- Email: [email protected]
Regional distribution of manganese superoxide dismutase 2 (Mn SOD2) expression in rodent and primate spiral ganglion cells
Hearing Research 253 (2009) 116–124
Contents lists available at ScienceDirect
Hearing Research journal homepage: www.elsevier.com/locate/heares
Research paper
Regional distribution of manganese superoxide dismutase 2 (Mn SOD2) expression in rodent and primate spiral ganglion cells Yu-Lan Mary Ying *, Carey D. Balaban Department of Otolaryngology, University of Pittsburgh, 200 Lothrop Street, Suite # 500, Pittsburgh, PA 15213, USA
a r t i c l e
i n f o
Article history: Received 28 January 2009 Received in revised form 2 April 2009 Accepted 2 April 2009 Available online 17 April 2009 Keywords: Spiral ganglion cells Manganese superoxide dismutase 2 Gradient Reactive oxygen species Primate Rodent Superoxide dismutase Immunohistochemistry
a b s t r a c t Manganese superoxide dismutase 2 (SOD2) is a key metabolic anti-oxidant enzyme for detoxifying free radicals inside mitochondria. This study documents a gradient in expression of SOD2 by spiral ganglion cells in basal versus apical turn of cochlea that is consistent with differential vulnerability of high frequency hearing to free radical damage. Immunohistochemical methods were used to identify distribution of SOD2 in temporal bone sections from mice, rats, macaques, and humans. In mice and rats, both the proportion of SOD2 immunopositive type 1 spiral ganglion cells and the intensity of immunoreactivity were elevated near cochlear apex. In macaques and humans, the proportion of SO2 immunopositive spiral ganglion cells was equal across cochlear turn, but the intensity of immunoreactivity remained highest for ganglion cells near cochlear apex. Strong SOD2 immunoreactivity was also observed in human type 1 spiral ganglion cells. The average area density of SOD2 immunoreactivity in ganglion cells for each species and cochlear turn showed an allometric relationship with body weight, which is consistent with a conserved basal metabolic characteristic. These findings suggest that spiral ganglion cell responses to ROS exposure may vary along cochlear spiral with lower response capacity at cochlear base contributing to cumulative susceptibility to high frequency hearing loss. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Multiple factors contribute to inner ear pathologies causing hearing loss. Genetic factors, age-related changes (presbycusis), acoustic trauma, infectious disease, and exposure to ototoxins (e.g., aminoglycosides and cisplatin) have been implicated as potential etiologic factors in clinical dysfunction. The histopathologic hallmarks of presbycusis include losses of hair cells, spiral ganglion cells, strial atrophy, and abnormalities of supporting structures within the cochlea (Schuknecht, 1964; Schuknecht and Gacek, 1993; Zimmermann et al., 1995). Degenerative changes in the organ of Corti and spiral ganglion cells in presbycusis are similar to the degeneration in noise trauma, toxic insults, infectious and immunologic influences and congenital lesions (Zimmermann et al., 1995). Specifically, high frequency sensorineural hearing loss is detected as an early sign of aminoglycoside toxicity, cisplatin toxicity or presbycusis. Hearing loss often progresses along a high to low frequency gradient that corresponds to progressive pathophysiologic changes from the basal to the apical cochlear turn. Different cochlear cell populations appear to have their own intrinsic susceptibilities to free radical damage. For example, Sha et al. (2001) reported that outer hair cells are most vulnerable to * Corresponding author. Tel.: +1 412 647 8050/2298; fax: +1 412 647 0108. E-mail addresses: [email protected] (Y.-L.M. Ying), [email protected] (C.D. Balaban). 0378-5955/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2009.04.006
free radical mediated ototoxicity followed by inner hair cells; supporting cells are the least susceptible to damage. Recent studies have regarded hair cells within the organ of Corti as primary sites of tissue insult in acoustic trauma and in presbycusis (Wang et al., 2002; Chen and Fechter, 2003), and tend to assume that degenerative changes in spiral ganglion cells or the cochlear nerve are secondary effects. However, there is long-standing evidence that degeneration of the spiral ganglion cells can be an independent and primary phenomenon with no obvious initiating event in the organ of Corti (Schuknecht, 1955; Schuknecht and Gacek, 1993). Because significant aminoglycoside destruction of spiral ganglion cells can occur without any effect on hair cells (Stone et al., 1998), pathological effects at the level of spiral ganglion cells may affect hearing independent of hair cell status. The basal half of the cochlea appears to have a predilection for the most severe spiral ganglion degeneration (Zimmermann et al., 1995; Nelson and Hinojosa, 2006). Basal turn outer hair cells are also more susceptible to aminoglycoside and cisplatin damage than apical outer hair cells (Sha et al., 2001). These phenomena parallel the common clinical observation of high frequency hearing loss and raise the question of which mechanism might be responsible for differential cellular vulnerability along the cochlear spiral. The cochlear nerve serves as the link between sensory hair cells and the central nervous system. Preservation of spiral ganglion cells is essential for success of cochlear implantation because stimulation of particular type I ganglion cells is of primary importance
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
for speech recognition (Zimmermann et al., 1995). Therefore, there is a need to develop therapeutic strategies to preserve cochlear nerve function in the face of age-related pathophysiologic changes and ototoxic processes in the inner ear. Although the cellular mechanisms underlying cochlear degeneration are far from fully known, the available evidence implicates the formation and accumulation of reactive oxygen species (ROS) may play a significant role in cochlear cell loss over time. For example, antioxidant treatments to prevent degeneration of deafferented CN VIII spiral ganglion cells in deafened guinea pigs in the context of free radical formation that signals cell death pathways has been reported (Maruyama et al., 2007, 2008). A family of superoxide dismutases (SODs) plays an important role in the initial cellular defense against generation of ROS (Freeman and Crapo, 1982). Copper/Zinc SOD 1 (SOD1) is widely distributed in the nucleus and cytoplasm, and comprises 90% of total SOD activity. Manganese SOD 2 (SOD2) is localized in the mitochondria. Extracellular SOD is localized in the extracellular matrix (Gao et al., 2008). Null mutant sod1 transgenic mice display early hearing loss, accompanied by degeneration of spiral ganglion neurons and hair cells (McFadden et al., 1999). By contrast, only limited data are available concerning the distribution of mitochondrial SOD2 in the auditory neurons. For example, Jiang et al. (2007) showed decreased SOD2 immunofluorescence with age (by 18 months) in aging male CBA/J mice. Two roles have been suggested for mitochondrial SOD2 in protection from ROS damage. First, mitochondrial DNA (mtDNA), located near the site of oxidative phosphorylation in inner mitochondria membrane, is believed to be especially vulnerable to accumulated effects of ROS after toxin exposure and during aging (Miquel et al., 1980). Studies have shown increased mtDNA mutations lead to signs of accelerated aging including early onset hearing loss, and mtDNA damage is present in cochleae from aged individuals (Fischel-Ghodsian et al., 1997). Second, SOD2 is a rate-limiting component of a pathway that activates mitochondrial uncoupling proteins (UCPs), which then uncouple cellular respiration from ATP production and reduce mitochondrial superoxide radical production (Echtay et al., 2002; Borecky et al., 2001). The high sequence homology between plant UCPs and mammalian UCP2 and UCP3 (Borecky et al., 2001) suggests a general and fundamental role of the SOD2-UCP pathway in eukaryotic cell survival. This study demonstrates that SOD2 is expressed highly in spiral ganglion cells in regions more resistant to oxidative stress (e.g., apex) and at lower levels in regions more susceptible to oxidative stress (e.g., base). These findings raise the hypothesis that regional differences in SOD2 expression by spiral ganglion cells may contribute to site-specific (base to apex) vulnerability to oxidative stress.
2. Materials and methods The animal use protocol for normal tissue collection was approved by the University of Pittsburgh Institutional Animal Care and Use Committee. One adult (4 week old) male CD-1 mouse, three adult (4 week old) male C57BL/6 mice, four adult (8 week old) Wistar rats and one adult (8 week old) Long Evans rat, and two male young adult 3–5 kg macaques were euthanized with sodium pentobarbital (100 mg/kg) and perfused transcardially with 0.1 M PBS, followed by paraformaldehyde-lysine-periodate fixative (McLean and Nakane, 1974). Temporal bones were post-fixed in 4% paraformaldehyde for 24 h at RT, decalcified in 10% formic acid to chemical testing criterion around 30 days, neutralized in overnight 5% sodium sulfate and embedded in paraffin with standard methods (Kammerman
117
et al., 1992). The temporal bones or whole mouse heads were sectioned at five microns in the modiolar plane. After deparaffinization, sections were incubated overnight with primary polyclonal antibody raised to full length rat SOD2 protein (Abcam Inc., ab13534) at 1:1000 dilution. This antibody has a broad spectrum of species reactivity (Drosophila and vertebrate) and its specificity has been confirmed by western blotting at 1/2000 dilution to a single band (Saha and Pahan, 2007; Bidmon et al., 1998; Liu et al., 1993). A biotinylated secondary anti-rabbit antibody and standard ABC-peroxidase reagents (Vector Laboratories) were applied. After buffer rinses, the slides were incubated in diaminobenzidine chromogen. Normal serum was substituted for primary antibody as negative controls. Archival celloidin-embedded human temporal bone sections from two human subjects (three ears), were obtained from the University of Pittsburgh Otopathology Laboratory. One subject (54 year old female) died of renal failure, and the other subject (4 year old male) died of pneumonia. The clinical histories had no indications of otopathology and there was no histopathologic evidence of autolysis. The specimens were collected within 24 hour of death, fixed by immersion in 10% formalin, decalcified in 5% trichloroacetic acid, dehydrated in graded concentrations of ethanol, embedded in celloidin, sectioned horizontally at 30 lm and stored in 70% ethanol. Immunohistochemical procedures for archival human temporal bone specimens followed our previous published protocols, using the same antibodies and reagents as the paraffin embedded animal specimens (Ganbo et al., 1997). 2.1. Quantitative methods The proportion of immunopositive spiral ganglion cells in the basal, middle and apical turns of each near mid-modiolar section was determined by counting all immunopositive and immunonegative cell bodies containing a nucleus and nucleolus from a minimum of two sections from each temporal bone. The proportion of SOD2 immunopositive spiral ganglion cells was then calculated for samples from each cochlear turn as the ratio of immunopositive cell bodies to the sum of immunopositive and immunonegative cell bodies. For quantification of SOD2 immunoreactivity, digital images of spiral ganglion cells were obtained in Metamorph software (Version 6.1, Molecular Devices) with a Spot RT Monochrome camera (Model 2.1.1, Diagnostic Instruments, Inc., Sterling Heights, MI, USA), and a Nikon Eclipse E600N microscope using either a 20 or 100 objective. Quantitative analysis was performed within each section only. All sections of the cochlear tissue were immunoprocessed and chromogenically reacted for identical times. The percentage cell body area that contains punctate dark staining SOD2 immunoreactivity (see Fig. 1) was quantified using the intensity thresholding function in Metamorph software from cells outlined with a digitizing pad on the image. The percentage of the SOD2 immunopositive soma area and soma area were logged automatically in a spreadsheet. Percentage immunoreactive somatic area is defined as the ratio of immunopositive punctate staining representative of Mn SOD2 localized in mitochondria of a cell (value obtained from thresholding function in Metamorph software) to total somatic cell body area. This approach to assessing cellular metabolic capacity is adapted from the allometry literature that examined the scaling of relative mitochondrial volume densities of cells with respect to body mass (Else and Hulbert, 1985). Because dense punctate SOD2 immunoreactive deposits represent mitochondrial expression, the somatic immunopositive area in a cell is regarded as a measure of cellular mitochondrial SOD2 capacity. The percentage somatic immunopositive area reflects the proportion of the soma that contains mitochondria expressing SOD2. Stromal cells and early haematopoietic cells in bone marrow of
118
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
Fig. 1. The similarity in appearance of manganese SOD2 immunoreactive spiral ganglion cells across species is shown in photomicrographs from (A) mouse cochlea, (B) rat cochlea, (C) macaque cochlea and (D) Human cochlea. The bold arrows indicate SOD2 immunopositive neurons. The thin arrows indicate cells that were classified as SOD2 immunonegative. The calibration bar represents 20 lm. Note the larger size and more widespread immunostaining of primate and human ganglion cells.
the same cochlear tissue served as positive controls for comparison (Southgate et al., 2006; Lechpammer et al., 2005). Statistical analyses were performed using Systat 11 software (SYSTAT, Inc.). Data on the proportion of immunopositive spiral ganglion cells and intensity of immunoreactivity at different cochlear locations (i.e., apical, middle, and basal) from different animal species were analyzed using analysis of variance (ANOVA) and least significant difference tests. 3. Results 3.1. SOD2 distribution 3.1.1. Cellular distribution of SOD2 Consistent with its primary localization in mitochondria, SOD2 immunoreactivity appeared predominantly as granular, dense accumulations within the cytoplasm in mouse, rat, macaque and human spiral ganglion cells (Fig. 1). This punctate appearance was independent of cell size and was consistent across species. Many cells also displayed a light but homogenous cytoplasmic immunoreaction. Since SOD2 is transcribed from nuclear DNA, the cytoplasmic staining likely represents translated SOD2 polypeptide prior to its translocation into mitochondria (Culotta et al., 2006). The individual spiral ganglion cells varied in the degree of granular SOD2 immunoreactivity. Some cells (thin arrows) were SOD2immunonegative, with no evidence of punctate staining. The majority of ganglion cells, though, showed varying amounts of intense granular immunoreactive elements (thick arrows), presumably the mitochondria. These cellular populations were defined as immunonegative and immunopositive, respectively, for quantification of regional staining in the sections that follow. The percentage of the cell area containing dark punctate staining was also quantified. In general, the quantitative data analyses indicated that there was no relationship between soma size and
pattern of SOD2 immunoreactivity. There were equal distributions of small and large size immunopositive cell bodies for all animal species across the cochlear turns. 3.1.2. Mice The proportion of immunopositive spiral ganglion cells and intensity of immunoreactivity within those cells were greatest in the apical turn (Fig. 2B–D) of the mouse cochlea. One-way repeated measures analysis of variance (F(2, 4) = 15.3, p < 0.05), followed by least significant differences tests (Fig. 3), demonstrated that the proportion of SOD2 immunopositive spiral ganglion cells was significantly greater (p < 0.05) in the apical turn (0.89 ± 0.12), than in either the middle turn (0.64 ± 0.13) or the basal turn (0.49 ± 0.21). The percentage area of somata containing intense punctuate immunoreactivity was assessed from a sample of 496 immunopositive spiral ganglion cells from all three cochlear turns of two mice (both ears). One-way analysis of variance, (F(2, 493) = 62.90, p < 0.01), followed by least significant difference tests, indicated that the percentage of the soma containing intense immunoreactivity was greatest in the apical cochlear turn (19.81 ± 0.74% area), followed by cells in the basal turn (11.19 ± 0.98% area) and cells in the middle turn (7.26 ± 0.9% area). Each paired comparison differed significantly (Fisher’s least significant differences tests, p < 0.01). 3.1.3. Rats Our material replicated the intense SOD2 immunoreactivity observed in the spiral ligament, spiral prominence, spiral limbus, stria vascularis, organ of Corti region, and spiral ganglion that was reported previously by Lai et al. (1996) and Rarey and Yao (1996). However, there was a previously unrecognized base-toapex gradient in the distribution of SOD2 immunoreactivity spiral ganglion cells (Fig. 4). The intensity of SOD2 immunoreactivity and proportion of immunopositive neurons appeared to be greater among spiral ganglion cells located at the cochlear apex
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
119
Fig. 2. Regional differences in manganese SOD2 immunoreactivity in the mouse cochlea. (A) This low magnification view of the cochlea (calibration bar = 500 lm) shows the base-to-apex gradient in SOD2 immunoreactivity. Higher magnification views (calibration bar = 20 lm) of the spiral ganglion cells in the basal turn (B), middle (C) and apical (D) turns show the distribution in more detail.
Fig. 3. The proportion of immunopositive spiral ganglion cells in mice as a function of cochlear turn. This plot quantifies a base-to-apex gradient in the proportion spiral ganglion cells displaying SOD2 immunopositivity, according to criteria described in association with Fig. 1.
(Fig. 4D) than at the base (Fig. 4B), with roughly intermediate findings in the middle turn (Fig. 4C). Quantitative analyses indicated that the distribution of SOD2 immunoreactivity in the rat cochlea was similar to the pattern observed in the mouse cochlea (Fig. 5). One-way analysis of variance (F(2, 6) = 25.7, p < 0.01), followed by least significant differences tests, showed that the proportion of SOD2 immunopositive spiral ganglion cells was greatest (p < 0.05) in the apical cochlear turn (0.63 ± 0.11), than in either the middle (0.41 ± 0.15), or the basal (0.17 ± 0.03) turn. The percentage area of somata containing intense punctate immunoreactivity was assessed from a sample of 494 immunopositive spiral ganglion cells from all three cochlear turns of two rats (both ears). One-way analysis of variance revealed a highly significant difference in the immunopositive percentage of somatic area across cochlear turns (F(2, 491) = 67.83, p = 0.01). Least significant difference tests demonstrated that the percentage area of somata containing intense punctate SOD2 immunoreactivity was significantly greater (p < 0.01) in spiral ganglion cells of the apical cochlear turn (35.96 ± 1.96% area) compared to the basal turn (9.79 ± 1.77% area). However, although the percentage immunopositive somatic area of spiral ganglion cells did not differ significantly between the apical and middle (35.65 ± 1.88% area) cochlear turns, the middle turn value is significantly greater than the basal turn value (p = 0.01).
120
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
Fig. 4. Regional differences in manganese SOD2 immunoreactivity in the rat cochlea. (A) This low magnification view of the rat cochlea (calibration bar = 500 lm) illustrates the differences in SOD2 immunoreactivity as a function of the location of the ganglion cells. Higher magnification views (calibration bar = 20 lm) of the spiral ganglion cells in the basal turn (B), middle (C) and apical (D) turns illustrate the differences in more detail.
spiral ganglion cells at the apex had the greatest percentage immunoreactive area (40.13 ± 1.93% area), which differed significantly (Least significant differences test, p < 0.05) from ganglion cells in either the middle turn (33.78 ± 1.63% area) or the basal turn (25.17 ± 1.99% area). The percentage immunoreactive somatic area also differed significantly (p < 0.01) between the middle turn and the basal turn.
Fig. 5. The proportion of immunopositive spiral ganglion cells in rats as a function of cochlear location. This plot quantifies the differences along spiral ganglion in the proportion ganglion cells displaying SOD2 immunopositivity, according to criteria described in association with Fig. 1.
3.1.4. Macaques Temporal bone specimens from macaques displayed the most intense SOD2 immunostaining of spiral ganglion cells at the cochlear apex (Fig. 6B). However, unlike cochleae from mice and rats, the proportion of immunopositive spiral ganglion cells was similar across cochlear turns. One-way repeated measures analysis of variance (F(2, 7) = 0.22, p = 0.81), followed by least significant differences tests, demonstrated that the proportion of SOD2 immunopositive spiral ganglion cells was equal in all turns (apical turn: 0.87 ± 0.02, middle turn: 0.88 ± 0.02, basal turn: 0.87 ± 0.02). The percentage area of somata containing intense punctuate immunoreactivity was sampled from 375 immunopositive spiral ganglion cells from different cochlear turns. Statistical analyses demonstrated a clear gradient from apex-to-base in the percentage of somatic area containing intense punctate immunoreactivity (one way ANOVA, F(2, 372) = 14.66, p < 0.01). The immunopositive
3.1.5. Archival human temporal bones Intensely SOD2 immunopositive spiral ganglion cells were observed in all turns of the cochlea (Fig. 7B–D) in temporal bones from two individuals (three ears). Virtually all spiral ganglion cells displayed punctate somatic SOD2 immunoreactivity and no differences were noted in the proportion of immunopositive spiral ganglion cells located at the apex compared to the middle or basal turn of cochlea. A total of 677 immunopositive spiral ganglion cells measured to determine the percentage of somatic area containing intense punctate immunoreactivity as a function of cell location along the cochlear spiral. One way ANOVA showed a highly significant difference in this metric of immunoreactivity across cochlear turns (F(2, 674) = 20.45, p = 0.01). Consistent with the other species, immunopositive spiral ganglion cells in the apical turn displayed the highest level percentage of somatic area containing SOD2 immunoreactivity (71.20 ± 2.10% area), which was significantly greater (Least significant difference tests, p < 0.01) than spiral ganglion cells in either the basal turn (62.58 ± 1.79% area) or the middle turn (55.59 ± 1.33% area). However, as observed in mice, the immunopositive percentage of somatic area was significantly greater for spiral ganglion cells in the basal turn than the middle turn (Least significant differences test, p < 0.01). 3.2. Interspecies comparisons It is well known that the basal metabolic rate shows an allometric relationship with the mass of organisms (Kleiber, 1932; Schmidt-Nielsen, 1984). This power function relationship is expressed most commonly as BMR = aMb, where BMR is the basal metabolic rate, M is body mass, and b is the scaling exponent (approximately 0.75 for whole body mass). More recently, there has been considerable interest in the extension of implications of
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
121
Fig. 6. Regional differences in manganese SOD2 immunoreactivity in the macaque cochlea. (A) This low magnification photograph (calibration bar = 500 lm) illustrates the differences in SOD2 immunoreactivity as a function of the location of the ganglion cells. Higher magnification micrographs (calibration bar = 50 lm) of the spiral ganglion cells in the apical turn (B) and basal turn (C) show the differences in more detail.
Fig. 7. Regional differences in manganese SOD2 immunoreactivity in the human cochlea. (A) This low magnification micrograph (calibration bar = 500 lm) illustrates the differences in SOD2 immunoreactivity as a function of the location of the ganglion cells. Higher magnification photographs (calibration bar = 100 lm) of the spiral ganglion cells in the basal turn (B), middle (C) and apical (D) turns document the differences in more detail.
these relationships to the levels of cells and organelles (West et al., 2002). The analysis that follows suggests that an allometric relationship also holds for the absolute baseline spiral ganglion cell expression of immunoreactive SOD2 across species. A metric for the absolute immunoreactive SOD2 per cell was calculated by multiplying the cell body area by the product of the population mean percentage of somatic area occupied by intense punctate immunoreactivity (including immunonegative cells) for each species. Spiral
ganglion cells in human specimens have the highest absolute SOD2 immunopositivity per cell followed by macaque, rat, and mouse. The exponent for the allometric relationship (b) was then identified as the slope of the linear relationship described by log S = log (a) + b log(M), where S is average absolute SOD2 expression per cell and M is the body mass of each species in question. These linear regression analyses for each cochlear turn showed strong linear fits with exponent estimates (b) of 0.33 (r2 = 0.88) for spiral ganglion
122
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
cells in the apical cochlear turn, 0.45 (r2 = 0.90) for spiral ganglion cells in the middle cochlear turn, and 0.47 (r2 = 0.92) for spiral ganglion cells in the basal turn. Thus, the absolute SOD2 expression per spiral ganglion cell shows an allometric relationship with body mass. Furthermore, ganglion cells at the apical turn have higher level of absolute SOD2 immunoreactivity than middle and basal turns across all species. However, neither the total number of ganglion cells (data from Nadol, 1988) nor the size of spiral ganglion cell somata demonstrated an allometric scaling with body mass.
4. Discussion This study provides the first evidence for base-to-apex gradient in spiral ganglion cell expression of SOD2 across species from rodents to primates. Across all species, there appears to be less cellular SOD2 immunopositivity in ganglion cells at the cochlear base than ganglion cells at the cochlear apex. The ganglion cells from middle cochlear turns, however, can be similar in SOD2 expression to either cells at the apex or the base in different species. Both the proportion of SOD2 immunopositive and intensity of SOD2 immunoreactive spiral ganglia cells are higher in apex compared to base of cochlea in mice and rats. Although almost all spiral ganglion cells are SOD2 immunopositive in macaque and human specimens, the percentage area of the ganglion cell bodies showing intense SOD2 immunoreactivity was higher in apical than basal turn of the cochlea. The appearance of middle turn ganglion cells, though, varied across species. While the proportion of SOD2 immunopositive spiral ganglion cells in the middle turn is greater than in the basal turn in rodents and equal in primates, the intensity of immunoreactivity among immunopositive spiral ganglion cells in the middle turn can be greater (rat and macaques) or less (mouse and humans) than the basal turn. More importantly, the intensity of SOD2 immunoreactivity is never greater for spiral ganglion cells in the middle turn than those in the apical turn of the cochlea. One interesting and novel finding is that the area of intense somatic SOD2 immunoreactivity in spiral ganglion cells scales as a power function of body weight across the species examined in the study. This type of allometric scaling relationship was describe initially for whole body data from mammals and birds by Kleiber (Kleiber, 1932) in the 1930s, such that basal metabolic rate (BMR) is a power function of body mass (M) of the form BMR = B0M3/4 (with Bo being a normalization coefficient). It has been extended subsequently to a wide range of organisms from smallest microbes to the largest vertebrates and plants (Hemmingsen, 1950; Niklas, 1994). The scaling exponent for basal metabolism of organisms is invariably close to 3=4 , with many other physiological variables scale with exponents that are typically multiples of 1=4 (Schmidt-Nielsen, 1984). Based on the linear regression analysis, the scaling exponents (re: body weight) were 0.33 for the apical cochlear turn, 0.45 for the middle cochlear turn, and 0.47 for the basal cochlear turn. These exponents are greater than the exponents for the allometric relationship between cochlear size of extant mammals (slant height and diameters of first two turns) and body weight (slope = 0.159) described by Spoor et al. (2002), but are within the range that has been reported in the literature for the relationship between basilar papilla length and body mass in birds (Gleich et al., 2005), for the relationship between areas of vestibular neuroepithelial area and body weight in mammals (Desai et al., 2005), and for components of mammalian cardiovascular and respiratory systems (West et al., 1997). It is likely that the allometric relationship for cellular SOD2 expression reflects fundamental metabolic demand consequences of scaling both neural and non-neural structures within the cochlea. In this regard, it will be of particular interest to determine the differential contributions of ROS demand from tissues such as the organ of Corti, stria
vascularis and limbus to the cellular SOD2 expression by spiral ganglion cells. Because intense, punctate somatic deposits of SOD2 immunoreactivity likely represent mitochondrial expression, it is reasonable to assume that the percentage of the cell area containing these punctuate, intense deposits of SOD2 immunoreactivity is an indicator of the current (in our tissue, basal level) superoxide metabolic capacity of a cell. Hence, the similar allometric exponent values for each cochlear turn suggest that regional SOD2 expression from basal to apical turn could be a conserved bioenergetic feature that reflects a common mammalian pattern of free radical load across cochlear turns. The relative expression of SOD2 may be indicative of the relative demand for ROS metabolism by cochlear mitochondria. Previous studies showed higher SOD2 immunolocalization at relatively metabolic active sites of the cochlea (e.g., stria vascularis) which are likely to suffer tissue injury from superoxide radicals generated from vascular endothelial cells or during ischemia-reperfusion incidents (Lai et al., 1996; Suzuki et al., 1993). This inference reflects the general principle that SODs appear to be distributed preferentially in vulnerable cell population that is exposed to reasonably high ROS loads. Further, because SOD2 is a key enzymatic step in detoxifying the free radical cascade inside the mitochondria, it is attractive to hypothesize that the different levels of SOD2 expression may be one determinant of site-specific cochlea vulnerability to ROS damage at the cochlear base. A corollary hypothesis is that the capacity of spiral ganglion cells to respond to ROS challenges may vary along the cochlear spiral, with a lower SOD2 response capacity in cells located at the base than those at the apex. Increased levels of superoxide anions and other radicals have been linked directly to regional differences in sensory cell damage and high frequency hearing threshold shifts. For example, Sha et al. (2001) reported that enhanced susceptibility of basal outer hair cells to ROS injury is associated with a significantly lower level of the potent, endogenous antioxidant glutathione (i.e., oxidative stress capacity) in basal outer hair cells. A similar phenomenon has also been documented for spiral ganglion neurons. Le and Keithley (2007) presented evidence that dietary antioxidants differentially affect the age-related loss of spiral ganglion cells in the apical turn of the cochlea of beagles. The basal turn had the largest decrease in neuronal density, and the apical turn had a smaller loss when compared to cochleae from young dogs. The addition of antioxidants to the diet for the last three years of life of the animal reduced the magnitude of these ganglion cell losses in the apical but not the basal turn. These findings are consistent with the hypothesis that differential susceptibility to ROS load of spiral ganglion cells may reflect differences in metabolic capacity to handle ROS challenges. Regional differences in SOD2 could be one contributing factor to differences in ROS metabolism. The significance of the innate apical-to-basal gradient of decreasing SOD2 expression in mammals in the absence of ototoxic challenge may suggests a selection bias in the evolutionary process of cochlear design that favors higher SOD2 expression in the apex corresponding to greater ROS load in apical cochlear turn. A variety of molecular, structural and environmental factors may contribute to different ROS exposures as a function of location in the cochlea. For example, neurotrophin-3 (NT-3) expression shows a similar base-to-apex gradient in the mouse inner ear (Sugawara et al., 2007). Because endogenous NT-3 can enhance both free radical production by neurons and potentiate neuronal injury during ischemic events (Bates et al., 2002), the observed patterns may represent a dynamic balance between NT3 ROS potentiation and SOD2 ROS catabolism along the cochlear spiral. Secondly, a difference in endolymph and perilymph clearance capacity between the apex and base could also affect ROS clearance and, hence, the SOD2
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
123
Fig. 8. This schematic diagram illustrates the role of manganese SOD2 in mitochondrial superoxide metabolism. Manganese SOD2 (Mn SOD2) converts superoxide radicals to hydrogen peroxide, which forms a substrate for catalase, glutathione peroxidases (GPXs) and catalase. The results of a Fenton reaction can also activate mitochondrial uncoupling proteins 2 and 3 (UCP2 and UCP3), which reduce ROS production.
metabolic activity/expression. Alternatively, higher expression at the cochlear apex could be a protective mechanism against disproportionate low frequency environmental noise exposure (i.e., cardiac pulse and circulatory blood flow, HVAC systems and fluorescent light noise in a room) for humans and animals living in buildings. The conserved and allometrically scaled gradient of basal SOD2 expression across rodents and primates is significant because SOD2 is a rate-limiting component of mitochondrial metabolic response pathways to intracellular ROS load that can impact on cell survival (Fig. 8). Dismutation of superoxide into hydrogen peroxide by manganese Mn SOD2 in the mitochondria is upstream to many other important metabolic enzymes, including catalase and glutathione peroxidase that convert hydrogen peroxide into water. Myeloperoxidase converts hydrogen peroxide to hypochlorous acid, a strong oxidizing agent that can protect cells against pathogens but may also contribute to neuronal degeneration (Green et al., 2004; Choi et al., 2005). Finally, SOD2 is upstream to highly conserved, mitochondrial UCPs in a signaling cascade that reduces ROS production by the respiratory chain. The high degree of sequence homology across plants and animals implies that UCPs are not only proteins with specialized function in a specific mammalian species, but their metabolic physiological roles may be more general (Borecky et al., 2001). Therefore, as proposed originally by Schuknecht (1955), it is highly possible that partially independent metabolic factors underlie the differential vulnerability of hair cells and spiral ganglion cells, through different molecular vulnerabilities, without imposing a degenerative change upon the other or other supporting cochlear tissue. The different levels of SOD2 expression in spiral ganglion cells as a function of location in the cochlea raises the possibility that the reserve capacity of fundamental eukaryotic ROS defense mechanisms may vary along the
cochlear spiral, with higher SOD2 present in the spiral ganglion cells at the cochlear apex compared to those at the base. Financial disclosure None. Acknowledgments The authors thank Gloria Limetti and Jean Betsch for their technical assistance to the project. This project was supported by the Pennsylvania Lions Hearing Research Foundation and American Otologic Society Research Fellowship. References Bates, B., Hirt, L., Thomas, S.S., Akbarian, S., Le, D., Amin-Hanjani, S., Whalen, M., Jaenisch, R., Moskowitz, M.A., 2002. Neurotropin-3 promotes cell death induced in cerebral ischemia, oxygen-glucose deprivation, and oxidative stress: possible involvement of oxygen free radicals. Neurobiol. Dis. 9, 24–37. Bidmon, H.J., Wu, J., Buchkremer-Ratzmann, I., Mayer, B., Witte, O.W., Zilles, K., 1998. Transient increase of manganese-superoxide dismutase in remote brain areas after focal photothrombotic cortical lesion. Stroke 29, 203–210. discussion 211. Borecky, J., Maia, I.G., Arruda, P., 2001. Mitochondrial uncoupling proteins in mammals and plants. Biosci. Rep. 21 (2), 201–212. Chen, G.-D., Fechter, L.D., 2003. The relationship between noise-induced hearing loss and hair cell loss in rats. Hear. Res. 177, 81–90. Choi, D.K., Pennathur, S., Perier, C., Tieu, K., Teismann, P., Wu, D.C., Jackson-Lewis, V., Vila, M., Vonsattel, J.P., Heinecke, J.W., Przedborski, S., 2005. Ablation of the inflammatory enzyme myeloperoxidase mitigates features of Parkinson’s disease in mice. J. Neurosci. 25 (28), 6594–6600. Culotta, V.C., Yang, M., O’Halloran, T.V., 2006. Activation of superoxide dismutases: putting the metal to the pedal. Biochim. Biophys. Acta 1763, 747–758. Desai, S.S., Ali, H., Lysakowski, A., 2005. Comparative morphology of rodent vestibular periphery. II. Cristae ampullares. J. Neurophysiol. 93, 267–280.
124
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
Echtay, K.S., Roussel, D., St-Pierre, J., Jekabsons, M.B., Cadenas, S., Stuart, J.A., Harper, J.A., Roebuck, S.J., Morrison, A., Pickering, S., Clapham, J.C., Brand, M.D., 2002. Superoxide activates mitochondrial uncoupling proteins. Nature 415, 96–99. Else, P.L., Hulbert, A.J., 1985. Mammals: an allometric study of metabolism at tissue and mitochondrial level. Am. J. Physiol. Regul. Integr. Comp. Physiol. 248, 415–421. Fischel-Ghodsian, N., Bykhovskaya, Y., Taylor, K., Kahen, T., Cantor, R., Ehrenman, K., Smith, R., Keithley, E.M., 1997. Temporal bone analysis of patients with presbycusis reveals high frequency of mitochondrial mutations. Hear. Res. 110, 147–154. Freeman, B.A., Crapo, J.D., 1982. Biology of disease. Free radicals and tissue injury. Lab. Invest. 47, 412–426. Ganbo, T., Sando, I., Balaban, C.D., Suzuki, C., Sudo, M., 1997. Immunohistochemistry of lymphocytes and macrophages in human celloidin-embedded temporal bone sections with acute otitis media. Ann. Otol. Rhinol. Laryngol. 106 (8), 662–668. Gao, F., Kinnula, V.L., Myllarniemi, M., Oury, T.D., 2008. Extracellular superoxide dismutase in pulmonary fibrosis. Antioxid. Redox. Signal. 10 (2), 343–354. Gleich, O., Dooling, R.J., Manley, G.A., 2005. Audiogram, body mass, and basilar papilla length: correlations in birds and predictions for extinct archosaurs. Naturewissenschaften 92, 595–598. Green, P.S., Mendez, A.J., Jacob, J.S., Crowley, J.R., Growdon, W., Hyman, B.T., Heinecke, J.W., 2004. Neuronal expression of myeloperoxidase is increased in Alzheimer’s disease. J. Neurochem. 90, 724–733. Hemmingsen, AM., 1950. Rep. Stenographic Mem. Hosp. Copenh. 4, 1–58. Jiang, H., Talaska, A.E., Schacht, J., Sha, S.H., 2007. Oxidative imbalance in the aging inner ear. Neurobiol. Aging 28, 1605–1612. Kammerman, J.R., Prophet, E.B., Barnes, C.F., 1992. Orthopedic histotechnology. In: Prophet, E.B., Mills, B., Arrington, J.B., Sobin, L.H. (Eds.), Armed Forces Institute of Pathology Laboratory Methods in Histotechnology. American Registry of Pathology, Washington, DC., pp. 72–73. Kleiber, M., 1932. Body size and metabolism. Hilgardia 6, 315–353. Lai, M.T., Ohmichi, T., Egusa, K., Okada, S., Masuda, Y., 1996. Immunohistochemical localization of manganese superoxide dismutase in rat cochlea. Eur. Arch. Otorhinolaryngol. 253 (4–5), 273–277. Le, T., Keithley, E.M., 2007. Effects of antioxidants on the aging inner ear. Hear. Res. 226, 194–202. Lechpammer, S., Epperly, M.W., Zhou, S., Nie, S., Glowacki, J., Greenberger, J.S., 2005. Adipocyte differentiation in Sod2( / ) and Sod2(+/+) murine bone marrow stromal cells is associated with low antioxidant pools. Exp. Hematol. 33, 1201–1208. Liu, X.H., Kato, H., Nakata, N., Kogure, K., Kato, K., 1993. An immunohistochemical study of copper/zinc superoxide dismutase in rat hippocampus after transient cerebral ischemia. Brain Res. 625 (1), 29–37. Maruyama, J., Yamagata, T., Ulfendahl, M., Bredberg, G., Altschuler, R.A., Miller, J.M., 2007. Effects of antioxidants on auditory nerve function and survival in deafened guinea pigs. Neurobiol. Dis. 25, 309–318. Maruyama, J., Miller, J.M., Ulfendahl, M., 2008. Glial cell line-derived neurotrophic factor and antioxidants preserve the electrical responsiveness of the spiral ganglion neurons after experimentally induced deafness. Neurobiol. Dis. 29, 14–21. McFadden, S.L., Ding, D., Reaume, A.G., Flood, D.G., Salvi, R.J., 1999. Age-related cochlear hair cell loss is enhanced in mice lacking copper/zinc superoxide dismutase. Neurobiology 20, 1–8.
McLean, I.W., Nakane, P.K., 1974. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077–1083. Miquel, J., Economos, A.C., Fleming, J., Johnson Jr., J.E., 1980. Mitochondrial role in cell aging. Exp. Gerontol. 15, 575–591. Nadol, J.B., 1988. Comparative anatomy of the cochlea and auditory nerve in mammals. Hear. Res. 34, 253–266. Nelson, E.G., Hinojosa, R., 2006. Presbycusis: a human temporal bone study of individuals with downward sloping audiometric patterns of hearing loss and review of the literature. Laryngoscope 116 (9, Part 3), 1–12. Niklas, K.J., 1994. Plant Allometry: The Scaling of Form and Process. University of Chicago Press, Chicago. Rarey, K.E., Yao, X., 1996. Localization of Cu/Zn-SOD and Mn SOD in the rat cochlea. Acta Otolaryngol. 116 (6), 833–835. Saha, R.N., Pahan, K., 2007. Differential regulation of Mn-Superoxide dismutase in neurons and astroglia by HIV-1 gp120: implications for HIV-associated dementia. Free Radic. Biol. Med. 42, 1866–1878. Schmidt-Nielsen, K., 1984. Scaling: Why is Animal Size So Important? Cambridge University Press, Cambridge, UK. Schuknecht, H.F., 1955. Presbycusis. Laryngoscope 65 (5), 402–419. Schuknecht, H.F., 1964. Further observations on the pathology of presbycusis. Arch Otolaryngol. 80, 369–382. Schuknecht, H.F., Gacek, M.R., 1993. Cochlear pathology in presbycusis. Ann. Oto. Rhino. Laryngol. 102, 1–16. Sha, S.H., Taylor, R., Forge, A., Schacht, J., 2001. Differential vulnerability of basal and apical hair cells is based on intrinsic susceptibility to free radicals. Hear. Res. 155, 1–8. Southgate, T.D., Sheard, V., Milsom, M.D., Ward, T.H., Mairs, R.J., Boyd, M., Fairbairn, L.J., 2006. Radioprotective gene therapy through retroviral expression of manganese superoxide dismutase. J. Gene Med. 8 (5), 557–565. Spoor, M., Bajpal, S., Hussain, S.T., Kumar, K., Thewissen, J.G.M., 2002. Vestibular evidence for evolution of aquatic behavior in early cetaceans. Nature 417, 163– 166. Stone, M., Schachern, P.A., Paparella, M.M., 1998. Loss of spiral ganglion cells as primary manifestation of aminoglycoside ototoxicity. Hear. Res. 115, 217–223. Sugawara, M., Murtie, J.C., Stankovic, K.M., Liberman, M.C., Corfas, G., 2007. Dynamic patterns of neurotrophin 3 expression in the postnatal mouse inner ear. J. Comp. Neurol. 501 (1), 30–37. Suzuki, K., Tatsumi, H., Satoh, S., Send, T., Nakata, T., Fuji, J., Taniguchi, N., 1993. Manganese-superoxide dismutase in endothelial cells: localization and mechanism of induction. Am. J. Physiol. 265, H1173–H1178. Wang, Y., Hirose, K., Liberman, M.C., 2002. Dynamics of noise-induced cellular injury and repair in the mouse cochlea. J. Assoc. Res. Otolaryngol. 3, 248–268. West, G.B., Brown, J.H., Enquist, B.J., 1997. A general model for the origin of allometric scaling laws in biology. Science 276, 122–126. West, G.B., Woodruff, W.H., Brown, J.H., 2002. Allometric scaling of metabolic rate from molecules and mitochondria to cells and mammals. PNAS 99 (1), 2473– 2478. Zimmermann, C.E., Burgess, B.J., Nadol, J.B., 1995. Patterns of degeneration in the human cochlear nerve. Hear. Res. 90 (1–2), 192–201.
Contents lists available at ScienceDirect
Hearing Research journal homepage: www.elsevier.com/locate/heares
Research paper
Regional distribution of manganese superoxide dismutase 2 (Mn SOD2) expression in rodent and primate spiral ganglion cells Yu-Lan Mary Ying *, Carey D. Balaban Department of Otolaryngology, University of Pittsburgh, 200 Lothrop Street, Suite # 500, Pittsburgh, PA 15213, USA
a r t i c l e
i n f o
Article history: Received 28 January 2009 Received in revised form 2 April 2009 Accepted 2 April 2009 Available online 17 April 2009 Keywords: Spiral ganglion cells Manganese superoxide dismutase 2 Gradient Reactive oxygen species Primate Rodent Superoxide dismutase Immunohistochemistry
a b s t r a c t Manganese superoxide dismutase 2 (SOD2) is a key metabolic anti-oxidant enzyme for detoxifying free radicals inside mitochondria. This study documents a gradient in expression of SOD2 by spiral ganglion cells in basal versus apical turn of cochlea that is consistent with differential vulnerability of high frequency hearing to free radical damage. Immunohistochemical methods were used to identify distribution of SOD2 in temporal bone sections from mice, rats, macaques, and humans. In mice and rats, both the proportion of SOD2 immunopositive type 1 spiral ganglion cells and the intensity of immunoreactivity were elevated near cochlear apex. In macaques and humans, the proportion of SO2 immunopositive spiral ganglion cells was equal across cochlear turn, but the intensity of immunoreactivity remained highest for ganglion cells near cochlear apex. Strong SOD2 immunoreactivity was also observed in human type 1 spiral ganglion cells. The average area density of SOD2 immunoreactivity in ganglion cells for each species and cochlear turn showed an allometric relationship with body weight, which is consistent with a conserved basal metabolic characteristic. These findings suggest that spiral ganglion cell responses to ROS exposure may vary along cochlear spiral with lower response capacity at cochlear base contributing to cumulative susceptibility to high frequency hearing loss. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Multiple factors contribute to inner ear pathologies causing hearing loss. Genetic factors, age-related changes (presbycusis), acoustic trauma, infectious disease, and exposure to ototoxins (e.g., aminoglycosides and cisplatin) have been implicated as potential etiologic factors in clinical dysfunction. The histopathologic hallmarks of presbycusis include losses of hair cells, spiral ganglion cells, strial atrophy, and abnormalities of supporting structures within the cochlea (Schuknecht, 1964; Schuknecht and Gacek, 1993; Zimmermann et al., 1995). Degenerative changes in the organ of Corti and spiral ganglion cells in presbycusis are similar to the degeneration in noise trauma, toxic insults, infectious and immunologic influences and congenital lesions (Zimmermann et al., 1995). Specifically, high frequency sensorineural hearing loss is detected as an early sign of aminoglycoside toxicity, cisplatin toxicity or presbycusis. Hearing loss often progresses along a high to low frequency gradient that corresponds to progressive pathophysiologic changes from the basal to the apical cochlear turn. Different cochlear cell populations appear to have their own intrinsic susceptibilities to free radical damage. For example, Sha et al. (2001) reported that outer hair cells are most vulnerable to * Corresponding author. Tel.: +1 412 647 8050/2298; fax: +1 412 647 0108. E-mail addresses: [email protected] (Y.-L.M. Ying), [email protected] (C.D. Balaban). 0378-5955/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2009.04.006
free radical mediated ototoxicity followed by inner hair cells; supporting cells are the least susceptible to damage. Recent studies have regarded hair cells within the organ of Corti as primary sites of tissue insult in acoustic trauma and in presbycusis (Wang et al., 2002; Chen and Fechter, 2003), and tend to assume that degenerative changes in spiral ganglion cells or the cochlear nerve are secondary effects. However, there is long-standing evidence that degeneration of the spiral ganglion cells can be an independent and primary phenomenon with no obvious initiating event in the organ of Corti (Schuknecht, 1955; Schuknecht and Gacek, 1993). Because significant aminoglycoside destruction of spiral ganglion cells can occur without any effect on hair cells (Stone et al., 1998), pathological effects at the level of spiral ganglion cells may affect hearing independent of hair cell status. The basal half of the cochlea appears to have a predilection for the most severe spiral ganglion degeneration (Zimmermann et al., 1995; Nelson and Hinojosa, 2006). Basal turn outer hair cells are also more susceptible to aminoglycoside and cisplatin damage than apical outer hair cells (Sha et al., 2001). These phenomena parallel the common clinical observation of high frequency hearing loss and raise the question of which mechanism might be responsible for differential cellular vulnerability along the cochlear spiral. The cochlear nerve serves as the link between sensory hair cells and the central nervous system. Preservation of spiral ganglion cells is essential for success of cochlear implantation because stimulation of particular type I ganglion cells is of primary importance
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
for speech recognition (Zimmermann et al., 1995). Therefore, there is a need to develop therapeutic strategies to preserve cochlear nerve function in the face of age-related pathophysiologic changes and ototoxic processes in the inner ear. Although the cellular mechanisms underlying cochlear degeneration are far from fully known, the available evidence implicates the formation and accumulation of reactive oxygen species (ROS) may play a significant role in cochlear cell loss over time. For example, antioxidant treatments to prevent degeneration of deafferented CN VIII spiral ganglion cells in deafened guinea pigs in the context of free radical formation that signals cell death pathways has been reported (Maruyama et al., 2007, 2008). A family of superoxide dismutases (SODs) plays an important role in the initial cellular defense against generation of ROS (Freeman and Crapo, 1982). Copper/Zinc SOD 1 (SOD1) is widely distributed in the nucleus and cytoplasm, and comprises 90% of total SOD activity. Manganese SOD 2 (SOD2) is localized in the mitochondria. Extracellular SOD is localized in the extracellular matrix (Gao et al., 2008). Null mutant sod1 transgenic mice display early hearing loss, accompanied by degeneration of spiral ganglion neurons and hair cells (McFadden et al., 1999). By contrast, only limited data are available concerning the distribution of mitochondrial SOD2 in the auditory neurons. For example, Jiang et al. (2007) showed decreased SOD2 immunofluorescence with age (by 18 months) in aging male CBA/J mice. Two roles have been suggested for mitochondrial SOD2 in protection from ROS damage. First, mitochondrial DNA (mtDNA), located near the site of oxidative phosphorylation in inner mitochondria membrane, is believed to be especially vulnerable to accumulated effects of ROS after toxin exposure and during aging (Miquel et al., 1980). Studies have shown increased mtDNA mutations lead to signs of accelerated aging including early onset hearing loss, and mtDNA damage is present in cochleae from aged individuals (Fischel-Ghodsian et al., 1997). Second, SOD2 is a rate-limiting component of a pathway that activates mitochondrial uncoupling proteins (UCPs), which then uncouple cellular respiration from ATP production and reduce mitochondrial superoxide radical production (Echtay et al., 2002; Borecky et al., 2001). The high sequence homology between plant UCPs and mammalian UCP2 and UCP3 (Borecky et al., 2001) suggests a general and fundamental role of the SOD2-UCP pathway in eukaryotic cell survival. This study demonstrates that SOD2 is expressed highly in spiral ganglion cells in regions more resistant to oxidative stress (e.g., apex) and at lower levels in regions more susceptible to oxidative stress (e.g., base). These findings raise the hypothesis that regional differences in SOD2 expression by spiral ganglion cells may contribute to site-specific (base to apex) vulnerability to oxidative stress.
2. Materials and methods The animal use protocol for normal tissue collection was approved by the University of Pittsburgh Institutional Animal Care and Use Committee. One adult (4 week old) male CD-1 mouse, three adult (4 week old) male C57BL/6 mice, four adult (8 week old) Wistar rats and one adult (8 week old) Long Evans rat, and two male young adult 3–5 kg macaques were euthanized with sodium pentobarbital (100 mg/kg) and perfused transcardially with 0.1 M PBS, followed by paraformaldehyde-lysine-periodate fixative (McLean and Nakane, 1974). Temporal bones were post-fixed in 4% paraformaldehyde for 24 h at RT, decalcified in 10% formic acid to chemical testing criterion around 30 days, neutralized in overnight 5% sodium sulfate and embedded in paraffin with standard methods (Kammerman
117
et al., 1992). The temporal bones or whole mouse heads were sectioned at five microns in the modiolar plane. After deparaffinization, sections were incubated overnight with primary polyclonal antibody raised to full length rat SOD2 protein (Abcam Inc., ab13534) at 1:1000 dilution. This antibody has a broad spectrum of species reactivity (Drosophila and vertebrate) and its specificity has been confirmed by western blotting at 1/2000 dilution to a single band (Saha and Pahan, 2007; Bidmon et al., 1998; Liu et al., 1993). A biotinylated secondary anti-rabbit antibody and standard ABC-peroxidase reagents (Vector Laboratories) were applied. After buffer rinses, the slides were incubated in diaminobenzidine chromogen. Normal serum was substituted for primary antibody as negative controls. Archival celloidin-embedded human temporal bone sections from two human subjects (three ears), were obtained from the University of Pittsburgh Otopathology Laboratory. One subject (54 year old female) died of renal failure, and the other subject (4 year old male) died of pneumonia. The clinical histories had no indications of otopathology and there was no histopathologic evidence of autolysis. The specimens were collected within 24 hour of death, fixed by immersion in 10% formalin, decalcified in 5% trichloroacetic acid, dehydrated in graded concentrations of ethanol, embedded in celloidin, sectioned horizontally at 30 lm and stored in 70% ethanol. Immunohistochemical procedures for archival human temporal bone specimens followed our previous published protocols, using the same antibodies and reagents as the paraffin embedded animal specimens (Ganbo et al., 1997). 2.1. Quantitative methods The proportion of immunopositive spiral ganglion cells in the basal, middle and apical turns of each near mid-modiolar section was determined by counting all immunopositive and immunonegative cell bodies containing a nucleus and nucleolus from a minimum of two sections from each temporal bone. The proportion of SOD2 immunopositive spiral ganglion cells was then calculated for samples from each cochlear turn as the ratio of immunopositive cell bodies to the sum of immunopositive and immunonegative cell bodies. For quantification of SOD2 immunoreactivity, digital images of spiral ganglion cells were obtained in Metamorph software (Version 6.1, Molecular Devices) with a Spot RT Monochrome camera (Model 2.1.1, Diagnostic Instruments, Inc., Sterling Heights, MI, USA), and a Nikon Eclipse E600N microscope using either a 20 or 100 objective. Quantitative analysis was performed within each section only. All sections of the cochlear tissue were immunoprocessed and chromogenically reacted for identical times. The percentage cell body area that contains punctate dark staining SOD2 immunoreactivity (see Fig. 1) was quantified using the intensity thresholding function in Metamorph software from cells outlined with a digitizing pad on the image. The percentage of the SOD2 immunopositive soma area and soma area were logged automatically in a spreadsheet. Percentage immunoreactive somatic area is defined as the ratio of immunopositive punctate staining representative of Mn SOD2 localized in mitochondria of a cell (value obtained from thresholding function in Metamorph software) to total somatic cell body area. This approach to assessing cellular metabolic capacity is adapted from the allometry literature that examined the scaling of relative mitochondrial volume densities of cells with respect to body mass (Else and Hulbert, 1985). Because dense punctate SOD2 immunoreactive deposits represent mitochondrial expression, the somatic immunopositive area in a cell is regarded as a measure of cellular mitochondrial SOD2 capacity. The percentage somatic immunopositive area reflects the proportion of the soma that contains mitochondria expressing SOD2. Stromal cells and early haematopoietic cells in bone marrow of
118
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
Fig. 1. The similarity in appearance of manganese SOD2 immunoreactive spiral ganglion cells across species is shown in photomicrographs from (A) mouse cochlea, (B) rat cochlea, (C) macaque cochlea and (D) Human cochlea. The bold arrows indicate SOD2 immunopositive neurons. The thin arrows indicate cells that were classified as SOD2 immunonegative. The calibration bar represents 20 lm. Note the larger size and more widespread immunostaining of primate and human ganglion cells.
the same cochlear tissue served as positive controls for comparison (Southgate et al., 2006; Lechpammer et al., 2005). Statistical analyses were performed using Systat 11 software (SYSTAT, Inc.). Data on the proportion of immunopositive spiral ganglion cells and intensity of immunoreactivity at different cochlear locations (i.e., apical, middle, and basal) from different animal species were analyzed using analysis of variance (ANOVA) and least significant difference tests. 3. Results 3.1. SOD2 distribution 3.1.1. Cellular distribution of SOD2 Consistent with its primary localization in mitochondria, SOD2 immunoreactivity appeared predominantly as granular, dense accumulations within the cytoplasm in mouse, rat, macaque and human spiral ganglion cells (Fig. 1). This punctate appearance was independent of cell size and was consistent across species. Many cells also displayed a light but homogenous cytoplasmic immunoreaction. Since SOD2 is transcribed from nuclear DNA, the cytoplasmic staining likely represents translated SOD2 polypeptide prior to its translocation into mitochondria (Culotta et al., 2006). The individual spiral ganglion cells varied in the degree of granular SOD2 immunoreactivity. Some cells (thin arrows) were SOD2immunonegative, with no evidence of punctate staining. The majority of ganglion cells, though, showed varying amounts of intense granular immunoreactive elements (thick arrows), presumably the mitochondria. These cellular populations were defined as immunonegative and immunopositive, respectively, for quantification of regional staining in the sections that follow. The percentage of the cell area containing dark punctate staining was also quantified. In general, the quantitative data analyses indicated that there was no relationship between soma size and
pattern of SOD2 immunoreactivity. There were equal distributions of small and large size immunopositive cell bodies for all animal species across the cochlear turns. 3.1.2. Mice The proportion of immunopositive spiral ganglion cells and intensity of immunoreactivity within those cells were greatest in the apical turn (Fig. 2B–D) of the mouse cochlea. One-way repeated measures analysis of variance (F(2, 4) = 15.3, p < 0.05), followed by least significant differences tests (Fig. 3), demonstrated that the proportion of SOD2 immunopositive spiral ganglion cells was significantly greater (p < 0.05) in the apical turn (0.89 ± 0.12), than in either the middle turn (0.64 ± 0.13) or the basal turn (0.49 ± 0.21). The percentage area of somata containing intense punctuate immunoreactivity was assessed from a sample of 496 immunopositive spiral ganglion cells from all three cochlear turns of two mice (both ears). One-way analysis of variance, (F(2, 493) = 62.90, p < 0.01), followed by least significant difference tests, indicated that the percentage of the soma containing intense immunoreactivity was greatest in the apical cochlear turn (19.81 ± 0.74% area), followed by cells in the basal turn (11.19 ± 0.98% area) and cells in the middle turn (7.26 ± 0.9% area). Each paired comparison differed significantly (Fisher’s least significant differences tests, p < 0.01). 3.1.3. Rats Our material replicated the intense SOD2 immunoreactivity observed in the spiral ligament, spiral prominence, spiral limbus, stria vascularis, organ of Corti region, and spiral ganglion that was reported previously by Lai et al. (1996) and Rarey and Yao (1996). However, there was a previously unrecognized base-toapex gradient in the distribution of SOD2 immunoreactivity spiral ganglion cells (Fig. 4). The intensity of SOD2 immunoreactivity and proportion of immunopositive neurons appeared to be greater among spiral ganglion cells located at the cochlear apex
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
119
Fig. 2. Regional differences in manganese SOD2 immunoreactivity in the mouse cochlea. (A) This low magnification view of the cochlea (calibration bar = 500 lm) shows the base-to-apex gradient in SOD2 immunoreactivity. Higher magnification views (calibration bar = 20 lm) of the spiral ganglion cells in the basal turn (B), middle (C) and apical (D) turns show the distribution in more detail.
Fig. 3. The proportion of immunopositive spiral ganglion cells in mice as a function of cochlear turn. This plot quantifies a base-to-apex gradient in the proportion spiral ganglion cells displaying SOD2 immunopositivity, according to criteria described in association with Fig. 1.
(Fig. 4D) than at the base (Fig. 4B), with roughly intermediate findings in the middle turn (Fig. 4C). Quantitative analyses indicated that the distribution of SOD2 immunoreactivity in the rat cochlea was similar to the pattern observed in the mouse cochlea (Fig. 5). One-way analysis of variance (F(2, 6) = 25.7, p < 0.01), followed by least significant differences tests, showed that the proportion of SOD2 immunopositive spiral ganglion cells was greatest (p < 0.05) in the apical cochlear turn (0.63 ± 0.11), than in either the middle (0.41 ± 0.15), or the basal (0.17 ± 0.03) turn. The percentage area of somata containing intense punctate immunoreactivity was assessed from a sample of 494 immunopositive spiral ganglion cells from all three cochlear turns of two rats (both ears). One-way analysis of variance revealed a highly significant difference in the immunopositive percentage of somatic area across cochlear turns (F(2, 491) = 67.83, p = 0.01). Least significant difference tests demonstrated that the percentage area of somata containing intense punctate SOD2 immunoreactivity was significantly greater (p < 0.01) in spiral ganglion cells of the apical cochlear turn (35.96 ± 1.96% area) compared to the basal turn (9.79 ± 1.77% area). However, although the percentage immunopositive somatic area of spiral ganglion cells did not differ significantly between the apical and middle (35.65 ± 1.88% area) cochlear turns, the middle turn value is significantly greater than the basal turn value (p = 0.01).
120
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
Fig. 4. Regional differences in manganese SOD2 immunoreactivity in the rat cochlea. (A) This low magnification view of the rat cochlea (calibration bar = 500 lm) illustrates the differences in SOD2 immunoreactivity as a function of the location of the ganglion cells. Higher magnification views (calibration bar = 20 lm) of the spiral ganglion cells in the basal turn (B), middle (C) and apical (D) turns illustrate the differences in more detail.
spiral ganglion cells at the apex had the greatest percentage immunoreactive area (40.13 ± 1.93% area), which differed significantly (Least significant differences test, p < 0.05) from ganglion cells in either the middle turn (33.78 ± 1.63% area) or the basal turn (25.17 ± 1.99% area). The percentage immunoreactive somatic area also differed significantly (p < 0.01) between the middle turn and the basal turn.
Fig. 5. The proportion of immunopositive spiral ganglion cells in rats as a function of cochlear location. This plot quantifies the differences along spiral ganglion in the proportion ganglion cells displaying SOD2 immunopositivity, according to criteria described in association with Fig. 1.
3.1.4. Macaques Temporal bone specimens from macaques displayed the most intense SOD2 immunostaining of spiral ganglion cells at the cochlear apex (Fig. 6B). However, unlike cochleae from mice and rats, the proportion of immunopositive spiral ganglion cells was similar across cochlear turns. One-way repeated measures analysis of variance (F(2, 7) = 0.22, p = 0.81), followed by least significant differences tests, demonstrated that the proportion of SOD2 immunopositive spiral ganglion cells was equal in all turns (apical turn: 0.87 ± 0.02, middle turn: 0.88 ± 0.02, basal turn: 0.87 ± 0.02). The percentage area of somata containing intense punctuate immunoreactivity was sampled from 375 immunopositive spiral ganglion cells from different cochlear turns. Statistical analyses demonstrated a clear gradient from apex-to-base in the percentage of somatic area containing intense punctate immunoreactivity (one way ANOVA, F(2, 372) = 14.66, p < 0.01). The immunopositive
3.1.5. Archival human temporal bones Intensely SOD2 immunopositive spiral ganglion cells were observed in all turns of the cochlea (Fig. 7B–D) in temporal bones from two individuals (three ears). Virtually all spiral ganglion cells displayed punctate somatic SOD2 immunoreactivity and no differences were noted in the proportion of immunopositive spiral ganglion cells located at the apex compared to the middle or basal turn of cochlea. A total of 677 immunopositive spiral ganglion cells measured to determine the percentage of somatic area containing intense punctate immunoreactivity as a function of cell location along the cochlear spiral. One way ANOVA showed a highly significant difference in this metric of immunoreactivity across cochlear turns (F(2, 674) = 20.45, p = 0.01). Consistent with the other species, immunopositive spiral ganglion cells in the apical turn displayed the highest level percentage of somatic area containing SOD2 immunoreactivity (71.20 ± 2.10% area), which was significantly greater (Least significant difference tests, p < 0.01) than spiral ganglion cells in either the basal turn (62.58 ± 1.79% area) or the middle turn (55.59 ± 1.33% area). However, as observed in mice, the immunopositive percentage of somatic area was significantly greater for spiral ganglion cells in the basal turn than the middle turn (Least significant differences test, p < 0.01). 3.2. Interspecies comparisons It is well known that the basal metabolic rate shows an allometric relationship with the mass of organisms (Kleiber, 1932; Schmidt-Nielsen, 1984). This power function relationship is expressed most commonly as BMR = aMb, where BMR is the basal metabolic rate, M is body mass, and b is the scaling exponent (approximately 0.75 for whole body mass). More recently, there has been considerable interest in the extension of implications of
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
121
Fig. 6. Regional differences in manganese SOD2 immunoreactivity in the macaque cochlea. (A) This low magnification photograph (calibration bar = 500 lm) illustrates the differences in SOD2 immunoreactivity as a function of the location of the ganglion cells. Higher magnification micrographs (calibration bar = 50 lm) of the spiral ganglion cells in the apical turn (B) and basal turn (C) show the differences in more detail.
Fig. 7. Regional differences in manganese SOD2 immunoreactivity in the human cochlea. (A) This low magnification micrograph (calibration bar = 500 lm) illustrates the differences in SOD2 immunoreactivity as a function of the location of the ganglion cells. Higher magnification photographs (calibration bar = 100 lm) of the spiral ganglion cells in the basal turn (B), middle (C) and apical (D) turns document the differences in more detail.
these relationships to the levels of cells and organelles (West et al., 2002). The analysis that follows suggests that an allometric relationship also holds for the absolute baseline spiral ganglion cell expression of immunoreactive SOD2 across species. A metric for the absolute immunoreactive SOD2 per cell was calculated by multiplying the cell body area by the product of the population mean percentage of somatic area occupied by intense punctate immunoreactivity (including immunonegative cells) for each species. Spiral
ganglion cells in human specimens have the highest absolute SOD2 immunopositivity per cell followed by macaque, rat, and mouse. The exponent for the allometric relationship (b) was then identified as the slope of the linear relationship described by log S = log (a) + b log(M), where S is average absolute SOD2 expression per cell and M is the body mass of each species in question. These linear regression analyses for each cochlear turn showed strong linear fits with exponent estimates (b) of 0.33 (r2 = 0.88) for spiral ganglion
122
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
cells in the apical cochlear turn, 0.45 (r2 = 0.90) for spiral ganglion cells in the middle cochlear turn, and 0.47 (r2 = 0.92) for spiral ganglion cells in the basal turn. Thus, the absolute SOD2 expression per spiral ganglion cell shows an allometric relationship with body mass. Furthermore, ganglion cells at the apical turn have higher level of absolute SOD2 immunoreactivity than middle and basal turns across all species. However, neither the total number of ganglion cells (data from Nadol, 1988) nor the size of spiral ganglion cell somata demonstrated an allometric scaling with body mass.
4. Discussion This study provides the first evidence for base-to-apex gradient in spiral ganglion cell expression of SOD2 across species from rodents to primates. Across all species, there appears to be less cellular SOD2 immunopositivity in ganglion cells at the cochlear base than ganglion cells at the cochlear apex. The ganglion cells from middle cochlear turns, however, can be similar in SOD2 expression to either cells at the apex or the base in different species. Both the proportion of SOD2 immunopositive and intensity of SOD2 immunoreactive spiral ganglia cells are higher in apex compared to base of cochlea in mice and rats. Although almost all spiral ganglion cells are SOD2 immunopositive in macaque and human specimens, the percentage area of the ganglion cell bodies showing intense SOD2 immunoreactivity was higher in apical than basal turn of the cochlea. The appearance of middle turn ganglion cells, though, varied across species. While the proportion of SOD2 immunopositive spiral ganglion cells in the middle turn is greater than in the basal turn in rodents and equal in primates, the intensity of immunoreactivity among immunopositive spiral ganglion cells in the middle turn can be greater (rat and macaques) or less (mouse and humans) than the basal turn. More importantly, the intensity of SOD2 immunoreactivity is never greater for spiral ganglion cells in the middle turn than those in the apical turn of the cochlea. One interesting and novel finding is that the area of intense somatic SOD2 immunoreactivity in spiral ganglion cells scales as a power function of body weight across the species examined in the study. This type of allometric scaling relationship was describe initially for whole body data from mammals and birds by Kleiber (Kleiber, 1932) in the 1930s, such that basal metabolic rate (BMR) is a power function of body mass (M) of the form BMR = B0M3/4 (with Bo being a normalization coefficient). It has been extended subsequently to a wide range of organisms from smallest microbes to the largest vertebrates and plants (Hemmingsen, 1950; Niklas, 1994). The scaling exponent for basal metabolism of organisms is invariably close to 3=4 , with many other physiological variables scale with exponents that are typically multiples of 1=4 (Schmidt-Nielsen, 1984). Based on the linear regression analysis, the scaling exponents (re: body weight) were 0.33 for the apical cochlear turn, 0.45 for the middle cochlear turn, and 0.47 for the basal cochlear turn. These exponents are greater than the exponents for the allometric relationship between cochlear size of extant mammals (slant height and diameters of first two turns) and body weight (slope = 0.159) described by Spoor et al. (2002), but are within the range that has been reported in the literature for the relationship between basilar papilla length and body mass in birds (Gleich et al., 2005), for the relationship between areas of vestibular neuroepithelial area and body weight in mammals (Desai et al., 2005), and for components of mammalian cardiovascular and respiratory systems (West et al., 1997). It is likely that the allometric relationship for cellular SOD2 expression reflects fundamental metabolic demand consequences of scaling both neural and non-neural structures within the cochlea. In this regard, it will be of particular interest to determine the differential contributions of ROS demand from tissues such as the organ of Corti, stria
vascularis and limbus to the cellular SOD2 expression by spiral ganglion cells. Because intense, punctate somatic deposits of SOD2 immunoreactivity likely represent mitochondrial expression, it is reasonable to assume that the percentage of the cell area containing these punctuate, intense deposits of SOD2 immunoreactivity is an indicator of the current (in our tissue, basal level) superoxide metabolic capacity of a cell. Hence, the similar allometric exponent values for each cochlear turn suggest that regional SOD2 expression from basal to apical turn could be a conserved bioenergetic feature that reflects a common mammalian pattern of free radical load across cochlear turns. The relative expression of SOD2 may be indicative of the relative demand for ROS metabolism by cochlear mitochondria. Previous studies showed higher SOD2 immunolocalization at relatively metabolic active sites of the cochlea (e.g., stria vascularis) which are likely to suffer tissue injury from superoxide radicals generated from vascular endothelial cells or during ischemia-reperfusion incidents (Lai et al., 1996; Suzuki et al., 1993). This inference reflects the general principle that SODs appear to be distributed preferentially in vulnerable cell population that is exposed to reasonably high ROS loads. Further, because SOD2 is a key enzymatic step in detoxifying the free radical cascade inside the mitochondria, it is attractive to hypothesize that the different levels of SOD2 expression may be one determinant of site-specific cochlea vulnerability to ROS damage at the cochlear base. A corollary hypothesis is that the capacity of spiral ganglion cells to respond to ROS challenges may vary along the cochlear spiral, with a lower SOD2 response capacity in cells located at the base than those at the apex. Increased levels of superoxide anions and other radicals have been linked directly to regional differences in sensory cell damage and high frequency hearing threshold shifts. For example, Sha et al. (2001) reported that enhanced susceptibility of basal outer hair cells to ROS injury is associated with a significantly lower level of the potent, endogenous antioxidant glutathione (i.e., oxidative stress capacity) in basal outer hair cells. A similar phenomenon has also been documented for spiral ganglion neurons. Le and Keithley (2007) presented evidence that dietary antioxidants differentially affect the age-related loss of spiral ganglion cells in the apical turn of the cochlea of beagles. The basal turn had the largest decrease in neuronal density, and the apical turn had a smaller loss when compared to cochleae from young dogs. The addition of antioxidants to the diet for the last three years of life of the animal reduced the magnitude of these ganglion cell losses in the apical but not the basal turn. These findings are consistent with the hypothesis that differential susceptibility to ROS load of spiral ganglion cells may reflect differences in metabolic capacity to handle ROS challenges. Regional differences in SOD2 could be one contributing factor to differences in ROS metabolism. The significance of the innate apical-to-basal gradient of decreasing SOD2 expression in mammals in the absence of ototoxic challenge may suggests a selection bias in the evolutionary process of cochlear design that favors higher SOD2 expression in the apex corresponding to greater ROS load in apical cochlear turn. A variety of molecular, structural and environmental factors may contribute to different ROS exposures as a function of location in the cochlea. For example, neurotrophin-3 (NT-3) expression shows a similar base-to-apex gradient in the mouse inner ear (Sugawara et al., 2007). Because endogenous NT-3 can enhance both free radical production by neurons and potentiate neuronal injury during ischemic events (Bates et al., 2002), the observed patterns may represent a dynamic balance between NT3 ROS potentiation and SOD2 ROS catabolism along the cochlear spiral. Secondly, a difference in endolymph and perilymph clearance capacity between the apex and base could also affect ROS clearance and, hence, the SOD2
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
123
Fig. 8. This schematic diagram illustrates the role of manganese SOD2 in mitochondrial superoxide metabolism. Manganese SOD2 (Mn SOD2) converts superoxide radicals to hydrogen peroxide, which forms a substrate for catalase, glutathione peroxidases (GPXs) and catalase. The results of a Fenton reaction can also activate mitochondrial uncoupling proteins 2 and 3 (UCP2 and UCP3), which reduce ROS production.
metabolic activity/expression. Alternatively, higher expression at the cochlear apex could be a protective mechanism against disproportionate low frequency environmental noise exposure (i.e., cardiac pulse and circulatory blood flow, HVAC systems and fluorescent light noise in a room) for humans and animals living in buildings. The conserved and allometrically scaled gradient of basal SOD2 expression across rodents and primates is significant because SOD2 is a rate-limiting component of mitochondrial metabolic response pathways to intracellular ROS load that can impact on cell survival (Fig. 8). Dismutation of superoxide into hydrogen peroxide by manganese Mn SOD2 in the mitochondria is upstream to many other important metabolic enzymes, including catalase and glutathione peroxidase that convert hydrogen peroxide into water. Myeloperoxidase converts hydrogen peroxide to hypochlorous acid, a strong oxidizing agent that can protect cells against pathogens but may also contribute to neuronal degeneration (Green et al., 2004; Choi et al., 2005). Finally, SOD2 is upstream to highly conserved, mitochondrial UCPs in a signaling cascade that reduces ROS production by the respiratory chain. The high degree of sequence homology across plants and animals implies that UCPs are not only proteins with specialized function in a specific mammalian species, but their metabolic physiological roles may be more general (Borecky et al., 2001). Therefore, as proposed originally by Schuknecht (1955), it is highly possible that partially independent metabolic factors underlie the differential vulnerability of hair cells and spiral ganglion cells, through different molecular vulnerabilities, without imposing a degenerative change upon the other or other supporting cochlear tissue. The different levels of SOD2 expression in spiral ganglion cells as a function of location in the cochlea raises the possibility that the reserve capacity of fundamental eukaryotic ROS defense mechanisms may vary along the
cochlear spiral, with higher SOD2 present in the spiral ganglion cells at the cochlear apex compared to those at the base. Financial disclosure None. Acknowledgments The authors thank Gloria Limetti and Jean Betsch for their technical assistance to the project. This project was supported by the Pennsylvania Lions Hearing Research Foundation and American Otologic Society Research Fellowship. References Bates, B., Hirt, L., Thomas, S.S., Akbarian, S., Le, D., Amin-Hanjani, S., Whalen, M., Jaenisch, R., Moskowitz, M.A., 2002. Neurotropin-3 promotes cell death induced in cerebral ischemia, oxygen-glucose deprivation, and oxidative stress: possible involvement of oxygen free radicals. Neurobiol. Dis. 9, 24–37. Bidmon, H.J., Wu, J., Buchkremer-Ratzmann, I., Mayer, B., Witte, O.W., Zilles, K., 1998. Transient increase of manganese-superoxide dismutase in remote brain areas after focal photothrombotic cortical lesion. Stroke 29, 203–210. discussion 211. Borecky, J., Maia, I.G., Arruda, P., 2001. Mitochondrial uncoupling proteins in mammals and plants. Biosci. Rep. 21 (2), 201–212. Chen, G.-D., Fechter, L.D., 2003. The relationship between noise-induced hearing loss and hair cell loss in rats. Hear. Res. 177, 81–90. Choi, D.K., Pennathur, S., Perier, C., Tieu, K., Teismann, P., Wu, D.C., Jackson-Lewis, V., Vila, M., Vonsattel, J.P., Heinecke, J.W., Przedborski, S., 2005. Ablation of the inflammatory enzyme myeloperoxidase mitigates features of Parkinson’s disease in mice. J. Neurosci. 25 (28), 6594–6600. Culotta, V.C., Yang, M., O’Halloran, T.V., 2006. Activation of superoxide dismutases: putting the metal to the pedal. Biochim. Biophys. Acta 1763, 747–758. Desai, S.S., Ali, H., Lysakowski, A., 2005. Comparative morphology of rodent vestibular periphery. II. Cristae ampullares. J. Neurophysiol. 93, 267–280.
124
Y.-L.M. Ying, C.D. Balaban / Hearing Research 253 (2009) 116–124
Echtay, K.S., Roussel, D., St-Pierre, J., Jekabsons, M.B., Cadenas, S., Stuart, J.A., Harper, J.A., Roebuck, S.J., Morrison, A., Pickering, S., Clapham, J.C., Brand, M.D., 2002. Superoxide activates mitochondrial uncoupling proteins. Nature 415, 96–99. Else, P.L., Hulbert, A.J., 1985. Mammals: an allometric study of metabolism at tissue and mitochondrial level. Am. J. Physiol. Regul. Integr. Comp. Physiol. 248, 415–421. Fischel-Ghodsian, N., Bykhovskaya, Y., Taylor, K., Kahen, T., Cantor, R., Ehrenman, K., Smith, R., Keithley, E.M., 1997. Temporal bone analysis of patients with presbycusis reveals high frequency of mitochondrial mutations. Hear. Res. 110, 147–154. Freeman, B.A., Crapo, J.D., 1982. Biology of disease. Free radicals and tissue injury. Lab. Invest. 47, 412–426. Ganbo, T., Sando, I., Balaban, C.D., Suzuki, C., Sudo, M., 1997. Immunohistochemistry of lymphocytes and macrophages in human celloidin-embedded temporal bone sections with acute otitis media. Ann. Otol. Rhinol. Laryngol. 106 (8), 662–668. Gao, F., Kinnula, V.L., Myllarniemi, M., Oury, T.D., 2008. Extracellular superoxide dismutase in pulmonary fibrosis. Antioxid. Redox. Signal. 10 (2), 343–354. Gleich, O., Dooling, R.J., Manley, G.A., 2005. Audiogram, body mass, and basilar papilla length: correlations in birds and predictions for extinct archosaurs. Naturewissenschaften 92, 595–598. Green, P.S., Mendez, A.J., Jacob, J.S., Crowley, J.R., Growdon, W., Hyman, B.T., Heinecke, J.W., 2004. Neuronal expression of myeloperoxidase is increased in Alzheimer’s disease. J. Neurochem. 90, 724–733. Hemmingsen, AM., 1950. Rep. Stenographic Mem. Hosp. Copenh. 4, 1–58. Jiang, H., Talaska, A.E., Schacht, J., Sha, S.H., 2007. Oxidative imbalance in the aging inner ear. Neurobiol. Aging 28, 1605–1612. Kammerman, J.R., Prophet, E.B., Barnes, C.F., 1992. Orthopedic histotechnology. In: Prophet, E.B., Mills, B., Arrington, J.B., Sobin, L.H. (Eds.), Armed Forces Institute of Pathology Laboratory Methods in Histotechnology. American Registry of Pathology, Washington, DC., pp. 72–73. Kleiber, M., 1932. Body size and metabolism. Hilgardia 6, 315–353. Lai, M.T., Ohmichi, T., Egusa, K., Okada, S., Masuda, Y., 1996. Immunohistochemical localization of manganese superoxide dismutase in rat cochlea. Eur. Arch. Otorhinolaryngol. 253 (4–5), 273–277. Le, T., Keithley, E.M., 2007. Effects of antioxidants on the aging inner ear. Hear. Res. 226, 194–202. Lechpammer, S., Epperly, M.W., Zhou, S., Nie, S., Glowacki, J., Greenberger, J.S., 2005. Adipocyte differentiation in Sod2( / ) and Sod2(+/+) murine bone marrow stromal cells is associated with low antioxidant pools. Exp. Hematol. 33, 1201–1208. Liu, X.H., Kato, H., Nakata, N., Kogure, K., Kato, K., 1993. An immunohistochemical study of copper/zinc superoxide dismutase in rat hippocampus after transient cerebral ischemia. Brain Res. 625 (1), 29–37. Maruyama, J., Yamagata, T., Ulfendahl, M., Bredberg, G., Altschuler, R.A., Miller, J.M., 2007. Effects of antioxidants on auditory nerve function and survival in deafened guinea pigs. Neurobiol. Dis. 25, 309–318. Maruyama, J., Miller, J.M., Ulfendahl, M., 2008. Glial cell line-derived neurotrophic factor and antioxidants preserve the electrical responsiveness of the spiral ganglion neurons after experimentally induced deafness. Neurobiol. Dis. 29, 14–21. McFadden, S.L., Ding, D., Reaume, A.G., Flood, D.G., Salvi, R.J., 1999. Age-related cochlear hair cell loss is enhanced in mice lacking copper/zinc superoxide dismutase. Neurobiology 20, 1–8.
McLean, I.W., Nakane, P.K., 1974. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077–1083. Miquel, J., Economos, A.C., Fleming, J., Johnson Jr., J.E., 1980. Mitochondrial role in cell aging. Exp. Gerontol. 15, 575–591. Nadol, J.B., 1988. Comparative anatomy of the cochlea and auditory nerve in mammals. Hear. Res. 34, 253–266. Nelson, E.G., Hinojosa, R., 2006. Presbycusis: a human temporal bone study of individuals with downward sloping audiometric patterns of hearing loss and review of the literature. Laryngoscope 116 (9, Part 3), 1–12. Niklas, K.J., 1994. Plant Allometry: The Scaling of Form and Process. University of Chicago Press, Chicago. Rarey, K.E., Yao, X., 1996. Localization of Cu/Zn-SOD and Mn SOD in the rat cochlea. Acta Otolaryngol. 116 (6), 833–835. Saha, R.N., Pahan, K., 2007. Differential regulation of Mn-Superoxide dismutase in neurons and astroglia by HIV-1 gp120: implications for HIV-associated dementia. Free Radic. Biol. Med. 42, 1866–1878. Schmidt-Nielsen, K., 1984. Scaling: Why is Animal Size So Important? Cambridge University Press, Cambridge, UK. Schuknecht, H.F., 1955. Presbycusis. Laryngoscope 65 (5), 402–419. Schuknecht, H.F., 1964. Further observations on the pathology of presbycusis. Arch Otolaryngol. 80, 369–382. Schuknecht, H.F., Gacek, M.R., 1993. Cochlear pathology in presbycusis. Ann. Oto. Rhino. Laryngol. 102, 1–16. Sha, S.H., Taylor, R., Forge, A., Schacht, J., 2001. Differential vulnerability of basal and apical hair cells is based on intrinsic susceptibility to free radicals. Hear. Res. 155, 1–8. Southgate, T.D., Sheard, V., Milsom, M.D., Ward, T.H., Mairs, R.J., Boyd, M., Fairbairn, L.J., 2006. Radioprotective gene therapy through retroviral expression of manganese superoxide dismutase. J. Gene Med. 8 (5), 557–565. Spoor, M., Bajpal, S., Hussain, S.T., Kumar, K., Thewissen, J.G.M., 2002. Vestibular evidence for evolution of aquatic behavior in early cetaceans. Nature 417, 163– 166. Stone, M., Schachern, P.A., Paparella, M.M., 1998. Loss of spiral ganglion cells as primary manifestation of aminoglycoside ototoxicity. Hear. Res. 115, 217–223. Sugawara, M., Murtie, J.C., Stankovic, K.M., Liberman, M.C., Corfas, G., 2007. Dynamic patterns of neurotrophin 3 expression in the postnatal mouse inner ear. J. Comp. Neurol. 501 (1), 30–37. Suzuki, K., Tatsumi, H., Satoh, S., Send, T., Nakata, T., Fuji, J., Taniguchi, N., 1993. Manganese-superoxide dismutase in endothelial cells: localization and mechanism of induction. Am. J. Physiol. 265, H1173–H1178. Wang, Y., Hirose, K., Liberman, M.C., 2002. Dynamics of noise-induced cellular injury and repair in the mouse cochlea. J. Assoc. Res. Otolaryngol. 3, 248–268. West, G.B., Brown, J.H., Enquist, B.J., 1997. A general model for the origin of allometric scaling laws in biology. Science 276, 122–126. West, G.B., Woodruff, W.H., Brown, J.H., 2002. Allometric scaling of metabolic rate from molecules and mitochondria to cells and mammals. PNAS 99 (1), 2473– 2478. Zimmermann, C.E., Burgess, B.J., Nadol, J.B., 1995. Patterns of degeneration in the human cochlear nerve. Hear. Res. 90 (1–2), 192–201.