19 th INTERNATIONAL CONGRESS ON ACOUSTICS MADRID, 2-7 SEPTEMBER 2007

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1 19 th INTERNATIONAL CONGRESS ON ACOUSTICS MADRID, 2-7 SEPTEMBER 2007 HEAT-STRESS ACTUATED PROTECTIVE MECHANISM OF THE INNER EAR AGAINST NOISE-INDUCED HEARING LOSS PACS: Nd Murakoshi, Michio 1 ; Kitsunai, Yoko 1 ; Yoshida, Naohiro 2 ; Iida, Koji 1 ; Kumano, Shun 1 ; Kobayashi, Toshimitsu 2 ; Wada, Hiroshi 1 1 Department of Bioengineering and Robotics, Tohoku University; Aoba-yama, Sendai , Japan; michio@wadalab.mech.tohoku.ac.jp 2 Department of Otolaryngology, Tohoku University Graduate School of Medicine, 1-1 Seiryomachi, Sendai , Japan ABSTRACT Noise-induced hearing loss (NIHL) is irreversible damage to the ear caused by traumatic noise exposure. This is mainly due to mechanical damage to outer hair cells (OHCs), a type of sensory cell in the inner ear. Recently, prior conditioning with sublethal stressors has been reported to protect the inner ear from acoustic injury, realizing less hearing loss. To explore the stress-actuated protective mechanism of the inner ear against NIHL, the mechanical properties and the amount of filamentous actin (F-actin) of mouse OHCs were investigated before and after conditioning with heat stress. In addition, expression of heat shock protein 27 (HSP27) in the cochlea, which affects the formation of F-actin, was investigated. Heat stress caused an increase in Young s modulus of OHCs at 3 6 h after its application along with an increase in their amount of F-actin. This time course is similar to that found in a previous study in which heat stress was shown to suppress permanent threshold shift. Heat stress was also found to increase HSP27 in the cochlea. These results suggest that heat stress induces HSP27 expression and thus increases F-actin in OHCs, increasing their stiffness, resulting in protection of the ear against NIHL. INTRODUCTION Outer hair cells (OHCs) of the mammalian cochlea are capable of altering their cell length in response to changes in membrane potential, termed electromotility [1]. Due to this electromotility, OHCs can exert force on the basilar membrane, which results in cochlear amplification (Fig. 1). As a result, hearing in mammals is characterized by high sensitivity, wide dynamic range and sharp frequency selectivity. Unfortunately, however, they are susceptible to external stimuli. Hence, if the ear is overexposed to sufficiently intense and/or prolonged sound, Figure 1.- Human auditory system. OHCs are located on the basilar membrane in the cochlea and subject the membrane to force, leading to cochlear amplification. The cytoskeleton of the OHC consists of parallelly arranged F-actin and spectrin which cross-links the adjacent actin filaments.

2 OHCs are markedly damaged and the cochlear amplification is lost, causing permanent noiseinduced hearing loss (NIHL). Recently, it has become clear that OHCs can be protected from traumatic exposure by prior sublethal conditioning, such as nontraumatic sound exposure, heat stress, ischemia and physical restraint [2]. Although previous studies have suggested that the OHCs are structurally and/or functionally modified due to conditioning, the mechanisms underlying such structural and functional changes and consequent conditioning-related cochlear protection remain unknown. To determine the effects of conditioning on the structure and function of OHCs and to explore possible mechanisms of conditioning-related cochlear protection, Young s modulus, which indicates the elasticity of materials and is a factor determining stiffness, and the amount of filamentous actin (F-actin), which is a primary component of the cell structure (Fig. 1), of OHCs were measured before and after heat stress by atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM), respectively. In addition, changes in heat shock protein 27 (HSP27) within the cochlea, which is a factor affecting the formation of F-actin, were examined before and after such stress by Western blotting. MATERIALS AND METHODS CBA/JNCrj strain male mice, aged weeks (25 30 g), were used. The animals were anesthetized and placed in an aluminum boat floating in a hot water bath (46.5 C) to raise their rectal temperature up to 41.5 C. It was maintained at that temperature for 15 min. The animals were then transferred from the boat to a heating pad to fully recover from the anesthesia before being returned to the animal care facility. To measure Young s modulus of OHCs, the cochleae were detached from the animals in tissue culture medium 3, 6, 12, 24 and 48 h after 15-min heat stress. OHCs were isolated by triturating the organ of Corti in another tissue culture medium. As shown in Fig. 2, an indentation test was then performed using an AFM (NVB100, Olympus) [3]. For investigation of F-actin of OHCs, the cochleae were detached 3, 6, 12, 24, 48 and 96 h after 15-min heat stress. The organ of Corti was dissected and fixed with 4% paraformaldehyde and then stained with 0.3 µm rhodamine-phalloidin (Fig. 3). The images of 16 OHCs obtained at the OHC lateral wall using a CLSM (FV500, Olympus) were subjected to intensity analysis. To measure the expression level of HSP27 in the cochlea, the cochleae were detached 6 h after 15-min heat stress. The cochleae were kept in an Eppendorf tube containing 30 µl of lysis buffer (1% Nonidet P-40 non-ionic detergent, 100 µg/ml of phenylmethylsulfonyl fluoride and a 1:50 dilution of proteases inhibitor) and homogenized by sonication in ice. After 1-h lysis, samples were centrifuged at 2,900 g at 4ºC for 10 min and the supernatant was used as the cochlear sample. HSP27 expression was then analyzed by Western blotting Figure 2.- A photomicrograph of an isolated mouse OHC and the AFM cantilever. Young s modulus of mouse OHCs was measured by an indentation test using the AFM. Scale bar is 20 µm. 2

3 Figure 3.- Fluorescence image of F-actin, stained with rhodamine-phalloidin, at the lateral wall of OHCs. Scale bar is 10 µm. RESULTS Changes in Young s modulus of OHCs As shown in Fig. 4, the mean and standard deviation of Young s modulus of OHCs in the control group (n = 10) and of those in the anesthesia + heat groups with 3-h (n = 13), 6-h (n = 5), 12-h (n = 8), 24-h (n = 7) and 48-h (n = 12) intervals were 2.1 ± 0.5 kpa, 2.8 ± 0.8 kpa, 2.9 ± 0.6 kpa, 2.7 ± 1.0 kpa, 2.0 ± 0.3 kpa and 2.4 ± 0.6 kpa, respectively. Statistical analysis indicated significant differences between the control group and anesthesia + heat groups with the two shortest intervals, i.e., 3-h and 6-h intervals (P < 0.05 by Student s t-test). Young s modulus of the mouse OHCs increased by 3 h after heat stress, reached a peak at 6 h, and then began to decrease 12 h after such stress, at which point it was still greater than that of the control group. Young s modulus returned to the pre-conditioning level by 24 h. Figure 4.- The mean and standard deviation of Young s moduli of OHCs in the control group (n = 10) and the anesthesia + heat groups with 3-h (n = 13), 6-h (n = 5), 12-h (n = 8), 24-h (n = 7) and 48-h (n = 12) intervals. Statistical analysis indicated significant differences between the control group and the anesthesia + heat groups with 3-h and 6-h intervals, as shown by asterisks (P < 0.05 by Student s t-test). Changes in the amount of F-actin of OHCs Figure 5 shows the mean intensities of F-actin labeling at the lateral wall of OHCs obtained for the control group (n = 13) and the anesthesia + heat groups with 3-h (n = 10), 6-h (n = 8), 12-h (n = 6), 24-h (n = 10), 48-h (n = 10) and 96-h (n = 8) intervals. The mean intensity of F-actin increased by 3 h after heat stress, reached a peak at 12 h and then returned to the preconditioning level by h. Since such intensity is proportional to the amount of a fluorescent substance specifically conjugated with the specimens when the preparation and the observation are performed under the same conditions, the intensity of F-actin labeling indicates the amount of F-actin. 3

4 Figure 5.- The mean and standard deviation of the normalized average intensities of F-actin labeling in the control group (n = 13) and the anesthesia + heat groups with 3-h (n = 10), 6-h (n = 8), 12-h (n = 6), 24-h (n = 10), 48-h (n = 10) and 96-h (n = 8) intervals. Statistical analysis indicated significant differences between the control group and the anesthesia + heat groups with intervals of 3 h, 6 h, 12 h and 24 h, as shown by asterisks (P < 0.05 by Student s t-test). Changes in the expression level of HSP27 in the cochlea Figure 6A shows an example of the result of HSP27 expression within the cochlea examined by Western blotting. A band is visible at around 27 kda. Figure 6B shows the mean and standard deviation of the normalized intensity of the HSP27 band obtained for the control group (n = 14) and the anesthesia + heat group with a 6-h interval (n = 14). The intensity of this band increased 1.53-fold by 6 h after heat stress. A significant difference was found between the control and anesthesia + heat groups (P < 0.05 by Student s t-test). Figure 6.- HSP27 expression within the cochlea in the control group and anesthesia + heat group with a 6-h interval. (A) An example of Western blotting. Bands were visible at around 27 kda. (B) The mean of the normalized intensity of the HSP27 band in the control group (n = 14) and the anesthesia + heat group with a 6-h interval (n = 14). The asterisk indicates a statistically significant difference between the control group and anesthesia + heat group (P < 0.05, Student s t-test). 4

5 DISCUSSION Previous conditioning with sublethal stressors protects the ear from subsequent traumatic noise exposure [2, 4]. To elucidate the mechanisms underlying such cochlear protection, it is useful to consider how the structure of the cochlear cells is modified due to such conditioning. However, there are relatively few reports regarding the effects of conditioning on the structure of the cochlear cells. For example, a previous morphological study found an increase in the vesicle content in the presynaptic region of the OHC after sound conditioning, suggesting that OHCs were indeed involved in conditioning-related modulation of the cochlea [5]. Avinash and co-workers [6] reported that F-actin, which is a primary component of the structural filaments of OHCs, decreased following conditioning noise. On the contrary, Hu and Henderson [7] demonstrated that the amount of F-actin increased after animals were exposed to conditioning noise. Although these reports are inconsistent with each other, these reported phenomena suggest that conditioning probably modifies the stiffness of OHCs. In the present study, to clarify these uncertainties, Young s modulus and the amount of F-actin of OHCs in the mouse cochlea were investigated in parallel before and after conditioning with heat stress. As shown in Fig. 5, the amount of F-actin increased by 12 h and started to decrease at 24 h. The increase in Young s modulus of OHCs at 3 6 h after heat stress and its decrease at h showed tendencies similar to those observed in F-actin labelling of the OHC lateral wall (Figs. 4 and 5). These results suggest that an increase of F-actin due to heat stress presumably leads to an increase of Young s modulus of OHCs since F-actin is a primary component of the cytoskeleton of OHCs which contributes the mechanical properties of the cells, rendering the cells stiffer, thus resulting in protection of mammalian hearing from subsequent traumatic exposure. Although the exact mechanism of the increase of F-actin due to heat stress is unclear, the regulation of heat shock proteins (HSPs) is a likely candidate as a source of such increase. Among such HSPs, HSP27 is known as a regulator of F-actin polymerization, which acts as a barbed-end F-actin capping protein and inhibits actin polymerization. However, heat stress induces rapid phosphorylation of pre-existing HSP27 and leads to a loss of its actin capping activity [8], resulting in an acceleration of actin polymerization. As shown in Fig. 6, the expression level of HSP27 in the cochlea was confirmed to increase 6 h after heat stress. The expression and localization of HSP27 have been previously confirmed at the cuticular plate and the lateral walls of OHCs [9]. In the present study, therefore, the polymerization of F-actin in OHCs may possibly have been accelerated by increasing the expression level of HSP27 after conditioning with heat stress and caused the amount of F-actin in OHCs to increase, thereby leading to an increase in Young s modulus of OHCs. CONCLUSIONS Conditioning with heat stress caused an increase in Young s modulus of mouse outer hair cells (OHCs) at 3 6 h after heat stress. This factor returned to the pre-conditioning level by h after heat stress. The increase and decrease in Young s modulus of the OHCs showed tendencies similar to those in the amount of their filamentous actin (F-actin). The conditioning was also found to increase heat shock protein 27 (HSP27) in the cochlea. These results suggest that an increase in HSP27 due to heat stress may possibly lead to an increase in F- actin of OHCs, increasing their stiffness, thus resulting in protection of the ear against noiseinduced hearing loss. References: [1] W. E. Brownell, C. R. Bader, D. Bertrand, Y. de Ribaupierre: Evoked mechanical responses of isolated cochlear outer hair cells. Science 227 (1985) [2] N. Yoshida, A. Kristiansen, M. C. Liberman: Heat stress and protection from permanent acoustic injury in mice. Journal of Neuroscience 19 (1999) [3] M. Murakoshi, N. Yoshida, K. Iida, S. Kumano, T. Kobayashi, H. Wada: Local mechanical properties of mouse outer hair cells: atomic force microscopic study. Auris Nasus Larynx 33 (2006) [4] B. Canlon, E. Borg, A. Flock: Protection against noise trauma by pre-exposure to a low level acoustic stimulus. Hearing Research 34 (1998) [5] B. Canlon, P. Lofstrand, E. Borg: Ultrastructural changes in the presynaptic region of outer hair cells after acoustic stimulation. Neuroscience Letters 150 (1993)

6 [6] G. B. Avinash, A. L. Nuttall, Y. Raphael: 3-D analysis of F-actin in stereocilia of cochlear hair cells after loud noise exposure. Hearing Research 67 (1993) [7] B. H. Hu, D. Henderson: Changes in F-actin labeling in the outer hair cell and the Deiters cell in the chinchilla cochlea following noise exposure. Hearing Research 110 (1997) [8] J. I. Clark, P. J. Muchowski: Small heat-shock proteins and their potential role in human disease. Current Opinion in Structural Biology 10 (2000) [9] E. V. Leonova, D. A. Fairfield, M. I. Lomax, R. A. Altschuler: Constitutive expression of Hsp27 in the rat cochlea. Hearing Research 163 (2002)