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

In the study, we propose an innovative technology for photostimulation of meningeal lymphatic vessels and brain drainage during sleep under EEG control. Sleep is a natural state of activation of drainage and cleansing function of the brain, which is reduced in the 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 metabolism with the flow of food.

The technology we have developed from photobiomodulation of the MLD functions in brain drainage opens up new possibilities for non-pharmacological alternative therapy for a wide range of diseases of CNS that are associated with dysfunction of and suppression of brain drainage, including brain cordial brain injuries, Parkinson's disease, and multiple sclerosis, diabetes, et cetera. 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. 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 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 photo biomodulation or PBM process and stop the process if NREM sleep transitions to rapid eye movement sleep or wakefulness. 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 1, 050 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.