Introduction
Noise exposure is a major cause of hearing loss worldwide (
Hammer et al., 2014
- Hammer M.S.
- Swinburn T.K.
- Neitzel R.L.
Environmental noise pollution in the United States: developing an effective public health response.
). Noise exposure causes damage to diverse cochlear structures, including the spiral ganglia nerve fibers that normally form synaptic contacts with cochlear hair cells (
). These synapses enable the spiral ganglia to convey acoustic information from the cochlea to higher-order brain stem structures. Following intense noise exposure, hair cells release neurotransmitters that lead to excitotoxic damage in neurites, resulting in synaptic disruption and neurite degeneration that is evident after 24 hr (
Kujawa and Liberman, 2009
- Kujawa S.G.
- Liberman M.C.
Adding insult to injury: cochlear nerve degeneration after “temporary” noise-induced hearing loss.
,
Lin et al., 2011
- Lin H.W.
- Furman A.C.
- Kujawa S.G.
- Liberman M.C.
Primary neural degeneration in the Guinea pig cochlea after reversible noise-induced threshold shift.
,
). If noise exposure is moderate, neurite regeneration can occur, which can restore synaptic connectivity and auditory capacity (
Puel et al., 1998
- Puel J.L.
- Ruel J.
- Gervais d’Aldin C.
- Pujol R.
Excitotoxicity and repair of cochlear synapses after noise-trauma induced hearing loss.
). However, persistent noise exposure or intense acoustic trauma can result in permanent neurite degeneration (
).
Spiral ganglia neurite degeneration is linked to mitochondrial dysfunction. Following noise exposure, glutamate release induces the formation of mitochondria-derived reactive oxygen species (ROS) (
Jäger et al., 2000
- Jäger W.
- Goiny M.
- Herrera-Marschitz M.
- Brundin L.
- Fransson A.
- Canlon B.
Noise-induced aspartate and glutamate efflux in the guinea pig cochlea and hearing loss.
,
Ohlemiller et al., 1999
- Ohlemiller K.K.
- Wright J.S.
- Dugan L.L.
Early elevation of cochlear reactive oxygen species following noise exposure.
,
Puel et al., 1995
- Puel J.L.
- Saffiedine S.
- Gervais d’Aldin C.
- Eybalin M.
- Pujol R.
Synaptic regeneration and functional recovery after excitotoxic injury in the guinea pig cochlea.
,
Puel et al., 1998
- Puel J.L.
- Ruel J.
- Gervais d’Aldin C.
- Pujol R.
Excitotoxicity and repair of cochlear synapses after noise-trauma induced hearing loss.
,
Ruel et al., 2005
- Ruel J.
- Wang J.
- Pujol R.
- Hameg A.
- Dib M.
- Puel J.L.
Neuroprotective effect of riluzole in acute noise-induced hearing loss.
). Thus, impaired mitochondrial function may be an early step in NIHL.
Studies over the past decade have suggested that NAD
+ may be useful for blocking axonal degeneration; however, the idea that NAD
+ exerts axon-protective effects is controversial. Milbrandt and colleagues first showed that application of NAD
+ to sensory neurons prevents axonal degeneration elicited by transection (
Araki et al., 2004
- Araki T.
- Sasaki Y.
- Milbrandt J.
Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration.
). Although this study suggested that the effects of NAD
+ are transcription dependent and occur at micromolar concentrations, another study showed that the effects of NAD
+ are transcription independent and require application of millimolar concentrations to axons (
Wang et al., 2005
- Wang J.
- Zhai Q.
- Chen Y.
- Lin E.
- Gu W.
- McBurney M.W.
- He Z.
A local mechanism mediates NAD-dependent protection of axon degeneration.
). Other studies cast doubt on the idea that NAD
+-biosynthetic enzymes exert their axon-protective effects through NAD
+, since their protective effects do not correlate with their effects on NAD
+ levels (
Sasaki et al., 2009
- Sasaki Y.
- Vohra B.P.
- Lund F.E.
- Milbrandt J.
Nicotinamide mononucleotide adenylyl transferase-mediated axonal protection requires enzymatic activity but not increased levels of neuronal nicotinamide adenine dinucleotide.
). Additionally, the intracellular target of NAD
+ has been controversial. Initial studies suggested a role for the sirtuin SIRT1 in cultured neurons (
Araki et al., 2004
- Araki T.
- Sasaki Y.
- Milbrandt J.
Increased nuclear NAD biosynthesis and SIRT1 activation prevent axonal degeneration.
). However, this could not be replicated in
SIRT1 knockout animals (
Wang et al., 2005
- Wang J.
- Zhai Q.
- Chen Y.
- Lin E.
- Gu W.
- McBurney M.W.
- He Z.
A local mechanism mediates NAD-dependent protection of axon degeneration.
). The diverse inconsistencies seen in these and other studies make it unclear whether NAD
+ influences a physiologically relevant axon-degeneration pathway.
The inconsistencies seen in studies of NAD
+ may relate to the use of cultured neurons. Removal of neurons from their native environment and culturing them results in altered gene expression relative to neurons in vivo (
Díaz et al., 2002
- Díaz E.
- Ge Y.
- Yang Y.H.
- Loh K.C.
- Serafini T.A.
- Okazaki Y.
- Hayashizaki Y.
- Speed T.P.
- Ngai J.
- Scheiffele P.
Molecular analysis of gene expression in the developing pontocerebellar projection system.
). Additionally, Schwann cells and oligodendrocytes can be lost during culturing. These cells have a major role in regulating axonal integrity and influence axonal metabolism by transferring metabolites to axons (
Saab et al., 2013
- Saab A.S.
- Tzvetanova I.D.
- Nave K.A.
The role of myelin and oligodendrocytes in axonal energy metabolism.
). Since these cells are often lost during culturing, it is difficult to extrapolate studies on axon degeneration performed in vitro to axons in vivo that retain their interactions with diverse supporting cells. Thus, it remains unclear if NAD
+ exerts an axon protective effect, and if this effect is seen in animals.
It is difficult to determine if NAD
+ prevents axon degeneration in vivo. NAD
+ is readily degraded by serum hydrolases (
Chi and Sauve, 2013
Nicotinamide riboside, a trace nutrient in foods, is a vitamin B3 with effects on energy metabolism and neuroprotection.
), making it difficult to test its effects in animals. Additionally, NAD
+ is highly polar and, like other nucleotides and dinucleotides, is not readily taken up by cells (
Bortell et al., 2001
- Bortell R.
- Moss J.
- McKenna R.C.
- Rigby M.R.
- Niedzwiecki D.
- Stevens L.A.
- Patton W.A.
- Mordes J.P.
- Greiner D.L.
- Rossini A.A.
Nicotinamide adenine dinucleotide (NAD) and its metabolites inhibit T lymphocyte proliferation: role of cell surface NAD glycohydrolase and pyrophosphatase activities.
,
Yang et al., 2007
- Yang T.
- Chan N.Y.
- Sauve A.A.
Syntheses of nicotinamide riboside and derivatives: effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells.
). As a result, millimolar extracellular concentrations are needed to induce micromolar changes in intracellular NAD
+ concentrations (
Bortell et al., 2001
- Bortell R.
- Moss J.
- McKenna R.C.
- Rigby M.R.
- Niedzwiecki D.
- Stevens L.A.
- Patton W.A.
- Mordes J.P.
- Greiner D.L.
- Rossini A.A.
Nicotinamide adenine dinucleotide (NAD) and its metabolites inhibit T lymphocyte proliferation: role of cell surface NAD glycohydrolase and pyrophosphatase activities.
,
Yang et al., 2007
- Yang T.
- Chan N.Y.
- Sauve A.A.
Syntheses of nicotinamide riboside and derivatives: effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells.
). Nicotinamide, an NAD
+ precursor, has been tested for effects on axon degeneration in an encephalomyelitis model (
Kaneko et al., 2006
- Kaneko S.
- Wang J.
- Kaneko M.
- Yiu G.
- Hurrell J.M.
- Chitnis T.
- Khoury S.J.
- He Z.
Protecting axonal degeneration by increasing nicotinamide adenine dinucleotide levels in experimental autoimmune encephalomyelitis models.
). However, because nicotinamide inhibits sirtuins, NAD
+-dependent deacetylating/deacylating enzymes (
), it is unclear if the effects of nicotinamide reflect its ability to increase NAD
+ or its pleiotropic inhibitory effects. Thus, it remains unclear if increasing cellular NAD
+ levels is an effective approach for blocking axonal degeneration in vivo.
Here we examine whether augmentation of intracochlear NAD
+ levels protects mice from NIHL. Using Wld
S mice, which overexpress an NAD
+ biosynthetic enzyme (
Conforti et al., 2000
- Conforti L.
- Tarlton A.
- Mack T.G.
- Mi W.
- Buckmaster E.A.
- Wagner D.
- Perry V.H.
- Coleman M.P.
A Ufd2/D4Cole1e chimeric protein and overexpression of Rbp7 in the slow Wallerian degeneration (WldS) mouse.
), we show that genetic stabilization of NAD
+ levels markedly protects mice from NIHL. In order to test the effect of increasing intracellular NAD
+ levels, we used the NAD
+ precursor, nicotinamide riboside (NR). NR has been shown to inhibit axon degeneration elicited by axon transection in cultured neurons (
Sasaki et al., 2006
- Sasaki Y.
- Araki T.
- Milbrandt J.
Stimulation of nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy.
). We find that NR administration provides an efficient route to increase NAD
+ levels in the cochlea in mice and also protects mice from NIHL and noise-induced spiral ganglia neurite retraction. Importantly, we also show that the protective effects of NR can be achieved even when NR is administered after noise exposure, suggesting that NR might be clinically useful to prevent hearing loss after unexpected noise exposure in humans. We show that the effects of NR and the noise resistance of Wld
S mice are both promoted by the NAD
+-dependent mitochondrial sirtuin SIRT3. These data demonstrate the translational value of NR for pharmacologic augmentation of NAD
+ and activation of a SIRT3-dependent pathway that inhibits NIHL.
Discussion
Currently, there is no effective therapy to prevent hearing loss following noise exposure. In this study we show that noise exposure results in a drop in cochlear NAD+ levels, and pharmacologic augmentation of NAD+ using NR provides robust protection from acoustic trauma in a SIRT3-dependent manner. We show that NR preserves the synaptic contacts between the spiral ganglia neurites and hair cells, demonstrating that the effects of NR reflect preserved cochlear circuitry. These studies point to a NAD+/SIRT3 pathway that can be induced by NR to prevent NIHL and spiral ganglia neurite degeneration.
Our data identify SIRT3 as a target of NR and Wld
S in NIHL. The mitochondrial sirtuin SIRT3 is likely to be particularly responsive to pharmacologically administered NR, since NR preferentially increases mitochondrial NAD
+ levels (
Cantó et al., 2012
- Cantó C.
- Houtkooper R.H.
- Pirinen E.
- Youn D.Y.
- Oosterveer M.H.
- Cen Y.
- Fernandez-Marcos P.J.
- Yamamoto H.
- Andreux P.A.
- Cettour-Rose P.
- et al.
The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity.
). Mitochondrial proteins appear particularly prone to enzyme-inactivating acetylation on catalytic lysine residues (
Ghanta et al., 2013
- Ghanta S.
- Grossmann R.E.
- Brenner C.
Mitochondrial protein acetylation as a cell-intrinsic, evolutionary driver of fat storage: chemical and metabolic logic of acetyl-lysine modifications.
). SIRT3 maintains enzyme function by removing these acetylation marks (
Wagner and Payne, 2013
Widespread and enzyme-independent Nε-acetylation and Nε-succinylation of proteins in the chemical conditions of the mitochondrial matrix.
). Indeed, impaired SIRT3 function leads to enhanced ROS generation (
He et al., 2012
- He W.
- Newman J.C.
- Wang M.Z.
- Ho L.
- Verdin E.
Mitochondrial sirtuins: regulators of protein acylation and metabolism.
) and lower levels of reduced glutathione (
Someya et al., 2010
- Someya S.
- Yu W.
- Hallows W.C.
- Xu J.
- Vann J.M.
- Leeuwenburgh C.
- Tanokura M.
- Denu J.M.
- Prolla T.A.
Sirt3 mediates reduction of oxidative damage and prevention of age-related hearing loss under caloric restriction.
). Importantly, both ROS and impaired glutathione levels are linked to increased susceptibility to NIHL (
Someya et al., 2010
- Someya S.
- Yu W.
- Hallows W.C.
- Xu J.
- Vann J.M.
- Leeuwenburgh C.
- Tanokura M.
- Denu J.M.
- Prolla T.A.
Sirt3 mediates reduction of oxidative damage and prevention of age-related hearing loss under caloric restriction.
). The reduced cochlear NAD
+ levels seen after noise exposure may account for the increased mitochondria-derived ROS that is seen following acoustic trauma (
Ohlemiller et al., 1999
- Ohlemiller K.K.
- Wright J.S.
- Dugan L.L.
Early elevation of cochlear reactive oxygen species following noise exposure.
). Thus, activation of SIRT3 by augmenting intracellular NAD
+ levels provides a mechanism to counteract this central pathogenic mechanism in hearing loss.
The Wld
S allele ameliorates axonal degeneration in a variety of neurodegenerative models (
Kaneko et al., 2006
- Kaneko S.
- Wang J.
- Kaneko M.
- Yiu G.
- Hurrell J.M.
- Chitnis T.
- Khoury S.J.
- He Z.
Protecting axonal degeneration by increasing nicotinamide adenine dinucleotide levels in experimental autoimmune encephalomyelitis models.
,
Sajadi et al., 2004
- Sajadi A.
- Schneider B.L.
- Aebischer P.
Wlds-mediated protection of dopaminergic fibers in an animal model of Parkinson disease.
,
Samsam et al., 2003
- Samsam M.
- Mi W.
- Wessig C.
- Zielasek J.
- Toyka K.V.
- Coleman M.P.
- Martini R.
The Wlds mutation delays robust loss of motor and sensory axons in a genetic model for myelin-related axonopathy.
). However, therapeutic strategies that mimic Wld
S are currently unavailable. Administration of NAD
+ is not viable due to the poor solubility, cell impermeability, and serum instability of this compound (
Pitkänen, 1971
The hydrolysis of nicotinamide adenine dinucleotide phosphate by serum alkaline phosphatase.
). The recent development of a synthetic method to produce NR at quantity sufficient for administration to animals (
Yang et al., 2007
- Yang T.
- Chan N.Y.
- Sauve A.A.
Syntheses of nicotinamide riboside and derivatives: effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells.
) allows the testing of the physiologic effects of augmenting intracellular NAD
+ levels. Our finding that NR mimics the NIHL resistance seen in Wld
S animals raises the possibility that NR might also be useful for preventing axonal degeneration in other disease models that exhibit responsiveness to Wld
S expression.
The data supporting the role of NAD
+ in axon protection have been inconsistent. Although Wld
S animals show delayed axon degeneration for several weeks in a transection model (
Conforti et al., 2009
- Conforti L.
- Wilbrey A.
- Morreale G.
- Janeckova L.
- Beirowski B.
- Adalbert R.
- Mazzola F.
- Di Stefano M.
- Hartley R.
- Babetto E.
- et al.
Wld S protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice.
), application of NAD
+ to transected axons in culture delays degeneration for only a few hours or days (
Wang et al., 2005
- Wang J.
- Zhai Q.
- Chen Y.
- Lin E.
- Gu W.
- McBurney M.W.
- He Z.
A local mechanism mediates NAD-dependent protection of axon degeneration.
) and requires millimolar concentrations. Additionally, inconsistent results using cultured neurons have made it unclear whether NAD
+ would function to inhibit axon degeneration in animals. Poor bioavailability has prevented testing the role of NAD
+ in animals using disease-relevant axon degeneration models. Our data using NR demonstrate that NAD
+ and an NAD
+ target, SIRT3, control axon degeneration in animals. It will be important to perform thorough analysis of NR and SIRT3 in other forms of axon degeneration in animals to define which of these can be blocked by NR.
Although our data identify a NAD
+/SIRT3 pathway that influences neurite degeneration, other pathways are also likely to affect neurite degeneration. First, it is likely that other sirtuins are activated by increased NAD
+ levels and contribute to the axon-protective effects of NAD
+, possibly by enhancing SIRT3 transcription, as has been described for SIRT1 (
Amat et al., 2009
- Amat R.
- Planavila A.
- Chen S.L.
- Iglesias R.
- Giralt M.
- Villarroya F.
SIRT1 controls the transcription of the peroxisome proliferator-activated receptor-gamma Co-activator-1α (PGC-1α) gene in skeletal muscle through the PGC-1α autoregulatory loop and interaction with MyoD.
). Additionally, transection axon injury results in a loss of the axonal NAD
+ biosynthetic enzyme NMNAT-2 (
). This is expected to result in an increase in NMN levels (
). Expression of Wld
S may delay transection-induced degeneration by converting this accumulated NMN to NAD
+. However, in addition to the loss of the protective effects of NAD
+, loss of NMNAT-2 could lead to the accumulation of NAD
+ metabolic precursors that may have their own, potentially toxic, effects in axons (
Di Stefano et al., 2014
- Di Stefano M.
- Nascimento-Ferreira I.
- Orsomando G.
- Mori V.
- Gilley J.
- Brown R.
- Janeckova L.
- Vargas M.E.
- Worrell L.A.
- Loreto A.
- et al.
A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration.
). NR could have direct protective effects by being converted to mitochondrial NAD
+ and subsequently to mitochondrial NADPH, which is required to regenerate reduced glutathione, a neuroprotective agent in NIHL (
Bellomo et al., 1987
- Bellomo G.
- Mirabelli F.
- DiMonte D.
- Richelmi P.
- Thor H.
- Orrenius C.
- Orrenius S.
Formation and reduction of glutathione-protein mixed disulfides during oxidative stress. A study with isolated hepatocytes and menadione (2-methyl-1,4-naphthoquinone).
,
Yamasoba et al., 1998
- Yamasoba T.
- Nuttall A.L.
- Harris C.
- Raphael Y.
- Miller J.M.
Role of glutathione in protection against noise-induced hearing loss.
). NR or NAD
+ may also influence CNS auditory pathways. Lastly, axon protection mediated by overexpression of NMNAT enzymes may be in part due to chaperone-like functions of these proteins. This activity may contribute to their axon-protective effects in addition to their NAD
+ biosynthetic function (
Zhai et al., 2008
- Zhai R.G.
- Zhang F.
- Hiesinger P.R.
- Cao Y.
- Haueter C.M.
- Bellen H.J.
NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration.
). Nevertheless, our data suggest that NR mediates its effects through SIRT3 to induce substantial protective effects even in the absence of these additional mechanisms.
It is currently unclear why there are marked person-to-person differences in susceptibility to NIHL (
Davis et al., 2003
- Davis R.R.
- Kozel P.
- Erway L.C.
Genetic influences in individual susceptibility to noise: a review.
). Polymorphisms exist in the SIRT3 gene that affects its activity (
Hirschey et al., 2011
- Hirschey M.D.
- Shimazu T.
- Jing E.
- Grueter C.A.
- Collins A.M.
- Aouizerat B.
- Stančáková A.
- Goetzman E.
- Lam M.M.
- Schwer B.
- et al.
SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome.
) and may in turn contribute to susceptibility to NIHL. NR is used as a dietary supplement and is a natural component of milk and certain other foods (
Bogan and Brenner, 2008
Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+ precursor vitamins in human nutrition.
), raising the possibility that variability in dietary NR consumption could also contribute to the susceptibility to hearing loss in humans. Since NR exhibits minimal toxicity (
Yang et al., 2007
- Yang T.
- Chan N.Y.
- Sauve A.A.
Syntheses of nicotinamide riboside and derivatives: effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells.
,
Bieganowski and Brenner, 2004
- Bieganowski P.
- Brenner C.
Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans.
,
Cantó et al., 2012
- Cantó C.
- Houtkooper R.H.
- Pirinen E.
- Youn D.Y.
- Oosterveer M.H.
- Cen Y.
- Fernandez-Marcos P.J.
- Yamamoto H.
- Andreux P.A.
- Cettour-Rose P.
- et al.
The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity.
), NR supplementation may be a valuable approach for reducing the prevalence of NIHL.
Experimental Procedures
Chemicals
NR was synthesized as previously described (
Yang et al., 2007
- Yang T.
- Chan N.Y.
- Sauve A.A.
Syntheses of nicotinamide riboside and derivatives: effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells.
). Other chemicals and reagents were purchased from Sigma except as indicated below.
Animals
Eight- to ten-week-old animals were used for all experiments to eliminate the effects of aging in C57BL/6 animal backgrounds as previously described (
Someya et al., 2010
- Someya S.
- Yu W.
- Hallows W.C.
- Xu J.
- Vann J.M.
- Leeuwenburgh C.
- Tanokura M.
- Denu J.M.
- Prolla T.A.
Sirt3 mediates reduction of oxidative damage and prevention of age-related hearing loss under caloric restriction.
). C57BL/6, BalbC, and CBA mice were purchased from Jackson Laboratories. Wld
S tg/+ mice were provided backcrossed on a C57BL/6 background and were further backcrossed a minimum of five generations on the C57BL/6 background (Jackson Laboratories). These mice were a kind gift of Dr. Michael Coleman (Babraham Institute, University of Cambridge).
For experiments using SIRT3 knockout mice, male and female SIRT3−/− mice were purchased from the Mutant Mouse Resource Centers at the University of North Carolina-Chapel Hill (Chapel Hill, NC). These mice were developed from embryonic stem cells (ESCs) (Omni bank number OST341297) with a retroviral promoter trap that functionally inactivates one allele of the SIRT3 gene. SIRT3−/− mice were backcrossed five generations onto the C57BL/6 background.
The transgene
SIRT3-overexpressing vector was constructed by introducing a C-terminal FLAGed cDNA encoding the mouse mitochondrial form of SIRT3 (
Cooper et al., 2009
- Cooper H.M.
- Huang J.Y.
- Verdin E.
- Spelbrink J.N.
A new splice variant of the mouse SIRT3 gene encodes the mitochondrial precursor protein.
) into the pCall2 vector (
Lobe et al., 1999
- Lobe C.G.
- Koop K.E.
- Kreppner W.
- Lomeli H.
- Gertsenstein M.
- Nagy A.
Z/AP, a double reporter for cre-mediated recombination.
). The vector pCall2-mSIRT3 was double digested with XmnI and SfiI, and the resulting 7.9 kb fragment was gel purified and used to microinject C57BL/6 mouse embryos oocytes (Gladstone Transgenic Gene-Targeting Core). Several independent mouse lines carrying the
SIRT3 transgene were generated. First-generation offspring (F1) of the injected mice were crossed with wild-type mice and analyzed for germline incorporation of the transgene. Several F1 mice with germline incorporation were preserved as founding
SIRT3 transgenic mouse lines. The mouse line used in this manuscript is called line 28 and contains the transgene inserted in an intergenic region on chromosome 10 (NCBI Ref NT_039500.7).
All animal behavioral studies were conducted at the AAALAC-approved facility at Weill Cornell Medical College. Experiments were performed in accordance with protocols approved by the Weill Cornell Medical College Institutional Animal Care and Use Committee (New York, NY).
Cochlear Histology/Immunohistology
Mouse cochlea were prepared as previously described (
Whitlon et al., 2001
- Whitlon D.S.
- Szakaly R.
- Greiner M.A.
Cryoembedding and sectioning of cochleas for immunocytochemistry and in situ hybridization.
). Briefly, mouse cochlea were quickly dissected from the temporal bone following rapid decapitation. Once separated, the apex of the cochlea was gently fenestrated, and the cochlea were immediately fixed in 4% paraformaldehyde overnight at 4°C. The cochlea were then washed with three changes of PBS and incubated in a decalcification solution (10% EDTA/PBS [pH 7.4]) under constant rotation at 4°C for 7 days. Decalcification solution was changed daily. Cochlea were then washed in three changes of PBS and then treated with progressively increasing sucrose concentrations from 10%–30%. They were then incubated overnight in 30% sucrose at 4°C. The cochlea were then incubated an additional 24 hr in OCT compound (Tissue-Tek). Following this final incubation the cochlea were transferred to cryomolds, carefully aligning the modiolus parallel to the bottom of the mold and frozen over dry ice. Midmodiolar samples were then cut at a 10 μm thickness and mounted on glass slides (VWR superfrost plus). Sections were then dried for 2 hr prior to staining.
Slides were then postfixed with 1.5% paraformaldehyde for 5 min. Slides were washed and then incubated with 0.5% Triton X-100/PBS for 15 min. They were again washed and then blocked with 2% BSA/PBS. Sections were then incubated with phalloidin-488 (Invitrogen) as per the manufacturer’s instructions for 20 min. Slides were washed and then incubated with 1:1,000 rabbit anti-heavy neurofilament antibody overnight at 4°C. Slides were then washed and incubated with 1:1,000 Alexa Fluor 546 goat anti-rabbit antibody (Invitrogen) for 1 hr at room temperature. After a final wash, sections were mounted with ProLong Gold antifade reagent with DAPI (Life Technologies). Three-color epifluorescence imaging was then performed using a Nikon Eclipse Ti microscope with a Coolsnap HQ2 camera.
NAD+ Quantification
Mice were injected twice daily with NR (1,000 mg/kg) for 5 days prior to noise and injury and for 48 hr thereafter until they were sacrificed and cochlea harvested. Noise injury was performed as indicated below. Cochlea were isolated by microdissection from the otic capsule. Pairs of cochlea were homogenized with a micropestle and resuspended in 7% perchloric acid. Samples were incubated for 5 min on ice, and any remaining insoluble debris was pelleted and saved for protein quantification via bicinchoninic acid (BCA) assay. The supernatant was neutralized (pH 7) with NaOH and 0.1 M phosphate buffer. Sample was added to a 96-well plate and mixed with cycling assay buffer (5 mM Tris-HCl, 5 mM MgCl2, 50 mM KCl, 54 M resazurin, 2.25 mM lactate, 0.4 U/mL lactate dehydrogenase). Finally, 0.5 U diaphorase was added to each well, and the production of resorufin was recorded (530 nm excitation, 580 nm emission) every 30 s for 15 min. NAD+ concentration was calculated based on a standard curve, and the results were expressed as nmol NAD+ per mg of protein. Three technical replicates were performed for n = 7 mice per condition. Because of experiment-to-experiment variation in sample processing, samples were processed in parallel and normalized to the before-noise sample. The normalized values from each experiment were used to obtain the presented average values.
Auditory Testing
ABR testing was performed as previously described (
Willott, 2006
Measurement of the auditory brainstem response (ABR) to study auditory sensitivity in mice.
). Animals were tested following sedation with ketamine/xylazine (40 mg/kg and 10 mg/kg, respectively). Tone burst stimuli at 8,000, 16,000, and 32,000 kHz for 5 ms were used to elicit auditory evoked responses using an auditory brainstem recording system (Intelligent Hearing Systems, Miami, FL). An evoked response was determined by identifying waveforms at proper time intervals that grew increased in magnitude with increasing volume as described previously (
Willott, 2006
Measurement of the auditory brainstem response (ABR) to study auditory sensitivity in mice.
).
Noise Exposure
Animals were exposed to a 90 dB octave band for 2 hr in a cage placed in a soundproof chamber (MAC-2, Industrial Acoustics Company, Bronx NY). The mice were able to freely move throughout the cage. The octave band was generated using ToneGen software (NCH software, Greenwood Village, CO) routed through an Audiosource Amp100 amplifier driving two down-facing Fostex FT-96H speakers. The sound pressure level was confirmed at 0, 30, 60, and 90 min, and again just prior to completion of sound exposure using an Extech microphone 407736.
Article info
Publication history
Published: December 2, 2014
Accepted: November 4, 2014
Received in revised form: September 8, 2014
Received: July 21, 2014
Copyright
© 2014 Elsevier Inc. Published by Elsevier Inc.