodulation of lymphatic delivery of liposomes to the brain bypassing the blood-brain barrier: New perspectives for glioma therapy. Nanophotonics. 10 (12), 3215-3227 (2021).
- Semyachkina-Glushkovskaya, O., et al. Photomodulation of lymphatic delivery of Bevacizumab to the brain: The role of singlet oxygen. Adv Exp Med Biol. 1395, 53-57 (2022).
- Semyachkina-Glushkovskaya, O., et al. Transcranial photosensitizer-free laser treatment of glioblastoma in rat brain. Int J Mol Sci. 24 (18), 13696 (2023).
- Blázquez-Castro, A. Direct 1O2 optical excitation: A tool for redox biology. Redox Biol. 13, 39-59 (2017).
- Spitler, R., Berns, M. Comparison of laser and diode sources for acceleration of in vitro wound healing by low-level light therapy. J Biomed Opt. 19 (3), 038001 (2014).
- Sato, K., Watanabe, R., Hanaoka, H., Nakajima, T., Choyke, P., Kobayashi, H. Comparative effectiveness of light emitting diodes (LEDs) and Lasers in near infrared photoimmunotherapy. Oncotarget. 7 (12), 14324-14335 (2016).
- Keshri, G., Gupta, A., Yadav, A., Sharma, S., Singh, S. Photobiomodulation with pulsed and continuous wave near-infrared laser (810 nm, Al-Ga-As) augments dermal wound healing in immunosuppressed rats. PLoS One. 11 (11), e0166705 (2016).
- Kim, H., et al. Pulse frequency dependency of photobiomodulation on the bioenergetic functions of human dental pulp stem cells. Sci Rep. 7 (1), 15927 (2017).
- Chen, Z., et al. The pulse light mode enhances the effect of photobiomodulation on B16F10 melanoma cells through autophagy pathway. Lasers Med Sci. 38 (1), 71 (2023).
- Mezey, E., et al. An immunohistochemical study of lymphatic elements in the human brain. Proc Natl Acad Sci U S A. 118 (3), e2002574118 (2021).
- Chang, J., et al. Characteristic features of deep brain lymphatic vessels and their regulation by chronic stress. Research. 6, 0120 (2023).
- Prineas, L. W. Multiple sclerosis: Presence of lymphatic capillaries and lymphoid tissue in the brain and spinal cord. Science. 203 (4385), 1123-1125 (1979).
- Semyachkina-Glushkovskaya, O., et al. Pilot identification of the Live-1/Prox-1 expressing lymphatic vessels and lymphatic elements in the unaffected and affected human brain. bioRxiv. , (2021).
- Semyachkina-Glushkovskaya, O., Postnov, D., Kurths, J. Blood-brain barrier, lymphatic clearance, and recovery: Ariadne’s thread in labyrinths of hypotheses. Int J Mol Sci. 19 (12), 3818 (2018).
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