The FBN rat model of aging: investigation of ABR waveforms and ribbon synapse changes.

Age-related hearing loss is experienced by one-third of individuals aged 65 years and older and can be socially debilitating. Historically, there has been poor correlation between age-related threshold changes, loss of speech understanding, and loss of cochlear hair cells. We examined changes in ribbon synapse number at four different ages in Fisher Brown Norway rats, an extensively studied rat model of aging. In contrast to previous work in mice/Wistar rats, we found minimal ribbon synapse loss before 20 months, with significant differences in 24- and 28-month-old rats at 4 kHz. Significant outer HC loss was observed at 24 and 28 months in low- to mid-frequency regions. Age-related reductions in auditory brainstem response wave I amplitude and increases in threshold were strongly correlated with ribbon synapse loss. Wave V/I ratios increased across age for click, 2, 4, and 24 kHz. Together, we find that ribbon synapses in the Fisher Brown Norway rat cochlea show resistance to aging until ∼60% of their life span, suggesting species/strain differences may underpin decreased peripheral input into the aging central processor.

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea.

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

Protection by low-dose kanamycin against noise-induced hearing loss in mice: dependence on dosing regimen and genetic background.

We recently demonstrated that sub-chronic low-dose kanamycin (KM, 300 mg/kg sc, 2×/day, 10 days) dramatically reduces permanent noise-induced hearing loss (NIHL) and hair cell loss in 1 month old CBA/J mice (Fernandez et al., 2010, J. Assoc. Res. Otolaryngol. 11, 235-244). Protection by KM remained for at least 48 h after the last dose, and appeared to involve a cumulative effect of multiple doses as part of a preconditioning process. The first month of life lies within the early 'sensitive period' for both cochlear noise and ototoxic injury in mice, and CBA/J mice appear exquisitely vulnerable to noise during this period (Ohlemiller et al., 2011; Hearing Res. 272, 13-20). From our initial data, we could not rule out 1) that less rigorous treatment protocols than the intensive one we applied may be equally-or more-protective; 2) that protection by KM is tightly linked to processes unique to the sensitive period for noise or ototoxins; or 3) that protection by KM is exclusive to CBA/J mice. The present experiments address these questions by varying the number and timing of fixed doses (300 mg/kg sc) of KM, as well as the age at treatment in CBA/J mice. We also tested for protection in young C57BL/6J (B6) mice. We find that nearly complete protection against at least 2 h of intense (110 dB SPL) broadband noise can be observed in CBA/J mice at least for ages up to 1 year. Reducing dosing frequency to as little as once every other day (a four-fold decrease in dosing frequency) appeared as protective as twice per day. However, reducing the number of doses to just 1 or 2, followed by noise 24 or 48 h later greatly reduced protection. Notably, hearing thresholds and hair cells in young B6 mice appeared completely unprotected by the same regimen that dramatically protects CBA/J mice. We conclude that protective effects of KM against NIHL in CBA/J mice can be engaged by a wide range of dosing regimens, and are not exclusive to the sensitive period for noise or ototoxins. While we cannot presently judge the generality of protection across genetic backgrounds, it appears not to be universal, since B6 showed no benefit. Classical genetic approaches based on CBA/J × B6 crosses may reveal loci critical to protective cascades engaged by kanamycin and perhaps other preconditioners. Their human analogs may partly determine who is at elevated risk of acquired hearing loss.

Different cellular and genetic basis of noise-related endocochlear potential reduction in CBA/J and BALB/cJ mice.

The acute and permanent effects of noise exposure on the endocochlear potential (EP) and cochlear lateral wall were evaluated in BALB/cJ (BALB) inbred mice, and compared with CBA/J (CBA) and C57BL/6 (B6) mice. Two-hour exposure to broadband noise (4-45 kHz) at 110 dB SPL leads to a approximately 50 mV reduction in the EP in BALB and CBA, but not B6. EP reduction in BALB and CBA is reliably associated with characteristic acute cellular pathology in stria vascularis and spiral ligament. By 8 weeks after exposure, the EP in CBA mice has returned to normal. In BALBs, however, the EP remains depressed by an average approximately 10 mV, so that permanent EP reduction contributes to permanent threshold shifts in these mice. We recently showed that the CBA noise phenotype in part reflects the influence of a large effect quantitative trait locus on Chr. 18, termed Nirep (Ohlemiller et al., Hear Res 260:47-53, 2010b). While CBA "EP susceptibility" alleles are dominant to those in B6, examination of (B6 × BALB) F1 hybrid mice and (F1 × BALB) N2 backcross mice revealed that noise-related EP reduction and associated cell pathology in BALBs are inherited in an autosomal recessive manner, and are dependent on multiple genes. Moreover, while N2 mice formed from B6 and CBA retain strong correspondence between acute EP reduction, ligament pathology, and strial pathology, N2s formed from B6 and BALB include subsets that dissociate pathology of ligament and stria. We conclude that the genes and cascades that govern the very similar EP susceptibility phenotypes in BALB and CBA mice need not be the same. BALBs appear to carry alleles that promote more pronounced long term effects of noise on the lateral wall. Separate loci in BALBs may preferentially impact stria versus ligament.

Multiple ligament knee reconstruction clinical follow-up and gait analysis.

Multiple ligament knee injuries are serious and rare injures that have not been studied using advanced gait analysis techniques. The purpose of this study was to perform clinical follow-up and gait analysis on patients with multiple knee ligament reconstruction. Twenty-four patients who underwent a multi-ligament knee reconstruction by a single surgeon volunteered to participate in this study. We performed complete clinical exam including instrumented ACL exam (KT-1000), and radiological exam including weight-bearing and PCL stress radiographs (TELOS) at minimum 2 years post index surgery. In addition, we performed complete three-dimensional gait analysis on 18 patients. We used a 10-camera, high speed (120 Hz) motion analysis system in conjunction with a multi-axis strain-gage force plate which calculated knee joint kinetics and kinematics while subjects performed flat-ground walking and stair-descent tasks. Kinematic and kinetic variables were compared between reconstructed and contralateral knees and unmatched, healthy control knees. All knee joint moments were normalized to subjects' weight. Clinical: Average knee joint flexion/extension 123.6 +/- 15.5/1.7 +/- 3.5, respectively. Average KT-1000 side-to-side difference was 1.2 +/- 2.0 mm, TELOS side-to-side difference on stress radiographs was 4.0 +/- 3.1 mm. Median IKDC score was 67 (range 13-94). Fifty-three percent of patients exhibited radiographic evidence of osteoarthritis (OA) on the operative side; one patient on the contralateral knee. During gait analysis, patients exhibited significantly reduced total knee joint range of motion, and external knee flexion moment in the reconstructed knee compared to the contralateral knee and healthy control knees. The magnitude of these differences was greater while descending a step. Finally, patients who had radiographic evidence of knee joint OA had significantly lower magnitude external knee flexion moment compared to those who did not have OA at the time of follow-up. Greater than 2 years after reconstruction, patients with multi-ligament knee injuries are able to return to daily activities. Gait analysis data suggests that patients may be experiencing higher magnitude changes in sagittal plane kinematics and kinetics during demanding functional tasks (stair decent). Changes in walking gait biomechanics may help explain why this group is experiencing unilateral knee joint degeneration.