This research was supported by a grant from the Russian Science Foundation (No. 23-75-30001).

All procedures were performed in accordance with the &#34;Guide for the Care and Use of Laboratory Animals&#34;, 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 (N&#186; 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
<ol>
	<li>Cut a piece of f...

Enhancing Brain Drainage During Sleep with Transcranial Photobiomodulation

MLVs are an important target for the development of innovative technologies for modulation of the brain&#39;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...

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 A&#946; 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&#39;s drainage, which reduces the quality of research results17.
A&#946; is a metabolic product of normal neural activity18. As it was established in cultured rat cortical neurons, A&#946; is released from them at high rates into the extracellular space (2-4 molecules/neuron/s for A&#946;)19. There is evidence that the dissolved form of A&#946;, located in the extracellular and perivascular spaces, is most toxic to neurons and synapses20. The soluble A&#946; is rapidly cleared from the human brain during 1-2.5 h21. MLVs are the tunnels for removal of the soluble A&#946; from the brain1,7 that declines with age, leading to the accumulation of A&#946; in the aged brain1,22. There is evidence that extracellular abnormalities of A&#946; levels in the brain correlate with cognitive performance in aging and are associated with the development of Alzheimer&#39;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 C57BL/6 mice of different ages to stimulate lymphatic clearance of A&#946; from the brain into the peripheral lymphatic system (the deep cervical lymph nodes, dcLNs).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1% Tween20</td>
			<td>Helicon,&#160; Russia</td>
			<td>SB-G2009-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Catheter</td>
			<td>Scientific Commodities Inc., USA</td>
			<td>PE-10, 0.28 mm ID &#215; 0.61 mm OD</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 chamber</td>
			<td>Binder, Germany</td>
			<td>CB-S 170</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscop</td>
			<td>Nikon, Japan</td>
			<td>A1R MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental acrylic</td>
			<td>Zermack, Poland-Russia</td>
			<td>Villacryl S, V130V4Z05</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill</td>
			<td>Foredom, Russia</td>
			<td>SR W-0016</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps</td>
			<td>Stoelting, USA</td>
			<td>52100-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Evans Blue dye</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>206334</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton</td>
			<td>Hamilton Bonaduz AG, Switzerland</td>
			<td>29 G needle</td>
			<td></td>
		</tr>
		<tr>
			<td>Ibuprofen</td>
			<td>Sintez OJSC, Russia</td>
			<td>N/A</td>
			<td>&#160;Analgesic drug</td>
		</tr>
		<tr>
			<td>Insulin needle</td>
			<td>INSUPEN, Italy</td>
			<td>31 G, 0.25 mm x 6 mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Levomekol antibacterial ointment</td>
			<td>Nizhpharm</td>
			<td>D06C </td>
			<td>For external use at a dose of 40 mg/g, 1 time per day</td>
		</tr>
		<tr>
			<td>Micro forceps</td>
			<td>Stoelting, USA</td>
			<td>52102-02P</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Gyrozen, South Korea</td>
			<td>GZ-1312</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Stoelting, USA</td>
			<td>53311</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-sharp tweezer</td>
			<td>Stoelting, USA</td>
			<td>52108-83P</td>
			<td></td>
		</tr>
		<tr>
			<td>PINNACLE system</td>
			<td>Pinnacle Technology, USA</td>
			<td>8400-K3-SL</td>
			<td>System for recording EEG (2 channels) and EMG (1 channel) of mice</td>
		</tr>
		<tr>
			<td>Shaving machine</td>
			<td>Braun</td>
			<td>Series 3310s</td>
			<td></td>
		</tr>
		<tr>
			<td>Single and multi-channel pipettes</td>
			<td>Eppendorf, Austria</td>
			<td>Epp 3120 000.020, Epp 3122 000.019</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Kraspharma, Russia</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Soldering station</td>
			<td>AOYUE, China</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic frame</td>
			<td>Stoelting, USA</td>
			<td>51500</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight dissecting scissors</td>
			<td>Stoelting, USA</td>
			<td>52132-10P</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetracycline</td>
			<td>JSC Tatkhimfarmpreparaty, Russia</td>
			<td>N/A</td>
			<td>Eye ointment</td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Stoelting, USA</td>
			<td>52100-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic cell disrupter</td>
			<td>Biobase, China</td>
			<td>USD-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Wound retractor</td>
			<td>Stoelting, USA</td>
			<td>52125</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylanit</td>
			<td>Nita-Farm, Russia</td>
			<td>N/A</td>
			<td>Muscle relaxant</td>
		</tr>
		<tr>
			<td>Zoletil 100</td>
			<td>Virbac Sante Animale, France</td>
			<td>N/A</td>
			<td>General anesthesia</td>
		</tr>
	</tbody>
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photobiomodulation under electroencephalographic controls of sleep for stimulation of lymphatic removal of toxins from mouse brain

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&#39;s and Parkinson&#39;s diseases, brain tumors, traumatic brain injuries, and intracranial hemorrhages. Sleep is a natural state when the brain&#39;s drainage processes are most active. Therefore, stimulation of the brain&#39;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 (A&#946;)) 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&#39;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.

Author Spotlight: Developing Photo-Stimulation Technology for Toxin Removal in Sleep-Related Brain Diseases

Photobiomodulation Under Electroencephalographic Controls of Sleep for Stimulation of Lymphatic Removal of Toxins from Mouse Brain

In the study we propose a new way of technology for photo-stimulation of many germ fighting vessels and brain drainage doing sleep and control. Sleep is a natural state of activation of drainage and cleansing function of the brain which is reduced in large numbers of diseases of CNS. It's necessary to create technologies for treating brain diseases during sleep in order to most effectively stimulate the cleansing of its tissues from toxins and metabolists with the flow of food.The technology we have developed for photo-biomulation of the Amylin functions in brain drainage opens up new possibilities from non-pharmacological alternative preferred for a wide prevention of diseases of CNS. They are shaded with a special version of brain drainage including brain or cord injuries, Parkinson's disease and multiple sclerosis, diabetes, et cetera.

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 A&#946; 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,<sup class="x...

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 C57BL/6 mice of different ages to stimulate lymphatic clearance of A&#946; from the brain into the peripheral lymphatic system (the deep cervical lymph nodes, dcLNs).

The meningeal lymphatic vessels (MLVs) play an important role in the removal of toxins from the brain. The development of innovative technologies for the ...

photobiomodulation-under-electroencephalographic-controls-sleep-for

Research

JoVE Journal

Neuroscience

Hardware Assembly to Perform Transcranial Photobiomodulation for Brain Drainage in Aged Mice

To begin the hardware assembly, solder the light emitting diode or LED to the printed circuit board or PCB, and place the LED and magnets on the 3D frame. Then connect the resistor between an LED anode and the five volt port on the Arduino. Next, connect an LED cathode to the metal oxide semiconductor field effect transistor or MOSFET drain, and secure the MOSFET source to the ground.After connecting the pull down resistor between the MOSFET gate and the ground, proceed to pin the MOSFET gate to pin three on the Arduino. Then connect a liquid crystal display or LCD keypad shield to the Arduino. And insert the Arduino board with LCD keypad shield MOSFET and LED connector into the case.Finally, download the Arduino sketch and open it via the Arduino integrated development environment. Select the correct communication port and flash the firmware.

EEG and Catheter Implantation in Mouse Brain for Transcranial Photobiomodulation to Stimulate Meningeal Lymphatic Vessel Functions

Place the anesthetized mouse in a stereotaxic frame over a heating pad and apply ophthalmic ointment to the eyelids to prevent the eyeballs from drying. Shave the head in the area from the nasal bones to the occipital bones, and disinfect the exposed skin with alternating rounds of chlorhexidine and alcohol three times each. Using straight dissecting scissors, cut off the scalp, hold it with micro forceps, and clean the skull from fascia.Using a drill with a 1.3 millimeter diameter, make two holes in the temporal bones on each side along the coordinates for the two screw pairs. Place the electroencephalographic, or EEG screws, with wire leads in alcohol for 15 minutes, followed by the saline solution. Place four silver plated screws with electrodes into the holes to a depth of one millimeter.Fix the screws on the surface of the skull using dental acrylic, followed by attaching an EEG recording sensor to the animal's nose. After 30 minutes, use curved tweezers to fix the EMG electrodes on the back of the orbicularis oculi muscle with dental acrylic. Connect and fix the EEG electrodes to the silver-plated recess of the sensor.Finally, place the mouse on a heating pad to maintain body temperature until the animal fully recovers from anesthesia. After seven days, use dental acrylic and Dumont forceps to fix a metal plate with a diameter of five millimeters on the occipital bone of the mouse skull. To prepare the chronic catheter, mark a segment on the insulin needle two millimeters from the side of its beveled end.Fix the insulin needle in the holder from the side of the beveled tip to the marked segment. Place a two-centimeter PE-10 polyethylene catheter over the entire remaining length of the needle. Fill the catheter with a saline solution and cover it with a plastic cap.For the implantation, drill a 1.5 millimeter diameter trepanation hole at the appropriate coordinates. Place the PE-10 polyethylene catheter in a stereotactic holder and insert it into the mouse's skull. Afterward, fix it with dental acrylic and let it harden for 15 minutes.Connect any commercial EEG recording system to the connector on the mouse's head, and set the requirement value of the pulse width modulation, or PWM duty cycle. Monitor the EEG signal and wait for delta rhythm activity. If non-rapid eye movement, or NREM, sleep is seen, initiate the photobiomodulation, or PBM process and stop the process if NREM sleep transitions to rapid eye movement sleep or wakefulness.

Confocal Imaging to Assess Lymphatic Removal of Beta-Amyloid from Mouse Brain After Transcranial Photobiomodulation

For confocal imaging, first connect a 10-centimeter catheter to an insulin needle. Using a Hamilton syringe with a 29-gauge needle, prepare five microliters of fluorescent beta amyloid for infusion into the catheter. After fixing the mouse's hand, connect the catheter through a needle to the implanted chronic catheter.Connect the catheter to a micro injector and place the mouse in an individual box. Set the injection rate to 0.1 microliters per minute in the micro injector menu, and press the Start button to inject FA beta into the right lateral ventricle. After FA beta administration, make photobiomodulation, or PBM, using an LED for 61 minutes following the algorithm.Then intravenously inject any tracer for labeling the cerebral vessels via the tail. Post euthanization, use sharp straight scissors to make a small transverse incision in the skin along the trachea, holding the skin with straight non-sharp tweezers. Then with straight scissors, make a longitudinal incision along the entire length of the neck.Employing curved tweezers, take up the salivary glands and carefully separate them from the connective tissue. Place a wound retractor on the open section of the incision and fix it to push back the surrounding tissues. Make use of two curved tweezers from both sides of the neck to examine the area between the trachea and the cleidomastoid muscle.With straight tweezers having blunt ends, take off the deep cervical lymph node on each side and cut it from the connective tissue. Finally, place the nodes in a Petri dish with a saline solution and cover them with horizontally oriented cover glass to perform confocal microscopy. Light doses of 30 joules per square centimeter showed the most significant effect for promoting lymphatic removal of FA beta from the brains of awake adult male mice.The pulse mode at 1050 nanometers proved most effective in enhancing the removal of FA beta compared to other wavelengths and the continuous mode at the same wavelength. A 10-day course of transcranial PBM during NREM sleep significantly reduced soluble FA beta levels in aged mice, aligning them with levels found in younger adult mice. This effect was not observed when the treatment was applied during wakefulness.

228 Views.  Saratov State University.  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 C57BL/6 mice of different ages to stimulate lymphatic clearance of Aβ from the brain into the peripheral lymphatic system (the deep cervical lymph nodes, dcLNs).

Enhancing Brain Drainage During Sleep with Transcranial Photobiomodulation

Summary