The mechanoelectrical transducer channel is not required for regulation of cochlear blood flow during loud sound exposure in mice

Abstract

The mammalian cochlea possesses unique acoustic sensitivity due to a mechanoelectrical 'amplifier', which requires the metabolic support of the cochlear lateral wall. Loud sound exposure sufficient to induce permanent hearing damage causes cochlear blood flow reduction, which may contribute to hearing loss. However, sensory epithelium involvement in the cochlear blood flow regulation pathway is not fully described. We hypothesize that genetic manipulation of the mechanoelectrical transducer complex will abolish sound induced cochlear blood flow regulation. We used salsa mice, a Chd23 mutant with no mechanoelectrical transduction, and deafness before p56. Using optical coherence tomography angiography, we measured the cochlear blood flow of salsa and wild-type mice in response to loud sound (120 dB SPL, 30 minutes low-pass filtered noise). An expected sound induced decrease in cochlear blood flow occurred in CBA/CaJ mice, but surprisingly the same sound protocol induced cochlear blood flow increases in salsa mice. Blood flow did not change in the contralateral ear. Disruption of the sympathetic nervous system partially abolished the observed wild-type blood flow decrease but not the salsa increase. Therefore sympathetic activation contributes to sound induced reduction of cochlear blood flow. Additionally a local, non-sensory pathway, potentially therapeutically targetable, must exist for cochlear blood flow regulation.

Figure 1

En face reconstruction of the ventral view of lateral wall blood flow in the middle turn of a murine left cochlea using OCTA. The middle turn organ of Corti (not visible) runs mediolaterally from low to high frequency along the cochlear spiral, between the two dashed lines, and includes the approximate location of a typical region of interest selection for flow analysis. This image is an average of 5 consecutive scans in the absence of sound.

Figure 2

Characterization of hearing function in the salsa mouse. (a) Example ABR time domain response to a 16 kHz tone, for an 8 week old CBA/CaJ mouse. The threshold was identified to be 30 dB SPL. (b) Example ABR time domain response to a 16 kHz tone for an 8 week old salsa mouse. There was no identifiable response at the highest level tested (90 dB SPL). (c) 2f1-f2 amplitudes (L1 = L2 + 10 dB=60 dB SPL, red circles solid line), ABR thresholds for 16, 24 and 32 kHz, for an example heterozygous salsa mouse (blue plusses solid line) and homozygous salsa mice (N = 10, red arrows). There was no ABR response and no 2f1-f2 signal for the salsa mice. (d) CAP and CM measurements for an example CBA/CaJ mouse (CAP: blue crosses, CM: blue circles) and salsa mice (N = 7). There was no measurable CAP at any attenuation value for the salsa mice (red arrows). No CM signal specific to the cochlea was measured (red squares solid line). (e) IHC (N = 8, red line) and OHC (N = 7, blue line) survival for 8 week old salsa mice. 98.8 ± 2.5%, 98.9 ± 1.6% and 96.4 ± 4.5% of the OHCs and 100% of the IHCs survived at the 14, 18 and 25 kHz locations respectively. (f) An example of an 8–14 kHz organ of Corti wholemount from an 8 week old salsa mouse, stained with Myosin 7 A (red). The OHCs and IHCs are largely intact in this location (labelled).

Figure 3

(a) Effect of genotype, stimulated ear, and sympathectomy on cochlear blood flow. Relative cochlear blood flow change during and after ipsilateral loud sound exposure in wild-type (N = 6, blue circles, solid line) and salsa (N = 7, red circles, solid line) mice, and during and after contralateral loud sound exposure in wild-type (N = 6, blue circles, dashed line) and salsa (N = 6, red circles, dashed line) mice. MET channel function was a statistically significant factor in cochlear blood flow change between the ipsilateral groups (rmANOVA, F = 7.448, p = <0.01). Neither MET channel function (rmANOVA, F = 1.076, p = 0.381) nor time (rmANOVA, F = 1.148, p = 0.348) significantly affected contralateral cochlear blood flow. Wild-type mice with the anterior branches of the SCG transected (N = 5, blue circles, dotted line) show significantly reduced cochlear blood flow decline during loud sound exposure compared to intact wild-type mice (rmANOVA, F = 3.043, p = 0.02). There was no statistically significant effect on cutting anterior branches of the SCG of salsa mice (N = 5 red circles, dotted line) compared to intact salsa mice (rmANOVA, F = 0.841, p = 0.510). Loud sound exposure is indicated using the shaded area of the graph. (b) Examples, selected at random, of OCTA scans for wild-type (upper panels) and salsa mice (lower panels) before (left) and after 25 minutes (right) of sound exposure. (c) as in Fig. 3b but for wild-type (upper panels) and salsa (lower panels) after sectioning of the anterior branches of the SCG. A sound induced flow artifact representing organ of Corti vibration (black rectangle, upper left panel) is shown, and was excluded from the ROI. For Fig. 3b,c, black symbols denote the position of the individual blood flow value on the graph in Fig. 3a. The color bar denotes arbitrary 8 bit pixel intensity in false color. For all during sound images (3 b, c, right hand panels), white arrows indicate example areas of blood flow change relative to before sound (3 b, c, left hand panels).