Near Infrared (NIr) Light Increases Expression of a Marker of Mitochondrial Function in the Mouse Vestibular Sensory Epithelium

The overall goal of the following experiment is to investigate the potential neuroprotective impact of near infrared light on mouse vestibular sensory epithelium. This is achieved by a simple, short-term transcranial application of near infrared light to the mice at various developmental stages. As a second step, the bony labyrinths of the treated animals are dissected, allowing isolation of the vestibular sensory epithelium.

Next, the expression of the antioxidant gene of interest is measured by R-T-P-C-R to assess the impact of the treatment on mitochondrial performance. Finally, densitometry is performed to assess the effect of brief near infrared light treatment on the expression of specific antioxidant genes in mice. The main advantage of this near infrared treatment technique is that it is simple and non-invasive, allowing for further downstream physiological, molecular and behavioral analysis post-treatment.

The implications of this technique extend towards the potential neuroprotection of the peripheral vestibular system during normal aging, or to the improvement of balanced performance following vestibular disease or deficit. Two to three days before starting the experiment, shave the fur on the head and neck regions of each mouse as closely as possible to prevent regrowth of the animal's hair before completion of the treatment regime. To maintain the pathogen free status of the animals, clean the instruments with 70%ethanol between each shaving.

Then on the day of the treatment for each animal intern, pick up the mouse at the proximal end of the tail and allow it to relax in the palm of one hand to minimize stress for the near infrared treated animals. For each animal, hold a 670 nanometer LED device, one to two centimeters away from the shaved area, and switch on the device for 90 seconds. For the sham group, repeat the treatment procedure in the same way, but with the device switched off for the near infrared blocked group, treat the animals in the same way as the near infrared treated group, but with the device covered with aluminum foil.

After repeating the treatments for each group at 24 hour intervals for five consecutive days, freeze freshly prepared, glycerol based artificial cerebral spinal fluid, or A CSF in a minus 80 degrees Celsius freezer for 45 minutes until an ice slurry forms.Next. For each mouse, use a rounded number 22 razor blade to make an incision along the sagittal skin of the skull, keeping the cranial vault brain and underlying vestibular apparatus as cool as possible with regular applications of ice cold A CSF over the tissue. Then use the pointed arm of a pair of standard pattern scissors to make a small incision in the skull at Lambda and cut along the sagittal suture.

Then gently slide one arm of a shallow URS beneath the parietal bone, and then secure and pull the parietal bone laterally and the occipital bone posteriorly until the brain is exposed. Then use a small stainless steel spatula to lift the brain away from the anterior and middle cranial fossa. To expose the vestibular cochlear nerve, transect the nerve to prevent unnecessary tension on the primary afferent axons that directly innervate the vestibular hair cells.

Then remove the brain in toto and observe the bony labyrinth containing the cochlea and the peripheral vestibular organs in the middle cranial fossa. Now make two small incisions beside each bony labyrinth and hold and pull the anterior semi-circular canal laterally to excise the entire structure. Then immediately immerse the excised labyrinth in a dissecting dish containing the ice cold A CSF solution while continuously perfusing with carbogen.

The trickiest part of this procedure is obtaining consistent quantities of viable tissue for each animal we recommend practice careful observation and specific lighting of the tissue. Next, under a stereo microscope, hold down the labyrinth by the cochlea and use forceps to secure the tissue to the bottom of the dish using straight fine forceps. Scratch a small opening in the bone above the anterior semi-circular canal ula.

Then reach immediately below the bone and flick outwards away from the ULA to gently enlarge the opening. Continue in this fashion until the utricle and the anterior and lateral pule are all exposed. Then use fine forceps to gently lift the utricle and pule away from the bony labyrinth until they are completely detached.

Finally, securely grasp the vestibular organs between the tip of the forceps and place them into freshly prepared lysis. Buffer in a screw top micro tube, gently swirl the forceps in the buffer to detach the vestibular organs. Then after confirming the forceps are clear of tissue under the stereo microscope, screw on the micro tubule lid and immediately freeze the sample in liquid nitrogen to compare the impact of near infrared treatment between young and older mice.

Messenger, RNA was reversed transcribed into complementary DNA, then the expression of antioxidant superoxide dismutase one or SOD one was analyzed as measured by densitometry a significant increase in beta actin normalized sod one expression of more than twofold in young, near infrared treated animals compared to young sham treated animals and young near infrared blocked animals was observed. Older near infrared treated animals also exhibited more than a twofold upregulation of sod one when compared with older near infrared blocked animals. Following this procedure, behavioral, molecular or physiological experiments can be performed to answer additional questions like, does near infrared light increase other gene or protein expression in the vestibular sensory epithelium?

Or does near infrared light improve the overall balance performance of treated mice?