Divergent Aging Characteristics in CBA/J and CBA/CaJ Mouse Cochleae
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Two inbred mouse strains, CBA/J and CBA/CaJ, have been used nearly interchangeably as ‘good hearing’ standards for research in hearing and deafness. We recently reported, however, that these two strains diverge after 1 year of age, such that CBA/CaJ mice show more rapid elevation of compound action potential (CAP) thresholds at high frequencies (Ohlemiller, Brain Res. 1277: 70–83, 2009). One contributor is progressive decline in endocochlear potential (EP) that appears only in CBA/CaJ. Here, we explore the cellular bases of threshold and EP disparities in old CBA/J and CBA/CaJ mice. Among the major findings, both strains exhibit a characteristic age (∼18 months in CBA/J and 24 months in CBA/CaJ) when females overtake males in sensitivity decline. Strain differences in progression of hearing loss are not due to greater hair cell loss in CBA/CaJ, but instead appear to reflect greater neuronal loss, plus more pronounced changes in the lateral wall, leading to EP decline. While both male and female CBA/CaJ show these pathologies, they are more pronounced in females. A novel feature that differed sharply by strain was moderate loss of outer sulcus cells (or ‘root’ cells) in spiral ligament of the upper basal turn in old CBA/CaJ mice, giving rise to deep indentations and void spaces in the ligament. We conclude that CBA/CaJ mice differ both quantitatively and qualitatively from CBA/J in age-related cochlear pathology, and model different types of presbycusis.
Keywordspresbycusis stria vascularis spiral ligament endocochlear potential hair cells outer sulcus cells marginal cells gender effects spiral ganglion
Age-related hearing loss is the major form of hearing loss and the single predominant neurodegenerative disease of aging (Schacht and Hawkins 2005; Ohlemiller and Frisina 2008; Frisina 2009; Schmiedt 2010). Assessments of human temporal bones have led to a general framework whereby three key inner ear components (hair cells, neurons, and strial cells) are taken to degenerate independently in a manner that reflects environmental and genetic risk factors that may be unique to each (Schuknecht 1974; Schuknecht and Gacek 1993). An individual may show hearing loss that principally reflects accelerated loss of hair cells (sensory presbycusis), loss of neurons (neural presbycusis), loss of strial cells with attendant endocochlear potential (EP) decline (strial presbycusis)—or some combination of these. Understanding of the interdependency of cochlear cell types for survival, along with shared risk factors by cell type, has benefited from the application of inbred mouse strains exhibiting very different aging characteristics. Many inbred and genetically engineered mutant lines usefully model specific aspects of human presbycusis (Willott 1991; Henry and McGinn 1992; Willott 2001; Ohlemiller 2006), and have helped clarify principles learned from other animals (Covell and Rogers 1957; Bohne et al. 1990; Tarnowski et al. 1991; Spicer and Schulte 2005a). As part of this process, inbred strains showing rapid progressive hearing loss such as C57BL/6 (B6) or BALB/c have typically been compared with a few ‘good hearing’ standard strains, most often CBA/J or CBA/CaJ (collectively ‘CBA’; e.g., Henry 1983; e.g., Erway et al. 1993; Hequembourg and Liberman 2001; Henry 2004). Both these strains hear well to at least 1 year of age, and have been used nearly interchangeably, or even mixed in the same studies. In fact, they diverged genetically over 80 years ago, and their genomes differ by more than 2,200 single-nucleotide polymorphisms (Bult et al. 2008). We recently reported that CBA/J appear much more vulnerable to noise in the first months of life (Fernandez et al. 2010). Moreover, after 1 year of age CBA/CaJ show more rapid hearing loss, in part due to EP decline that does not occur in CBA/J (Ohlemiller 2009). Here, we explore the cellular bases of threshold differences in old CBA/J and CBA/CaJ. As in a previous comparison of aging B6 and BALB/c mice (Ohlemiller et al. 2006), our strategy was to use CBA/J mice to indicate which age-related pathology has no impact on EP maintenance, and the pathology that is magnified in CBA/CaJ to indicate which changes are important for EP maintenance. We show here that these two strains model different forms of presbycusis. While CBA/J most closely model ‘uncomplicated’ sensory presbycusis (Sha et al. 2008), CBA/CaJ adds aspects of strial and neural presbycusis. These are more pronounced in females, suggesting potentially hormonally related mechanisms of loss in two distinct cell populations. We also report novel loss of outer sulcus cells (or ‘root’ cells) in spiral ligament of CBA/CaJ that presently appears unique to this strain.
Materials and methods
All procedures were approved by the Washington University Institutional Animal Care and Use Committee. Mice examined were offspring no more than five generations removed from CBA/J and CBA/CaJ breeders purchased from The Jackson Laboratory (JAX). The total sample included 105 CBA/J mice ranging in age from 2–27 months, and 124 CBA/CaJ mice ranging in age from 2–34 months. Differences in maximum age examined reflect lifes pan differences in the two strains, reportedly ranging 21–25 months in CBA/J and 24–29 months in CBA/CaJ (Fox et al. 1997). Samples were mixed by gender. Sample gender composition by data type is given in relevant sections.
Compound action potential (CAP) recordings were obtained from the left ear of 105 CBA/J and 124 CBA/CaJ mice. Animals were anesthetized (60 mg/kg sodium pentobarbital, IP) and positioned ventrally in a custom headholder. Core temperature was maintained at 37.5 ± 1.0°C using a thermostatically controlled heating pad in conjunction with a rectal probe (Yellow Springs Instruments Model 73A). An incision was made along the midline of the neck and soft tissues were blunt dissected and displaced laterally to expose the trachea and animal's left bulla. A tracheostomy was then made and the musculature over the bulla was cut posteriorly to expose the bone overlying the round window. Using a hand drill, a small hole was made over the round window. The recording electrode consisted of a fine silver wire coated in plastic except at the tip, which had been melted into a ball that could be inserted into round window antrum. Additional silver electrodes inserted into the neck musculature and hind leg served as reference and ground, respectively. Electrodes were led to a Grass P15 differential amplifier (100–3,000 Hz, ×100), then to a custom amplifier providing another ×1,000 gain, then digitized at 30 kHz using a Cambridge Electronic Design Micro1401 in conjunction with SIGNAL™ and custom signal averaging software operating on a 120 MHz Pentium PC. Sinewave stimuli generated by a Hewlett Packard 3325A oscillator were shaped by a custom electronic switch to 5 ms total duration, including 1 ms rise/fall times. The stimulus was amplified by a Crown D150A power amplifier and output to an Alpine SPS-OEOA coaxial speaker located 10 cm directly lateral to the left ear. Stimuli were presented freefield and calibrated using a B&K 4135 1/4 in. microphone placed where the external auditory meatus would normally be. Toneburst stimuli at each frequency and level were presented 100 times at 3/s. The minimum sound pressure level required for visual detection of a response (N1) was determined at 2.5, 5, 10, 20, 28.3, 40, and 56.6 kHz, using a 5 dB minimum step size.
Endocochlear potential recording
The EP was measured immediately after CAP recording in 89 CBA/J and 104 CBA/CaJ mice. Using a fine drill, a hole was made in the left cochlear capsule directly over scala media of the lower basal turn. Apical turn EPs were also measured in 68 CBA/J and 89 CBA/CaJ mice. Glass capillary pipettes (40–80 MΩ) filled with 0.15 M KCl were mounted on a hydraulic microdrive (Frederick Haer) and advanced until a stable positive potential was observed that did not change with increased electrode depth. The signal from the recording electrode was led to an AM Systems Model 1600 intracellular amplifier.
Tissue processing and sectioning
At the end of recording, animals were overdosed and perfused transcardially with cold 2.0% paraformaldehyde/2.0% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Each cochlea was rapidly isolated, immersed in the same fixative, and the stapes was immediately removed. Complete infiltration of the cochlea by fixative was ensured by making a small hole at the apex of the cochlear capsule, and gently circulating the fixative over the cochlea using a transfer pipet. After decalcification in sodium EDTA for 72 hours, cochleas were post-fixed in buffered 1% osmium tetroxide, dehydrated in an ascending acetone series, and embedded in Epon. Left cochleas were sectioned in the mid-modiolar plane at 4.0 μm, then stained with toluidine blue for bright field viewing with a Nikon Optiphot™ light microscope using a 100× oil objective and a calibrated grid ocular. Typically, 50 sections were obtained from each cochlea, spanning 200 μm centered on the modiolar ‘core’.
Morphometric analysis of mid-modiolar sections
For quantitative histologic evaluation by age and strain, cochleas were taken from ‘young’ (2–4 months) and ‘old’ (19–27 months) mixed-gender samples in each strain. ‘Young’ samples included eight CBA/J and 6 CBA/CaJ mice; ‘Old’ samples included 12 CBA/J and 13 CBA/CaJ. An additional ‘very old’ CBA/CaJ sample (28–34 months, n = 9) was evaluated for comparison with other CBA/CaJ, but could not be compared directly with CBA/J due to differences in lifespan. For each animal, five mid-modiolar sections distributed evenly over the 200-μm sectioned distance were analyzed by an observer blinded to strain and age. Strial and ligament measures were chosen from previous studies (Ohlemiller and Gagnon 2004a; Ohlemiller et al. 2006, 2008, 2009; Ohlemiller and Gagnon 2007), and included strial thickness, marginal cell density, intermediate cell density, basal cell density, strial capillary density, ligament thickness, density in ligament of types I, II, and IV fibrocytes, and the appearance of outer sulcus cells. Based on preliminary observations of anomalies in the outer sulcus cell region of spiral ligament in old CBA/CaJ, two additional metrics were devised as described below. DIC images used for illustration were obtained on a Zeiss LSM 700 laser scanning confocal microscope using ZEN™ software, then further processed using CANVAS™.
Estimates of spiral ganglion cell density were obtained in the cochlear lower basal turn, upper basal turn, and apical turn. Neuronal density was estimated by counting nucleated neuronal profiles within a 3,600-μm2 grid roughly centered on Rosenthal’s canal at each location.
Strial thickness was measured orthogonal to the midpoint in the lower base, upper base, and lower apex. Only nucleated profiles were included in strial cell counts. Because marginal cell density was of special interest among strial cell types, marginal cells were counted in the lower base, upper base, and lower apex, while other cell types were quantified only in the upper base. Marginal cells, intermediate cells, and basal cells were counted in an 80-μm linear segment of stria centered at the midpoint. No attempt was made to distinguish between lower and upper level intermediate cells, a distinction made by Spicer and Schulte (2005b). Unconnected capillary profiles were counted over the entire strial profile in the lower base, upper base, and lower apex (Fig. 13A). In a separate analysis (see Figs. 13B, C and 15C, F), capillaries were counted coincident with other strial cell counts in an 80 μm linear segment of stria in the upper base, and the luminal diameters of those capillaries were measured.
Ligament thickness was measured in the lower base, upper base, and lower apex. At each location, thickness was measured on an axis co-linear with the strial midline (coaxial with strial thickness measures). Types I, II, and IV fibrocytes were assessed in the upper base using a 1,600 μm2 area. These were identified based on location, an approach taken in previous studies (Hequembourg and Liberman 2001; Hirose and Liberman 2003; Lang et al. 2003). Only nucleated profiles were included.
Outer sulcus cells/root cells
OSCs were easily recognizable by light staining of their cytoplasm, and by their large, lightly stained, either circular or ovoid nuclei (Fig. 17; Duvall 1969; Spicer et al. 1996). These cells line the ligament wall between Claudius cells and spiral prominence epithelial cells, and are thought to be involved in spatial buffering and recirculation of K+ and Na+ (Marcus and Chiba 1999; Jagger et al. 2010). Depending on location, they may either directly border scala media, or be covered by Claudius and spiral prominence cells. Similar-appearing cells compose distinct ‘roots’ or ‘pegs’ (Duvall 1969) that do not border scala media, and project deep into the ligament. Each root is usually composed of multiple cells. It is presently not clear whether OSCs and root cells are biochemically or functionally distinct, and it is also possible that different basal/apical locations and even different superior/inferior positions in the ligament contain distinct types of these (Jagger et al. 2010). For present purposes, references to OSCs should be understood to include root cells. We noted that OSCs bordering scala media and those composing roots were often missing in old CBA/CaJ and devised two metrics to facilitate quantitative analysis. For the first metric, OSC nuclei were counted over the entire ligament profile in upper and lower basal turn. For the second metric, each section was blindly scored ‘present’ or ‘absent’ for obvious indentations or voids in the region where OSCs are normally observed. To ensure that empty capillary profiles were not scored as voids, apparent voids were always examined using a ×100 oil objective for an endothelial cell lining.
Hair cell counts
Right cochleas of eight old CBA/J and eight old CBA/CaJ mice were dissected using fine blades into half-turn segments, immersed in mineral oil in a depression slide, and examined as surface preparations by Nomarski optics using a ×20 oil objective and a calibrated grid ocular. The percent outer hair cells (OHCs) and inner hair cells (IHCs) missing (as judged by the absence of nuclei) was estimated in contiguous 200 μm segments, and data were recorded separately by cell type as a function of distance from the basal tip. For each hair cell type, cochlear distance versus percent present was plotted as a function of frequency based on Muller et al. (2005).
CAP thresholds were analyzed for each age group by two-way ANOVA (threshold × strain, frequency; SIGMASTAT™). OHC and IHC counts were separately analyzed by two-way ANOVA (percent present × strain, location). For other histologic metrics, estimates in each animal were averaged to yield an overall mean for that animal. Strial and ligament metrics obtained for more than one cochlear location (ganglion cell density, strial thickness, marginal cell density, strial capillary density, ligament thickness, OSC density) were analyzed by two-way ANOVA (metric × group, location). Metrics obtained only for the upper base were analyzed by one-way ANOVA. All ANOVAs were followed by Bonferroni multiple comparisons tests. Non-parametric data (presence versus absence of indentations or voids in ligament) were analyzed by Z test. For some analyses, linear or nonlinear regression was applied (SIGMASTAT™). For all tests, p < 0.05 was taken to denote a significant difference between groups, or regression slope significantly different from zero.
Hair cell loss
Spiral ganglion cell loss
EP reduction in old CBA/CaJ
Strial anatomic correlates of EP reduction
Since variation in the EP could account in part for CAP threshold variation by strain, we considered what anatomic changes in the cochlear lateral wall are most closely associated with EP decline, and whether these changes also show gender dependence. We begin with the stria vascularis.
Strial capillary density and size
Mean strial capillary size did not vary significantly by strain, age, or gender (not shown). Instead we noted a tendency for capillary sizes to redistribute in old animals, and for the manner of the redistribution to vary by group. As shown in Figure 13B, young mice irrespective of strain have strial capillaries that are equally likely to show diameters of 1–5 or 6–10 μm. In older mice, the proportion of capillaries with diameters of 6–10 μm is reduced, and the majority of remaining capillaries show smaller diameters. However, as made clearer in Figure 13C, which shows changes in capillary size with age, old CBA/CaJ mice retain a greater fraction of capillaries with diameters at least 6–10 μm in diameter. This trend is especially pronounced in old CBA/CaJ females, in which ∼11% of strial capillaries are larger than 10 μm (arrow in Fig. 13B). The old CBA/CaJ females show only a slight gain in the fraction of small capillaries, and uniquely, show a gain in the fraction of capillaries larger than 10 μm (arrows in Fig. 13C). In summary, capillary data suggest that neither reduction in strial capillary density nor reduced capillary size account for EP decline in old CBA/CaJ. Instead, retention of larger capillaries—including an increase in the fraction of large capillaries in females—may signal other changes that are tied to EP reduction.
Strial cell density
Spiral ligament histologic correlates of EP reduction
Loss of outer sulcus cells/root cells in CBA/CaJ
The OSCs most affected appeared to be those lying more superior in the ligament, immediately below the spiral prominence (Figs. 11 and 17A), and may represent a specific population. We further noted that these were most likely to lie in the zone where Claudius cells and spiral prominence epithelial cells typically meet in CBA/J cochleas, leaving no OSCs directly exposed to endolymph. In Figure 17, the point of termination of Claudius cells abutting the spiral ligament is compared in example old CBA/CaJ and CBA/J mice (compare points marked by arrowheads in Figure 17A, B). In the CBA/CaJ, a thin Claudius cell process ends well inferior to a spiral prominence epithelium that follows the outline of the void left by missing OSCs and their root processes. In the CBA/J, a more expansive network of Claudius cells clearly ends at the thin line of cells marking spiral prominence. Basal–apical variation in the extent of ‘exposure’ to endolymph of OSCs has been reported as a normal feature of gerbils and guinea pigs (Duvall 1969; Spicer et al. 1996). It was our impression, however, that these cells are less often exposed in CBA/J than in CBA/CaJ, even when young animals are compared.
Comparison of features in aging CBA/J and CBA/CaJ cochleae
Similar loss at low frequencies
CBA/CaJ show greater loss at high frequencies
Thresholds by gender
Females more susceptible than males
Hair cell loss
Similar IHC loss
CBA/J show more OHC loss
CBA/CaJ show more loss
EP by age
Only CBA/CaJ show EP decline
Increased fraction of capillaries <5 μm with age
CBA/CaJ females show increase in fraction of capillaries >10 μm with age
Strial marginal cells
Only CBA/CaJ show significant loss
Strial intermediate, basal cellsa
Similar density, little change with age
Thinner in CBA/CaJ
Ligament types I, II, IV fibrocytesa
Similar density, little change with age
Outer sulcus cells
Only CBA/CaJ show significant loss
CBA/CaJ versus CBA/J
We agree with a previous assessment (Sha et al. 2008) that CBA/J mice most closely approximate sensory presbycusis. The loss of predominantly outer hair cells in both base and apex (Fig. 4), also described by Sha et al. (2008), reasonably corresponds with the pattern of threshold shifts with age in CBA/J. According to our cell counts, old CBA/CaJ mice retain more OHCs than CBA/J, so that they cannot simply represent a more extreme version of the same aging model. We know of no other study that has specifically examined hair cell density in old CBA/CaJ mice. Previous studies have mixed CBA/J and CBA/CaJ (Spongr et al. 1997) or used other CBA/Ca substrains (Li 1992; Li and Hultcrantz 1994).
Comparative patterns of spiral ganglion cell loss in CBA/J and CBA/CaJ mice are nearly the inverse of their patterns of hair cell loss. That is, CBA/CaJs lose fewer OHCs than CBA/J, but more neurons, at both apical and basal ends of the cochlea (Figs. 4 and 5). Complementary loss of different cell types may help explain why the two strains show similar patterns of low frequency hearing loss with age (Figs. 1 and 3). Alternatively, of course, the rate of low frequency hearing loss may reflect a common degeneration of some other cell or structure we did not quantify. Since neuronal loss in CBA/CaJ appeared to outpace inner hair cell loss (compare Figs. 4 and 5), this loss was likely a combination of primary and secondary loss. The pattern of accelerated neuronal loss in both base and apex matches a trend noted for humans (Felder and Schrott-Fischer 1995). To our knowledge, the present study is the first suggesting gender bias in neuronal loss.
While CBA/CaJ mice lost fewer OHCs in the lower base than CBA/J, there was some loss. We therefore cannot say that hair cell loss played no role in progressive high frequency threshold shifts. It would appear more correct to say that CBA/CaJ mice add other forms of presbycusis to the sensory form displayed by CBA/J. Presently, no aging mouse model that has been well described remains free of significant hair cell loss over the normal life span (Spongr et al. 1997; Willott et al. 1998; Ohlemiller and Gagnon 2004a; Ohlemiller et al. 2008; Sha et al. 2008). Unlike spiral ganglion and stria vascularis, which possess redundant capacity, and for which moderate degeneration can apparently be tolerated without hearing loss (Schulte and Schmiedt 1992; El-Badry and McFadden 2009; Kujawa and Liberman 2009), OHCs are not present in excess. The predominant cochlear aging mode for mice may be sensory presbycusis, with strial and neural presbycusis added in some strains.
Threshold changes at high frequencies in both CBA/J and CBA/CaJ appear sexually dimorphic, with females showing more rapid hearing loss than males by 15 or 24 months, respectively (Fig. 3). In this regard, our data seem to depart from a previous study indicating that CBA/CaJ males sustain more rapid high frequency hearing loss (Henry 2004). At ages ranging 9–24 months, however, we likewise find poorer hearing in males, so that there appear to be two gender patterns over the CBA/CaJ lifespan. The oldest CBA/CaJ mice examined in the Henry (2004) study, 350 days of age, fell within a window that favors females in our data as well, and Henry would have missed a later transition. Notably, our data bring CBA/J and CBA/CaJ into register with another trend noted by Henry, namely that C57BL/6 J mice also feature more rapid hearing loss in females. CBA and B6 mice may not mimic a broadly supported trend in humans, whereby males fare more poorly with age (Jerger et al. 1993). However, they may usefully model the human phenomenon of accelerated hearing loss in females after menopause (Hederstierna et al. 2007) suggested to reflect loss of the protective effects of estrogen. Unfortunately, we know of only one estimate of age at menopause in mice, universally placing this event at 12–14 months (Silver 1995). It would be of interest to know whether the ∼9-month strain difference in the age at which male and female thresholds diverge (Fig. 3) reflects a later age at menopause in CBA/CaJ. Gender effects on thresholds in old CBA/CaJ parallel two anatomic trends. First, loss of cochlear neurons appears more pronounced in old females than in males (Fig. 5). Second, EP decline is significantly greater in females (Figs. 9 and 10). Therefore, neuronal loss and EP decline in CBA/CaJ may both be modulated by sex hormones or other gender-related characteristics (see below).
We cannot, of course, be sure that we have identified the essential cellular differences between aging CBA/J and CBA/CaJ that account for their threshold differences. We only sampled cochleas at fixed locations, although this strategy has proven successful in the past. The usual caveat—that cell counts need not correspond to cell functionality—must be injected. We found no clear anatomic correlate for more rapid hearing loss in female CBA/J mice. In CBA/CaJ, EP variation accounted for only 30% of threshold variation at most, and no clear pattern by gender was identified for marginal cell loss. Moderate loss of outer sulcus cells in the upper base of CBA/CaJ could certainly affect both EP and threshold (see below), but we detected no contribution to either EP decline or threshold elevation. Finally, we could not show correlation between neuronal density and thresholds in CBA/CaJ, and thus could not demonstrate that greater neuronal loss in old CBA/CaJ females helps explain their more severe hearing loss. Most evidence indicates that only substantial neuronal loss is manifested in behaviorally or physiologically determined threshold shifts (El-Badry and McFadden 2009; Kujawa and Liberman 2009). Nevertheless, the >50% loss of neurons exhibited in the lower base of old CBA/CaJ mice may be sufficient.
Correlates of EP decline in CBA/CaJ
Comparison with CBA/J revealed several properties of cochlear lateral wall in old CBA/CaJ mice that have been linked to explicit, or suggested, EP decline.
Strial thinning is a commonly reported aspect of cochlear aging (Kusunoki et al. 2004; Suzuki et al. 2006; Ishiyama et al. 2007), although it is often not clear when thinning is functionally meaningful. However, differences in strial thickness in our material (Fig. 12) actually favor CBA/CaJ, a trend that may reflect pathology not evident at the light microscope level.
Reduced capillary density, reduced capillary size, and changes in capillary structure have all been implicated in real or potential EP decline (Johnsson and Hawkins 1972; Gratton and Schulte 1995; Gratton et al. 1996; Thomopoulos et al. 1997). As we have noted however (Ohlemiller et al. 2006), neither >50% reductions in strial capillary density (Di Girolamo et al. 2001), nor more than doubling of capillary basement membrane thickness in C57BL/6 mice promote EP decline (Lang et al. 2002). By contrast, capillary loss in CBA/CaJ mice is generally less than 20% (Fig. 13A). Probably more important was strial capillary size redistribution with age (Fig. 13B, C). The fact that CBA/J mice showed the most pronounced shift toward smaller capillary sizes renders this an unlikely key factor in EP decline. Greater retention of larger capillaries in old CBA/CaJ—and particularly the increase in the fraction of the largest capillaries in old female CBA/CaJ—may be more significant. We previously noted a similar trend in old female BALB/c mice, in which average capillary size was greater in mice showing EP decline (Ohlemiller et al. 2006). A shift toward larger strial capillaries may point to another process that contributes to EP reduction, or perhaps to a compensatory response. Local stress responses may sense EP reduction, ion imbalance, or hypoxia, and increase capillary size in response. Abnormally large strial capillaries may emerge as an anatomic marker for the presence of EP decline.
Spiral ligament thickness and fibrocyte density
The finding that a relatively thin spiral ligament in CBA/CaJ (Fig. 16) coincides with a tendency toward EP decline mirrors a trend in two other mouse models we have described (Ohlemiller et al. 2006, 2009). BALB/c mice in particular combine somewhat low marginal cell density and a thin spiral ligament with slightly reduced EP (∼10 mV) from an early age, and these become more pronounced with time. There is disagreement about the existence of any causal link—and direction thereof—between pathology of the stria and spiral ligament (e.g., Hequembourg and Liberman 2001; Spicer and Schulte 2002; Wu and Marcus 2003; Ishiyama et al. 2007). Aging appears reliably associated with progressive reduction in ligament volume and loss of fibrocytes in both humans and animals (Wright and Schuknecht 1972; Spicer and Schulte 2002; Kusunoki et al. 2004; Ishiyama et al. 2007). However, comparison of the extent of ligament degeneration and strial pathology across multiple inbred mouse models suggests that these need not be closely related (Ohlemiller 2009). Disruption of K+ flux through the ligament can reduce the EP, but it is not clear this promotes strial degeneration (e.g., Minowa et al. 1999). A thin spiral ligament early in life may signal a developmental process that also limits marginal cell density, and promotes eventual EP decline.
The role of gender in hearing and EP decline
Our data suggest that CBA/CaJ mice begin life with fewer marginal cells than CBA/J (Fig. 14), then undergo further marginal cell loss with age. CBA/J mice, by contrast, do not show significant loss of marginal cells over their typical life span. Primary loss or dysfunction of marginal cells is emerging as the most frequently cited basis of EP decline in humans (Pauler et al. 1988; Schuknecht and Gacek 1993) and animal models (Spicer and Schulte 2005a; Ohlemiller et al. 2006; Ohlemiller 2009). That said, marginal cell loss did not seem to explain prominent gender differences in CBA/CaJ, and gender effects on marginal cell function must be considered. The idea that estrogen impacts strial marginal cell operation is supported by the literature (Konig et al. 2008; Motohashi et al. 2010). Interestingly, the effect appears to be one of inhibition (Lee and Marcus 2001). Inhibition of strial ion transport coincides with a suggested overall protective effect of estrogen on sensory cells (Vina et al. 2005; Charitidi et al. 2009). It is conceivable that effects of estrogen in females partly explain both reduction of the EP after 1 year and the acceleration of hearing loss after 2 years (that is, after menopause). Alternatively, sex-related differences other than strictly hormonal processes may underlie the gender trends we observe (Willott 2009). The apparent gender-skewing of EP decline in aging CBA/CaJ matches a trend suggested for humans (Gates et al. 1999; Gates and Mills 2005), so that the underlying mechanisms merit further study.
Loss of outer sulcus cells in CBA/CaJ
Loss of outer sulcus cells in CBA/CaJ adds a new twist to how threshold sensitivity and EP generation may be altered in these mice. In this and other mouse models we have examined, we have interpreted reduced marginal cell density as an indication that critical marginal cell functions in generating the EP are impaired. We suppose—but have yet to show—that fewer, larger, marginal cells maintain fewer critical pumps, transporters, and channels (Gratton et al. 1997), or alter the critical ‘stoichiometry’ of these among strial cell types (Diaz et al. 2007). We have not proposed or observed that marginal cell degeneration leaves exposed intermediate cells or alters the ion barrier of scala media. Published descriptions of aging gerbils, which also appear to model EP decline originating with marginal cell pathology, are likewise consistent with the maintenance of a continuous marginal cell covering. By contrast with marginal cells, outer sulcus cells and the root processes they compose project deep into the ligament (Duvall 1969; Spicer et al. 1996). OSC loss seems likely to uncover cells not normally exposed to endolymph, and not expressing tight junctions. OSC loss therefore probably violates the integrity of scala media. Sufficient loss of outer sulcus cells would act as an uncontrolled ion shunt, lowering the EP and reducing K+ currents through hair cells. While this certainly could explain some characteristics of CBA/CaJ mice, we could not link the severity of OSC degeneration to hearing loss. The spatially delimited pattern of OSC loss in these mice may reduce its impact on hearing. As we noted, the OSCs most affected appeared to be those lying where Claudius cells and spiral prominence epithelial cells typically meet, and appeared less often exposed in CBA/J than in CBA/CaJ. However, it has not been suggested that exposure of some OSCs represents a form of pathology. For now, loss of outer sulcus cells joins a long list of noted age-related cochlear changes—including capillary loss, ligament fibrocyte loss, limbus fibrocyte loss, organ of Corti supporting cell loss, clumping of neuronal cell bodies, pillar cell anomalies, and changes in Reissner’s membrane (Cohen et al. 1990; Adams and Schulte 1997; Hequembourg and Liberman 2001; Ohlemiller and Gagnon 2004a, 2004b)—whose significance for the incidence and severity of presbycusis is not clear.
The value of the nth aging model
Investigators in human presbycusis and its animal models must continually apply optimism that if we examine enough cases and models, a finite number of patterns will emerge. Even if a manageable number of patterns do emerge, they will certainly reflect a much larger set of genetic and environmental causes. Nevertheless, focusing on patterns may point the way to a manageable number of therapeutic targets. Unfortunately, the number of patterns divined from human temporal bones is discouragingly large (for an excellent review, see Nelson and Hinojosa 2006). Most discrepancies derive from the degree of presbycusis attributed to pathology of neurons versus hair cells versus strial cells. An implicit theme that runs through Schuknecht’s work (Schuknecht 1993; Schuknecht and Gacek 1993) is that these cells and structures can degenerate independently. In fact, these are rarely encountered in isolation, and temporal bone analyses rarely resolve how much hearing loss is attributable to each. The significance of their posited independent degeneration lies in its implications for underlying mechanisms. Invariant co-degeneration of neurons, hair cells, and strial cells (to an extent that all contribute to hearing loss) would suggest different disease mechanisms than would co-degeneration of only subsets of these. Just how much and what type of lateral wall degeneration is incompatible with a normal EP is currently best determined from comparisons of mouse models. Since the EP must be measured to ‘diagnose’ strial presbycusis, few models can be considered adequately characterized.
This work was supported by NIH R01 DC03454, DC08321 (KKO), P30 DC04665 (R. Chole) and Washington University Medical School Department of Otolaryngology.
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