The overall goal of the following experimental protocol, is to perform a transcranial photobiomodulation therapy using low levels of red laser in mice. This is accomplished by the direct contact of the laser probe on the mouse head. As a first step, in order to evaluation of the laser light transmission, carefully dissect the brain tissue out from the skull.
And then measure the transmitted light power through the skull plus the scalp and one millimeter slice of the brain tissue. In the therapy section, contact the tip of the laser probe directly on the scalp at the bregma. This spatial learning and memory functions are evaluated by Barnes maze task.
Subsequently, hippocampal ATP levels are determined by the spectrophotometric method. Laser transmission data show that approximately 1%of antecedent light on the scalp surface reached one millimeter depth from the cortical surface. The results from the Barnes maze task show an improvement of the spatial memory in the aged mice following two weeks of the transcranial red laser treatment.
Also, the results suggest an increased hippocampal ATP levels in the laser-treated aged mice. Transcranial photobiomodulation is a non-invasive therapeutic approach for the treatment of a wide range of neurological and psychiatric disorders and for improving healthy brain function. Indeed, it's believed that photobiomodulation therapy causes photodissociation of the nitric oxide from the cytochrome c oxidase in the mitochondria.
These in turn increasing mitrochondrial electron transport, which ultimately leads to an increase in ATP production. This method is a safe and cost-effective light-delivery approach that is performed by radiation of the head using external light sources including lasers and light-emitting diodes. Almost all transcranial photobiomodulation procedures are applied with red to near-infrared light at wavelength from 600 to 1100 nanometer, a power ranging from one to 500 milliwatt, and the fluence ranging from one to 20 joule per centimeter square.
In a deeply anesthetized mouse, carefully dissect the brain tissue out from the skull. First, fix the intact brain tissue on an agarose gel and prepare all the required materials including sodium chloride and glue. Then, spread a thin layer of glue on the surface of the vibratome mounting block.
Carefully attach the agarose block and adjust its position. Slightly match the vibratome blade to the upper surface of the block, and record the counter value. Fill the vibratome tank with ice-cold normal saline solution.
Adjust the speed and vibration frequency of the vibratome, and change the counter to appropriate value for obtaining a slice thickness of one millimeter. Turn on the vibratome and the push the start button and cut the brain transversally into a slice with a thickness of one millimeter. First, add a drop of water on the optical glass surface.
Then, put the brain slice on the glass. Add the drop of water on it, and carefully place the second optical glass. Note that a drop of water should be added to the sample and glass boundaries in order to prevent tissue drying and also light scattering from rough surfaces.
Transfer samples to the optical laboratory. Set up the optical devices and turn on the power meter. Wear eye protection goggles before starting to operate with the laser device.
Turn on the laser and have the beam focused on the mirror for guiding the beam to the photodiode's active area. First, place two blank optical glasses on the surface of the power meter, and read the transmitted light power from the display screen. Remove the blank glasses and gently place the brain sample on the surface of the power meter, and focus the beam on the tissue's respective area, and read the transmitted power.
This time, place a blank optical glass on the surface of the power meter and read the transmitted light power. Then, remove the blank glass and slightly place an optical glass with fresh skull and scalped tissue on the surface of the power meter. Focus the beam on the bregma and read the transmitted power.
Finally, turn off the laser device and the power meter. Therapy section of the current protocol includes the application of the Class 3B laser instruments and this requires proper training and safety guidelines. In order to adapt the animals to the new environment, bring mice from their home cages to the therapy room approximately 20 minutes prior to beginning the treatment.
First, insert the laser device plug into an electric protector. Cover the tip of the laser probe with a transparent nylon film in order to prevent any scratching to the probe surface. Carefully connect the laser probe to the channel of the device.
Wear eye protection goggles before starting to operate with the laser device. Turn on the laser device and wait for a few seconds for its warmup. After observing the Laser Ready sign on the device panel, adjust the treatment parameters including irradiation time and operation mode.
Before starting the treatment, determine the laser average power by contacting the tip of the probe to the active area of the power meter on the device. In the current protocol, the laser probe is placed on the bregma zone, which is approximately three millimeter rostral to a line drawn between the anterior base of the ears. In order to avoid direct radiation to the animal's eyes, first, place the tip of the laser probe on the head, then, turn on the laser and stably hold the probe until the completion of irradiation.
Then, turn off the laser device and disconnect the probe from the device. At the end of the procedure, clean the laser probe with an appropriate optical cleaner. The spatial learning and memory task is performed in a Barnes maze.
First, place the do not enter sign on the outside of the task room door. Attach visual spatial cues to the perimeter walls. Position a video camera above the maze platform.
Clean the surface of the maze platform with 70%ethanol in order to remove unwanted olfactory cues. Add a small amount of bedding from the animal's home cage in into the escape box to serve as an olfactory cue. Before starting the task, put each mouse in the new cage, and transfer the cage to the Barnes maze room.
To habituate, allow the animal to remain in the room for 30 minutes prior to the task. Then, remove the mouse from its cage and gently place the animal in the escape box, and allow it remain there for one minute. After one minute, gently remove the mouse from the escape box, place the animal in the center of the arena, and put the start chamber on it.
After a ten second period, lift the start chamber and allow the animal to explore the arena for three minutes. Quietly move to the computer area, wear earmuffs, and trigger a negative auditory stimulus consisting of a loud white noise. Then, begin videotaping and observe animal's behavior on the computer monitor.
Note that a black maze should be used for testing white mice. Also, a black mat should be placed under the maze in the case tracking system software is to be used. Set up the video tracking software program and extract the parameters of interest from the recorded videos.
For a biochemical assessment, dissect hippocampus tissues, homogenize them in a sample buffer, centrifuge it, and then assess its ATP levels using the spectrophotometric method. The 660 nanometer laser light transmission through the skull plus scalp of the aged mice was approximately 16%Also, the value of about 10%was measured as a laser transmittance through a one millimeter slice of aged brain tissue. There were no statistically significant differences in the log of motor activity in the open field test among all experimental groups.
Data from Barnes maze task showed that the latency times of the aged control animals are obviously longer than those of the young control group on the third and fourth days of the training session. However, the red laser treatment significantly reduced the latency time on day four. In the prop trial session, aged control animals spend significantly shorter times in the target quadrant compared with the young control animals.
However, the laser-treated aged mice spent significantly longer times in the target quadrants as compared to aged control mice. The hippocampal bioenergetics data reveals a decrease in ATP levels in the aged control animals. On the other hand, the red laser treatment significantly increases the mean ATP contents in the hippocampus of the aged mice.
We described protocol for laboratory conducting a transcranial photobiomudulation therapy procedure in mice. Our protocol can be adapted to any other laboratory animals that are frequently used in the translational neuroscience field, such as rabbit, dog, monkey, et cetera. Based on our investigation, low levels of red light can recover hippocampal disfunction in the aged brain by boosting ATP production, which is reflected in an enhanced spatial memory function.
In this method, based on which brain regions are effected by pathology, several physical and treatment parameters, including irradiation time, treatment interval, applied radiance, and fluence, are required to be optimally adjusted to achieve better results. Transcranial photobiomodulation therapy is proposed as a promising approach for improving brain metabolism and cognitive enhancement that could be a potential strategy for the age-related cognitive decline and neurodegenerative diseases.
