The authors wish to acknowledge Dr. Paul Witting and Ms. Genevieve Fong for their assistance with mRNA extraction and PCR, and the Garnett Passe and Rodney Williams Memorial Foundation for support.

Ethics Statement: All procedures outlined below were approved by the University of Sydney Animal Ethics Committee.
1. Animals
NOTE: 1 and 8 - 9 month old mice (C57/BL6) were obtained from the Animal Resources Centre (Perth, Australia). Mice were housed in the Bosch Rodent Facility at the University of Sydney.
<ol>
	<li>House mice in standard mouse cages on a 12/12 hr light/dark cycle with access to food and water ad libitum.</li>
	<li>Divide mice in each age group into near infrared (NIr) treated, or sham treated (control) groups for comparison.</li>
</ol>
2. Near Infrared (NIr) Irradiation and Sham Treatment
<ol>
	<li>Shave the fur on the head and neck region of the mouse with an electric razor as closely as possible to prevent quick regrowth of hair before the completion of the treatment regime. In order to maintain the pathogen-free status of the mice, use 70% ethanol to clean instruments between shaving animals and F10 veterinary disinfectant between animals from separate cages. Do this 2 - 3 days prior to the beginning of treatment so animals are not over-handled.</li>
	<li>Restrain mouse by holding the proximal end of the tail and allow it to relax in the palm of one hand or bench top so as to minimize stress which could lead to false results.</li>
	<li>Hold the NIr (670 nm) LED (light emitting diode) device 1 - 2 cm away from the exposed (shaved) area and switch on the device for 90 sec (Figure 1A). 
	NOTE: The temperature change as a result of 90 sec exposure was measured as &#60;0.2 &#176;C in 100 ml of water.</li>
	<li>Repeat steps 2.2 - 2.3 for the sham treatment group of animals but leave the device switched off (Figure 1B).</li>
	<li>Repeat steps 2.2 - 2.3 for the NIr-blocked treatment group of animals but cover the device with aluminium foil.</li>
	<li>Repeat steps 2.2 - 2.5&#160;daily at approximate 24 hr intervals for 5 consecutive days.</li>
	<li>Extract the vestibular sensory epithelium (3 cristae and 1 utricular macula) from both ears on the fifth day post-treatment (See Section 3 below).</li>
</ol>
3. Tissue Extraction20
<ol>
	<li>Prepare 300 ml of a glycerol-based artificial cerebrospinal fluid (ACSF) consisting of (in mM): 26 NaHCO3, 11 glucose, 250 glycerol, 2.5 KCl, 1.2 NaH2PO4, 1.2 MgCl2, and 2.5 CaCl2. Prior to the addition of CaCl2, gas the solution with carbogen (95% O2 and 5% CO2) to establish a pH of 7.4 and avoid calcium precipitation (cloudiness). Chill the solution in a -80 &#176;C freezer for 45 min so that an ice slurry is formed.</li>
	<li>Prepare the RNA isolation lysis buffer in labelled screw top microtubes (stops the popping of lids when tube pressure changes between freeze and thawing in liquid nitrogen) according to the instructions of the manufacturer or usual lab practices. Have liquid nitrogen ready in an aluminium dewar flask. 
	NOTE: Use protective clothing and eyeware when handling liquid nitrogen. Ensure that liquid nitrogen is used in a well-ventilated room as the volume of its gas form is approximately 700 times that of its liquid form and can cause asphyxiation.</li>
	<li>Deeply anaesthetize mice with ketamine (400 mg/kg) via an intra-peritoneal injection. Allow the hind-limb reflex to completely subside as indication that mice are fully anaesthetised.</li>
	<li>Decapitate mice with sharp stainless steel scissors and make an incision along the sagittal skin of the skull using a razor blade (rounded #22). At this point and throughout steps 3.5 - 3.9, keep the cranial vault, brain, and underlying vestibular apparatus as cool as possible by regular application of ice-cold ACSF over the tissue.</li>
	<li>Using the pointed arm of standard pattern scissors make a small incision in the skull at Lambda and cut along the sagittal suture.</li>
	<li>Gently slide one arm of a shallow rongeurs beneath the parietal bone keeping the blade as close as possible to the inferior surface of the bone without dragging the brain. Secure and pull away the parietal bone laterally and the occipital bone posteriorly until the brain is exposed.</li>
	<li>Use a small stainless steel spatula and lift the brain away from the anterior and middle cranial fossa to expose the vestibulocochlear nerve (CN VIII). Transect the nerve to prevent unnecessary tension on the primary afferent axons that directly innervate the vestibular hair cells.</li>
	<li>Remove the brain in toto after transection of CN VIII.</li>
	<li>Observe the bony labyrinth containing the cochlea and the peripheral vestibular organs in the middle cranial fossa. Make two small incisions beside each bony labyrinth and excise the entire structure by holding the anterior semicircular canal and pulling laterally.</li>
	<li>Immediately immerse excised labyrinths in a dissecting dish containing ice-cold ACSF solution (as described in step 3.1) while continuously perfusing with carbogen.</li>
	<li>Under a stereomicroscope, hold down the labyrinth by the cochlea and secure it to the bottom of the dish with forceps.
	<ol>
		<li>Use straight fine forceps to scratch a small opening in the bone above the anterior semicircular canal (SSC) ampulla.</li>
		<li>Gently enlarge this opening by reaching immediately below the bone and flicking outwards away from the ampulla. Exercise caution here to not push forceps into the opening and damage the fragile membranous labyrinth below. Continue in this fashion until the utricle, anterior and lateral ampullae are all exposed. If possible remove the posterior ampulla also.</li>
	</ol>
	</li>
	<li>Using fine forceps, gently lift the utricle and ampullae away from the bony labyrinth until they are completely detached. Where possible, hold them by their associated semicircular canals to avoid damaging the sensory epithelium. In some cases, the proximal part of the semicircular membranous duct may need to be cut with iris scissors to release the ampullae from the bone.</li>
	<li>Securely grasp the vestibular organs between the tip of forceps and place into the lysis buffer prepared earlier in step 3.2. Gently swirl the forceps around in the buffer to ensure vestibular organs have detached from the forceps. Check this is the case by bringing the forceps under the stereomicroscope.
	<ol>
		<li>Screw on the microtubule lid and freeze the sample in liquid nitrogen immediately.</li>
	</ol>
	</li>
</ol>
4. RNA Extraction and RT-PCR

<ol>
	<li>Follow standard methods of messenger RNA (mRNA) extraction according to manufacturer&#8217;s instructions or preferred lab protocols. 
	NOTE: A commercial kit that utilized carrier RNAs to help isolate small yields of mRNA was used in this protocol. Lower elution volumes can be used to increase final concentrations of mRNA. 
	NOTE: Negative &#8220;no enzyme&#8221; controls (NECs) and &#8220;no DNA template&#8221; controls (NTCs) must also be completed to ensure validity of observed effects.</li>
	<li>Apply standard methods of reverse transcription of mRNA to complementary DNA (cDNA) and amplification of target genes21-23.</li>
</ol>

<ol><li>Agrawal, Y., Carey, J. P., Della Santina,, C, C., Schubert, M. C., Minor, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disorders+of+balance+and+vestibular+function+in+US+adults%3A+data+from+the+National+Health+and+Nutrition+Examination+Survey%2C+2001-2004%5BTitle%5D)+AND+%22Archives+of+internal+medicine%22%5BJournal%5D)">Disorders of balance and vestibular function in US adults: data from the National Health and Nutrition Examination Survey, 2001-2004.</a> Archives of internal medicine. 169, 938-944 (2009).</li>
<li>Bessho, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+subthreshold+infrared+laser+treatment+for+drusen+regression+on+macular+autofluorescence+in+patients+with+age-related+macular+degeneration%5BTitle%5D)+AND+%22Retina%22%5BJournal%5D)">Effect of subthreshold infrared laser treatment for drusen regression on macular autofluorescence in patients with age-related macular degeneration.</a> Retina. 25, 981-988 (2005).</li>
<li>Olk, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapeutic+benefits+of+infrared+%28810-nm%29+diode+laser+macular+grid+photocoagulation+in+prophylactic+treatment+of+nonexudative+age-related+macular+degeneration%3A+two-year+results+of+a+randomized+pilot+study%5BTitle%5D)+AND+%22Ophthalmology%22%5BJournal%5D)">Therapeutic benefits of infrared (810-nm) diode laser macular grid photocoagulation in prophylactic treatment of nonexudative age-related macular degeneration: two-year results of a randomized pilot study.</a> Ophthalmology. 106, 2082-2090 (1999).</li>
<li>Rodanant, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Predictors+of+drusen+reduction+after+subthreshold+infrared+%28810+nm%29+diode+laser+macular+grid+photocoagulation+for+nonexudative+age-related+macular+degeneration%5BTitle%5D)+AND+%22American+journal+of+ophthalmology%22%5BJournal%5D)">Predictors of drusen reduction after subthreshold infrared (810 nm) diode laser macular grid photocoagulation for nonexudative age-related macular degeneration.</a> American journal of ophthalmology. 134, 577-585 (2002).</li>
<li>De Taboada, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcranial+laser+therapy+attenuates+amyloid-beta+peptide+neuropathology+in+amyloid-beta+protein+precursor+transgenic+mice%5BTitle%5D)+AND+%22Journal+of+Alzheimer%27s+disease+%3A+JAD%22%5BJournal%5D)">Transcranial laser therapy attenuates amyloid-beta peptide neuropathology in amyloid-beta protein precursor transgenic mice.</a> Journal of Alzheimer's disease : JAD. 23, 521-535 (2011).</li>
<li>Grillo, S. L., Duggett, N. A., Ennaceur, A., Chazot, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-invasive+infra-red+therapy+%281072+nm%29+reduces+beta-amyloid+protein+levels+in+the+brain+of+an+Alzheimer%27s+disease+mouse+model%5BTitle%5D)+AND+%22TASTPM.+Journal+of+photochemistry+and+photobiology.+B%2C+Biology%22%5BJournal%5D)">Non-invasive infra-red therapy (1072 nm) reduces beta-amyloid protein levels in the brain of an Alzheimer's disease mouse model.</a> TASTPM. Journal of photochemistry and photobiology. B, Biology. 123, 13-22 (2013).</li>
<li>Purushothuman, S., Johnstone, D. M., Nandasena, C., Mitrofanis, J., Stone, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photobiomodulation+with+near+infrared+light+mitigates+Alzheimer%27s+disease-related+pathology+in+cerebral+cortex+-+evidence+from+two+transgenic+mouse+models%5BTitle%5D)+AND+%22Alzheimer%27s+researc.%2C+%26+therapy%22%5BJournal%5D)">Photobiomodulation with near infrared light mitigates Alzheimer's disease-related pathology in cerebral cortex - evidence from two transgenic mouse models.</a> Alzheimer's researc., & therapy. 6, 2(2014).</li>
<li>Sommer, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((670+nm+laser+light+and+EGCG+complementarily+reduce+amyloid-beta+aggregates+in+human+neuroblastoma+cells%3A+basis+for+treatment+of+Alzheimer%27s+disease%5BTitle%5D)+AND+%22Photomedicine+and+laser+surgery%22%5BJournal%5D)">670 nm laser light and EGCG complementarily reduce amyloid-beta aggregates in human neuroblastoma cells: basis for treatment of Alzheimer's disease.</a> Photomedicine and laser surgery. 30, 54-60 (2012).</li>
<li>Moro, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photobiomodulation+preserves+behaviour+and+midbrain+dopaminergic+cells+from+MPTP+toxicity%3A+evidence+from+two+mouse+strains%5BTitle%5D)+AND+%22BMC+neuroscience%22%5BJournal%5D)">Photobiomodulation preserves behaviour and midbrain dopaminergic cells from MPTP toxicity: evidence from two mouse strains.</a> BMC neuroscience. 14, 40(2013).</li>
<li>Peoples, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photobiomodulation+enhances+nigral+dopaminergic+cell+survival+in+a+chronic+MPTP+mouse+model+of+Parkinson%27s+disease%5BTitle%5D)+AND+%22Parkinsonis.%2C+%26+related%22%5BJournal%5D)">Photobiomodulation enhances nigral dopaminergic cell survival in a chronic MPTP mouse model of Parkinson's disease.</a> Parkinsonis., & related. 18, 469-476 (2012).</li>
<li>Shaw, V. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuroprotection+of+midbrain+dopaminergic+cells+in+MPTP-treated+mice+after+near-infrared+light+treatment%5BTitle%5D)+AND+%22The+Journal+of+comparative+neurology%22%5BJournal%5D)">Neuroprotection of midbrain dopaminergic cells in MPTP-treated mice after near-infrared light treatment.</a> The Journal of comparative neurology. 518, 25-40 (2010).</li>
<li>Ying, R., Liang, H. L., Whelan, H. T., Eells, J. T., Wong-Riley, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pretreatment+with+near-infrared+light+via+light-emitting+diode+provides+added+benefit+against+rotenone-+and+MPP%2B-induced+neurotoxicity%5BTitle%5D)+AND+%22Brain+research%22%5BJournal%5D)">Pretreatment with near-infrared light via light-emitting diode provides added benefit against rotenone- and MPP+-induced neurotoxicity.</a> Brain research. 1243, 167-173 (2008).</li>
<li>Rajguru, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infrared+photostimulation+of+the+crista+ampullaris%5BTitle%5D)+AND+%22The+Journal+of+physiology%22%5BJournal%5D)">Infrared photostimulation of the crista ampullaris.</a> The Journal of physiology. 589, 1283-1294 (2011).</li>
<li>Chung, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+nuts+and+bolts+of+low-level+laser+%28light%29+therapy%5BTitle%5D)+AND+%22Annals+of+biomedical+engineering%22%5BJournal%5D)">The nuts and bolts of low-level laser (light) therapy.</a> Annals of biomedical engineering. 40, 516-533 (2012).</li>
<li>Desmet, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+and+experimental+applications+of+NIR-LED+photobiomodulation%5BTitle%5D)+AND+%22Photomedicine+and+laser+surgery%22%5BJournal%5D)">Clinical and experimental applications of NIR-LED photobiomodulation.</a> Photomedicine and laser surgery. 24, 121-128 (2006).</li>
<li>Huang, Y. Y., Chen, A. C., Carroll, J. D., Hamblin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biphasic+dose+response+in+low+level+light+therapy%5BTitle%5D)+AND+%22Dose-response+%3A+a+publication+of+International+Hormesis+Society%22%5BJournal%5D)">Biphasic dose response in low level light therapy.</a> Dose-response : a publication of International Hormesis Society. 7, 358-383 (2009).</li>
<li>Rojas, J. C., Gonzalez-Lima, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-level+light+therapy+of+the+eye+and+brain%5BTitle%5D)+AND+%22Eye+and+brain%22%5BJournal%5D)">Low-level light therapy of the eye and brain.</a> Eye and brain. 3, 49-67 (2011).</li>
<li>Vranceanu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Striated+organelle%2C+a+cytoskeletal+structure+positioned+to+modulate+hair-cell+transduction%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Striated organelle, a cytoskeletal structure positioned to modulate hair-cell transduction.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 4473-4478 (2012).</li>
<li>Johnson, K. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separate+and+combined+effects+of+Sod1+and+Cdh23+mutations+on+age-related+hearing+loss+and+cochlear+pathology+in+C57BL%2F6J+mice%5BTitle%5D)+AND+%22Hearing+research%22%5BJournal%5D)">Separate and combined effects of Sod1 and Cdh23 mutations on age-related hearing loss and cochlear pathology in C57BL/6J mice.</a> Hearing research. 268, 85-92 (2010).</li>
<li>Tung, V. W., Di Marco, S., Lim, R., Brichta, A. M., Camp, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+isolated+semi-intact+preparation+of+the+mouse+vestibular+sensory+epithelium+for+electrophysiology+and+high-resolution+two-photon+microscopy%5BTitle%5D)+AND+%22Journal+of+visualized+experiments+%3A+JoVE%22%5BJournal%5D)">An isolated semi-intact preparation of the mouse vestibular sensory epithelium for electrophysiology and high-resolution two-photon microscopy.</a> Journal of visualized experiments : JoVE. , e50471(2013).</li>
<li>Kirby, J., Menzies, F. M., Cookson, M. R., Bushby, K., Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+gene+expression+in+a+cell+culture+model+of+SOD1-related+familial+motor+neurone+disease%5BTitle%5D)+AND+%22Human+molecular+genetics%22%5BJournal%5D)">Differential gene expression in a cell culture model of SOD1-related familial motor neurone disease.</a> Human molecular genetics. 11, 2061-2075 (2002).</li>
<li>Parry, S. N., Ellis, N., Li, Z., Maitz, P., Witting, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myoglobin+induces+oxidative+stress+and+decreases+endocytosis+and+monolayer+permissiveness+in+cultured+kidney+epithelial+cells+without+affecting+viability%5BTitle%5D)+AND+%22Kidney+and+Blood+Pressure+Research%22%5BJournal%5D)">Myoglobin induces oxidative stress and decreases endocytosis and monolayer permissiveness in cultured kidney epithelial cells without affecting viability.</a> Kidney and Blood Pressure Research. 31, 16-28 (2008).</li>
<li>Saee-Rad, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+superoxide+dismutase+1%2C+dual-specificity+phosphatase+1%2C+and+transforming+growth+factor%2C+beta+1+genes+expression+in+keratoconic+and+non-keratoconic+corneas%5BTitle%5D)+AND+%22Molecular+vision%22%5BJournal%5D)">Analysis of superoxide dismutase 1, dual-specificity phosphatase 1, and transforming growth factor, beta 1 genes expression in keratoconic and non-keratoconic corneas.</a> Molecular vision. 19, 2501-2507 (2013).</li>
<li>Albert, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TRPV4+channels+mediate+the+infrared+laser-evoked+response+in+sensory+neurons%5BTitle%5D)+AND+%22Journal+of+neurophysiology%22%5BJournal%5D)">TRPV4 channels mediate the infrared laser-evoked response in sensory neurons.</a> Journal of neurophysiology. 107, 3227-3234 (2012).</li>
<li>Moro, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photobiomodulation+inside+the+brain%3A+a+novel+method+of+applying+near-infrared+light+intracranially+and+its+impact+on+dopaminergic+cell+survival+in+MPTP-treated+mice%5BTitle%5D)+AND+%22Journal+of+neuroscience%22%5BJournal%5D)">Photobiomodulation inside the brain: a novel method of applying near-infrared light intracranially and its impact on dopaminergic cell survival in MPTP-treated mice.</a> Journal of neuroscience. 120, 670-683 (2014).</li>
<li>Moreno, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infrared+neural+stimulation%3A+beam+path+in+the+guinea+pig+cochlea%5BTitle%5D)+AND+%22Hearing+research%22%5BJournal%5D)">Infrared neural stimulation: beam path in the guinea pig cochlea.</a> Hearing research. 282, 289-302 (2011).</li>
<li>Curthoys, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+red+thread+as+a+guide+in+the+vestibular+labyrinth%5BTitle%5D)+AND+%22The+Journal+of+physiology%22%5BJournal%5D)">A red thread as a guide in the vestibular labyrinth.</a> The Journal of physiology. 589, 1241-1241 (2011).</li>
<li>Chakrabarti, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+Dysfunction+during+Brain+Aging%3A+Role+of+Oxidative+Stress+and+Modulation+by+Antioxidant+Supplementation%5BTitle%5D)+AND+%22Aging+and+disease%22%5BJournal%5D)">Mitochondrial Dysfunction during Brain Aging: Role of Oxidative Stress and Modulation by Antioxidant Supplementation.</a> Aging and disease. 2, 242-256 (2011).</li>
<li>Petrosillo, G., De Benedictis, V., Ruggiero, F. M., Paradies, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decline+in+cytochrome+c+oxidase+activity+in+rat-brain+mitochondria+with+aging.+Role+of+peroxidized+cardiolipin+and+beneficial+effect+of+melatonin%5BTitle%5D)+AND+%22Journal+of+bioenergetics+and+biomembranes%22%5BJournal%5D)">Decline in cytochrome c oxidase activity in rat-brain mitochondria with aging. Role of peroxidized cardiolipin and beneficial effect of melatonin.</a> Journal of bioenergetics and biomembranes. 45, 431-440 (2013).</li>
<li>Zhu, H., Sun, A., Zou, Y., Ge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inducible+metabolic+adaptation+promotes+mesenchymal+stem+cell+therapy+for+ischemia%3A+a+hypoxia-induced+and+glycogen-based+energy+prestorage+strategy%5BTitle%5D)+AND+%22Arteriosclerosis%2C+thrombosis%2C+and+vascular+biology%22%5BJournal%5D)">Inducible metabolic adaptation promotes mesenchymal stem cell therapy for ischemia: a hypoxia-induced and glycogen-based energy prestorage strategy.</a> Arteriosclerosis, thrombosis, and vascular biology. 34, 870-876 (2014).</li>
</ol>

The authors declare they have no competing financial interests.

The representative results described here show that brief transcranial delivery of NIr light (90 sec/day for 5 days) is sufficient to raise the levels of antioxidant expression in older mice when compared with sham-treated mice. While emitted heat could represent a source of mitochondrial and/or neuronal activation, as reported for rat vestibular afferents24 &#8211; our measurement of heat emitted by the NIr LED device was &#60;0.2&#176;C over 90 sec, and as such is unlikely to cause the changes described here...

Declining balance performance and subsequent falls are common, and unfortunately often defining features of natural aging1. The impact of this decline can be both physical and social, and significantly reduces quality of life for older people. In response, physical therapies and rehabilitation have been the focus of research into falls but have not been associated with consistent reduction in the prevalence of repeated falls. At the same time, work investigating changes in the peripheral or central vestibular system (the system responsible for maintain balance) is scarce, and potential therapeutic strategies targeting these systems and the underlying causes of imbalance limited.
Recent work on age-associated neurodegenerative disorders including age-related macular degeneration2-4, Alzheimer&#8217;s disease models5-8, and Parkinson&#8217;s disease9-12 have shown neuroprotective effects of simple non-invasive application of near infrared (NIr) light. Further, in the vestibular system, NIr has been used to increase the activity of vestibular primary afferent neurons in vitro13. While the mechanism of NIr light is not well-understood, most studies using NIr have suggested that NIr stimulates mitochondria complex IV (cytochrome c oxidase)14-17 to facilitate cellular metabolism. In the vestibular sensory epithelium the subcuticular plate of type I hair cells is dense in mitochondria18 and as such may represent a site of action for therapeutic NIr treatment.
Here, a brief, non-invasive treatment regime of transcranially applied NIr that can be used to measure cellular metabolism (and by implication, mitochondrial function) in the mouse vestibular sensory epithelium is described. Also discussed is a preparation of the vestibular sensory epithelium and it is shown that NIr increases the expression of a ubiquitous anti-oxidant (superoxide dismutase 1) in the sensory epithelium &#8211; previously shown to be important for cochlea hair cell survival19.

<table><tbody><tr><td>Quantum WARP 10</td><td>Quantum Devices</td><td>2070N030-A</td><td></td></tr><tr><td>Screw top microtubules</td><td>Quality Scientific Plastics</td><td>520-GRD-Q</td><td></td></tr><tr><td>Ketamine</td><td>Parnell, Alexandria Australia</td><td></td><td></td></tr><tr><td>Standard pattern scissors</td><td>FST</td><td>14001-12</td><td></td></tr><tr><td>Carbon steel surgical blades #22</td><td>Livingstone</td><td>SBLDCL 22</td><td></td></tr><tr><td>Friedman-Pearson rongeurs</td><td>FST</td><td>16221-14</td><td></td></tr><tr><td>Stereo microscope</td><td>Leica Microsystems</td><td>A60S</td><td></td></tr><tr><td>Dumont #5 SF forceps</td><td>FST</td><td>11252-00</td><td></td></tr><tr><td>Isolate II RNA Micro Kit</td><td>Bioline</td><td>BIO-52075</td><td></td></tr></tbody></table>

near infrared (nir) light increases expression of a marker of mitochondrial function in the mouse vestibular sensory epithelium

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and exercise. However, these approaches do not address the underlying causes of balance decline. Using mice, the impact of near infrared light (NIr) on the metabolism of cells in the vestibular sensory epithelium was assessed. Data collected shows that this simple and safe intervention may protect these vulnerable cells from the deleterious effects of natural aging. mRNA was extracted from the isolated peripheral vestibular sensory epithelium (crista ampullaris and utricular macula) and subsequently transcribed into a cDNA library. This library was then probed for the expression of ubiquitous antioxidant (SOD-1). Antioxidant gene expression was then used to quantify cellular metabolism. Using transcranial delivery of NIr in young (4 weeks) and older (8 - 9 months) mice, and a brief treatment regime (90 sec/day for 5 days), this work suggests NIr alone may be sufficient to improve mitochondrial function in the vestibular sensory epithelium. Since there are currently no available, affordable, non-invasive methods of therapy to improve vestibular hair cell function, the application of external NIr radiation provides a potential strategy to counteract the impact of aging on cellular metabolism inthe vestibular sensory epithelium.

To compare the impact of NIr treatment in young (4 weeks) and older (8 - 9 months) mice we measured the expression of antioxidant superoxide dismutase 1 (SOD-1) in young (n = 16) and older (n = 20) mice that were NIr-treated, sham-treated, or NIr-blocked. Figure 2 shows a significant increase in &#946;-actin normalized SOD-1 expression of more than 2-fold in young NIr-treated animals compared to young sham-treated animals (p &#60; 0.01) and young NIr-blocked animals (p &#60; 0.01). Older NIr-treated anim...

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

Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

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

Strategies for attenuating decline in balance function with increasing age are predominantly focused on physical therapies including balance tasks and ...

near-infrared-nir-light-increases-expression-marker-mitochondrial

Research

JoVE Journal

Biology

9.9K Views.  University of Sydney. Mitochondrial dysfunction is a hallmark of cellular senescence. This paper uses non-invasive near-infrared (NIr) treatment to improve mitochondrial function in the aging mouse vestibular sensory epithelium.

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

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Neuroscience

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Biochemistry

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Medicine

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Measurement of Mitochondrial Membrane Potential In Vivo using a Genetically Encoded Voltage Indicator

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen species (ROS) generation, and the import of proteins encoded by the nucleus. Existing methods for measuring &#916;&#936;m typically use lipophilic cation dyes, such as Rhodamine 800 and tetramethylrhodamine methyl ester (TMRM), but these are limited by low specificity and are not well-suited for in vivo applications. To address these limitations, we have developed a novel protocol utilizing genetically encoded voltage indicators (GEVIs). Genetically encoded voltage indicators (GEVIs), which generate fluorescent signals in response to membrane potential changes, have demonstrated significant potential for monitoring plasma membrane and neuronal potentials. However, their application to mitochondrial membranes remains unexplored. Here, we developed protein-based mitochondrial-targeted GEVIs capable of detecting &#916;&#936;m fluctuations in cells and the motor cortex of living animals. The mitochondrial potential indicator (MPI)offers a non-invasive approach to study &#916;&#936;m dynamics in real-time, providing a method to investigate mitochondrial function under both normal and pathological conditions.

This protocol describes the application of mitochondria-targeted genetically encoded voltage indicators (GEVIs). These GEVIs offer a significant advantage over traditional mitochondrial membrane potential dyes by enabling specific, in vivo, and real-time monitoring of mitochondrial membrane potential.

Mitochondrial membrane potential (MMP, &#916;&#936;m) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen ...

Quantifying Mitophagy in C. elegans and a Liver Cancer Cell Line

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. 
Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.

Author Spotlight: Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes  

Exploring mitophagy through electron microscopy, genetic sensors, and immunofluorescence requires costly equipment, skilled personnel, and a significant time investment. Here, we demonstrate the efficacy of a commercial fluorescence dye kit in quantifying the mitophagy process in both Caenorhabditis elegans and a liver cancer cell line.

Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, ...

Imaging of Mitochondrial Peroxide in Living Yeast Cells Using mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, cancer, and normal aging. Here, an approach is described to assess mitochondrial function in living yeast cells at cellular and subcellular resolutions using a genetically encoded, minimally invasive, ratiometric biosensor. The biosensor, mitochondria-targeted HyPer7 (mtHyPer7), detects hydrogen peroxide (H2O2) in mitochondria. It consists of a mitochondrial signal sequence fused to a circularly permuted fluorescent protein and the H2O2-responsive domain of a bacterial OxyR protein. The biosensor is generated and integrated into the yeast genome using a CRISPR-Cas9 marker-free system, for&#160;more consistent expression compared to plasmid-borne constructs.
mtHyPer7 is quantitatively targeted to mitochondria, has no detectable effect on yeast growth rate or mitochondrial morphology, and provides a quantitative readout for mitochondrial H2O2 under normal growth conditions and upon exposure to oxidative stress. This protocol explains how to optimize imaging conditions using a spinning-disk confocal microscope system and perform quantitative analysis using freely available software. These tools make it possible to collect rich spatiotemporal information on mitochondria both within cells and among cells in a population. Moreover, the workflow described here can be used to validate other biosensors.

Author Spotlight: Advancing Mitochondrial Research - mtHyper7 Biosensor for Subcellular Analysis 

Hydrogen peroxide (H2O2) is both a source of oxidative damage and a signaling molecule. This protocol describes how to measure mitochondrial H2O2 using mitochondria-targeted HyPer7 (mtHyPer7), a genetically encoded ratiometric biosensor, in living yeast. It details how to optimize imaging conditions and perform quantitative cellular and subcellular analysis using freely available software.

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...

Analyzing Mitochondrial Morphology in C. elegans

Imaging and Quantifying Mitochondrial Morphology in C. elegans During Aging

Mitochondria, important cellular organelles found in most eukaryotic cells, are major sites of energy production through aerobic respiration. Beyond this well-known role as the 'cellular powerhouse,' mitochondria are also involved in many other essential cellular processes, including the regulation of cellular metabolism, proliferation, immune signaling, and hormonal signaling. Deterioration in mitochondrial function during aging or under mitochondrial stress is often characterized by distinct changes in mitochondrial morphology and volume. The nematode C. elegans is an ideal model for studying these changes due to its transparent body and short lifespan, which facilitate live microscopy throughout its lifetime. However, even within the C. elegans field, numerous transgenic constructs and methods for mitochondrial imaging are available, each with its own limitations. Here, single-copy, matrix-localized GFP constructs are presented as a robust and reliable method for imaging mitochondrial morphology in C. elegans. This study specifically focuses on experimentally controllable factors to minimize errors and reduce variability between replicates and across studies when performing mitochondrial imaging during the aging process. Additionally, mitoMAPR is recommended as a robust method to quantify changes in mitochondrial morphology across tissue types during aging.

This protocol provides a standardized approach for imaging mitochondrial morphology in multiple tissues of C. elegans during aging.

Mitochondria, important cellular organelles found in most eukaryotic cells, are major sites of energy production through aerobic respiration. Beyond this ...

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

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An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

In Vivo Imaging of Cx3cr1^gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

Measurement of Mitochondrial Membrane Potential In Vivo using a Genetically Encoded Voltage Indicator

Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Imaging and Quantifying Mitochondrial Morphology in C. elegans During Aging