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.
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.
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).
All procedures were performed in accordance with the "Guide for the Care and Use of Laboratory Animals", Directive 2010/63/EU on the Protection of Animals Used for Scientific Purposes, and the guidelines from the Ministry of Science and High Education of the Russian Federation (Nº 742 from 13.11.1984), which have been approved by the Bioethics Commission of the Saratov State University (Protocol No. 7, 22.09.2022).
1. Hardware assembly
2. Software guide (Figure 4)
3. Implantation of an EEG recording system (Figure 5)
4. Implantation of a plate for PBM
5. Preparation of a chronic catheter
6. Implantation of a chronic catheter into the right lateral ventricle
7. tPBM under EEG control of NREM sleep
8. Confocal imaging of lymphatic removal of Aβ from mouse brain
9. Analysis of Aβ in the lysates of brain tissues
In the first step, the study has focused on establishing the effective light dose (a 1050 nm LED) for stimulation of lymphatic removal of fluorescent Aβ from the brain to dcLNs in awake adult (2-3 month old, 26-29 g) male BALB/c mice. The light doses were selected randomly as 10 J/cm2, 20 J/cm2, and 30 J/cm2 based on our previous studies of tPBM effects on the removal of different dyes and the red blood cells from the brain7,8,9,10,11,12. The results of confocal imaging presented in Figure 8A–E clearly demonstrate the dose-related effects of tPBM on lymphatic removal of fluorescent Aβ from the brain to dcLNs. The light dose 30 J/cm2 was determined to be the most effective compared to the light doses 10 J/cm2 and 20 J/cm2 due to a higher intensity of a fluorescent signal from Aβ in dcLNs.
In the second step, the research was aimed at finding the wavelengths that are most effective for lymphatic elimination of fluorescent Aβ from the brain of adult (2-3 month old, 26-29 g) awake BALB/c male mice using the portable photo platforms with the different light wavelengths (880 nm, 1050 nm, 1300 nm) and modes (1050 nm in continues and pulse regimes) with a same dose (30 J/cm2). Figure 9A–E shows the confocal imaging results of dcLNs in the tested groups. The findings revealed that the light wavelength 1050 nm in pulse mode vs. other tested wavelengths (880 nm and 1300 nm) and the 1050 nm wavelength in continuous regime caused significantly higher removal of fluorescent Aβ from the brain to dcLNs.
In the final step, the effects of a 1050 nm LED in pulse mode on the level of soluble Aβ in the brain of aged mice before and after the 10 day course of tPBM under an EEG control of NREM and wakefulness were studied. The immunoassay analysis revealed that the soluble Aβ content in the brain was significantly higher in aged male BALB/c mice (16-18 months old, 30-33 g) compared with adult male BALB/c mice (2-3 months old, 26-29 g). The 10 day course of tPBM during deep sleep, but not while awake, effectively reduced the soluble Aβ level in the brains of aged mice to the level of soluble Aβ in the brains of adult mice (Figure 10).
Figure 1: Hardware assembly process. (A) 3D printed frame, (B) LED, (C) LED printed circuit board, (D) magnets, (E) metallic washer. Please click here to view a larger version of this figure.
Figure 2: LED control circuit. R1 – current regulation resistor, R2 – pull-down resistor for the MOSFET gate, Q1 – n-channel MOSFET. Please click here to view a larger version of this figure.
Figure 3: Case 3D view. (A) 3D printed cover plate, (B) 3D printed buttons, (C) LCD keypad shield for Arduino, (D) Arduino, (E) 3D printed case, (F) LED connector, (G) Channel MOSFET. Please click here to view a larger version of this figure.
Figure 4: Software. (A) Select button, (B) Left button, (C) Up/Down button, (D) Right button, (E) Selection indicator, (F) PWM duty cycle selection field, (G) RUN/OFF field. Please click here to view a larger version of this figure.
Figure 5: Implantation of an EEG recording system. (A) Skull preparation; (B) Implantation of screws; (C) Fixation of screws with the dental acrylic; (D) Fixation of the EEG recording sensor; (E) Implantation and fixation of the EMG electrodes on the back of the orbicularis oculi muscle; (F) The soldered electrodes to the silver-plated recess of the sensor. Please click here to view a larger version of this figure.
Figure 6: Fixation of a plate for PBM on the occipital bone of the skull. Please click here to view a larger version of this figure.
Figure 7: Implantation of a chronic catheter into the right lateral ventricle. Please click here to view a larger version of this figure.
Figure 8: The effects of different LED 1050 nm doses on lymphatic removal of fluorescent Aβ from the brain to dcLNs. (A–D) Representative images of dcLNs from the (A) control group and the PBM group with (B) 10 J/cm2, (C) 20 J/cm2, and (D) 30 J/cm2; (E) Quantitative analysis of the accumulation of fluorescent Aβ in dcLNs from the tested groups (n = 8, one-way ANOVA with Duncan post hoc test, **p < 0.01, *p < 0.05). Please click here to view a larger version of this figure.
Figure 9: The effects of different LED wavelengths on lymphatic removal of fluorescent Aβ from the brain to dcLNs. (A–D) Representative images of dcLNs from the PBM group with impulse mode 1050 nm (ImpMode_1050, A), 880 nm (ImpMode_880, B), 1300 nm (ImpMode_1300, C) and continuous mode 1050 nm (ContMode_1050 nm, D); (E) Quantitative analysis of accumulation of fluorescent Aβ in dcLNs from the tested groups (n = 8, one-way ANOVA with Duncan post hoc test, **p < 0.01). Please click here to view a larger version of this figure.
Figure 10: The PBM effects (LED 1050 nm in impulse mode, 30 J/cm2) during NREM and awake state on the level of soluble Aβ (pg/mL) in mouse brains of adult and aged mice. A one-way ANOVA with Duncan post hoc test, **p < 0.01, *p < 0.05. Please click here to view a larger version of this figure.
Supplementary Coding File 1: lcd1key.ino Please click here to download this File.
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 during deep sleep will be more effective than during wakefulness15,16. The new non-invasive and portable technology of tPBM under EEG control of sleep for stimulation of lymphatic removal of soluble Aβ from the brain of aged male mice is presented here.
The small size of the photo platform (7 mm x 11 mm) and its light weight (1 g) allow it to be firmly and securely mounted on the heads of mice, maintaining their natural motor activity. This also eliminates the need to use anesthesia during tPBM. The tPBM during deep sleep can be performed using any commercial device for an EEG control of sleep stages. Thus, our technology allows performing the study in a home cage, preserving the most natural conditions for animals.
In the first step, an LED wavelength of 1050 nm was randomly selected for the study of the effective dose of photo-effects on lymphatic removal of fluorescent Aβ from the brain to dcLNs in awake adult (2-3 month old) mice. The choice of this wavelength is related to the previous data on effective stimulation of MLVs with a 1267 nm laser8,10,27,28,29,30,31,32,33,34. This wavelength promotes the direct generation of the singlet oxygen in small quantities in brain tissue and in its meninges, which is one of the mechanisms underlying the PBM of MLVs8,28,34. However, the biological effects of both a 1267 nm laser and an LED 1050 nm are related to the generation of singlet oxygen excitation35. Indeed, the bands of a 1267 nm ± 20 nm laser and a 1065 nm ± 15 nm LED are related to formation of the singlet oxygen from the ground state of an oxygen molecule. A band of a 1267 nm laser pumps an oxygen molecule directly into the first excited singlet state, while a band of 1065 nm LED corresponds to additional vibrational energy, which decays very fast, releasing heat to the environment. Moreover, water absorption in the 1065 nm wavelength is tenfold lesser than in the 1267 nm wavelength, which makes it more favorable for biological research35. Furthermore, a 1050 nm LED is commercially available in the market and much cheaper compared with the rare and expensive 1267 nm laser. Note that the emission band of a used 1050 nm LED in this study was 40 nm wide, and it only partially overlaps with a singlet oxygen band. However, we demonstrated the significant effect of tPBM on the lymphatic removal of fluorescent Aβ from the brain to dcLNs using a 1050 nm LED (30 J/cm2). The light dose 30 J/cm2 was determined to be the most effective compared to the light doses 10 J/cm2 and 20 J/cm2 based on the higher intensity of a fluorescent signal from Aβ in dcLNs. Thus, a 1050 nm LED can be very useful for the application of the tPBM technique to humans, which is consistent with the common trend to use LEDs instead of lasers for PBM36,37.
In the second step, the research aimed to answer the question of which light wavelengths are most effective for lymphatic elimination of fluorescent Aβ from the brain of adult awake mice. This step of study was performed using a portable photo platform with different light wavelengths (880 nm, 1050 nm, 1300 nm) and modes (1050 nm in continuous and pulse regimes) with the same dose (30 J/cm2). These wavelengths were chosen due to the widespread use in clinical practice for tPBM (880 nm) and new expensive LEDs (1300 nm), which could potentially be clinically significant. In addition, the pulsed and continuous modes only for the 1050 nm LED as a light resource were compared. These data clearly demonstrate that only a 1050 nm LED in pulse mode vs. other wavelengths and the 1050 nm in continuous mode causes a significant lymphatic removal of Aβ from the brain to dcLNs. These results are consistent with the findings of other researchers, who also indicate the advantage of using a pulsed PBM to effectively achieve biological effects38,39,40.
In the final step, when the effective wavelength, dose and mode were selected, the study of the effects of a 1050 nm LED in pulse mode on the level of soluble Aβ in the brain of aged mice before and after the 10 day-course of tPBM under an EEG control of NREM and wakefulness was carried out. The immunoassay analysis revealed that the soluble Aβ content in the brain was significantly higher in aged mice (16-18 months old) compared with adult mice (2-3 months old). It is interesting to note that the 10-day course of tPBM during deep sleep, but not during awake, effectively reduced the soluble Aβ level in the brains of aged mice to the level of soluble Aβ in the brains of adult mice. An increase in the Aβ content in brain tissue of healthy mice and rats with age has also been noted in other studies25,26, which may be associated with an age-related decrease in the MLV functions leading to ineffective cleansing of brain tissue from this toxic protein1,22. The more effective tPBM of removal of soluble Aβ from brain tissue in sleeping mice compared to awake ones can be explained by the natural activation of brain tissue drainage during sleep. The MLV morphology changes significantly with age, leading to reduced lymphatic removal of metabolites from the aged brain22. However, tPBM during sleep helps restore the lymphatic removal of soluble Aβ from the brain tissue of aging mice to the level of adult animals.
Considering the scattering of light energy when passing through the skull, tPBM limits its effects only on MLVs without penetrating deep into the brain tissues. However, despite the fact that the lymphatic vessels have not yet been found directly in the brain tissues of humans and animals, growing evidence is emerging indicating the presence of the cerebral lymphatic system41,42,43,44. This explains the results showing the removal of toxins (blood and Aβ) from the deep structures of the brain (the ventricular system, the hippocampus) to MLVs and further to the periphery (dcLNs)5,7. Over the course of a century, knowledge has accumulated, indicating a close connection between the brain's drainage and the peripheral lymphatic system45. Even in the absence of a generally recognized lymphatic network in the central nervous system, there are facts indicating lymphatic removal of macromolecules from the deep parts of the brain to the periphery1,2,3,4,5,6,7 This also explains why tPBM, acting only on MLVs, stimulates the clearance of blood and Aβ from the brain5,7.
In summary, this study presents a portable technology of tPBM under EEG control of sleep designed to photo-stimulate the removal of toxins using the example of soluble Aβ from the brain of aged 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 natural motor activity of mice. These data open up new prospects for the development of non-invasive and clinically promising photo-technologies for correcting age-related changes in the MLV functions and brain's drainage processes and for the effective cleansing of brain tissues from metabolites and toxins. The proposed technology for photostimulation of the brain's drainage and lymphatic removal of toxins from the brain has limited effects on MLVs due to the scattering of light energy when passing through the skull. Therefore, the technology can be used to develop new methods for treating brain diseases associated with MLV dysfunction. The most pronounced stimulating photo-effects on MLVs are observed in deep (NREM) sleep, which requires the use of PBM under EEG control. This creates certain difficulties (technical, programming) for simultaneous control of the onset of deep sleep and the supply of light exposure.
The authors have nothing to disclose.
This research was supported by a grant from the Russian Science Foundation (No. 23-75-30001).
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 |
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