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.
