G Model

ARTICLE IN PRESS

NSR 3723 1–6

Neuroscience Research xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Neuroscience Research journal homepage: www.elsevier.com/locate/neures

Inhaled hydrogen gas therapy for prevention of noise-induced hearing loss through reducing reactive oxygen species

1

2

3 4 5 6

Q1

Takaomi Kurioka a , Takeshi Matsunobu a,∗ , Yasushi Satoh b , Katsuki Niwa a , Akihiro Shiotani a a b

Department of Otolaryngology, National Defense Medical College, Saitama, Japan Department of Anesthesiology, National Defense Medical College, Saitama, Japan

7

8 20

a r t i c l e

i n f o

a b s t r a c t

9 10 11 12 13 14

Article history: Received 18 March 2014 Received in revised form 25 July 2014 Accepted 22 August 2014 Available online xxx

15

19

Keywords: Hydrogen Noise-induced hearing loss (NIHL) Reactive oxygen species (ROS)

21

1. Introduction

16 17 18

22 23 24 25 26 27 28 29 30 31 32 33

Reactive oxygen species (ROS) that form in the inner ear play an important role in noise-induced hearing loss (NIHL). Recent studies have revealed that molecular hydrogen (H2 ) has great potential for reducing ROS. In this study, we examined the potential of hydrogen gas to protect against NIHL. We tested this hypothesis in guinea pigs with 0.5%, 1.0% and 1.5% H2 inhalation in air for 5 h a day after noise exposure, for five consecutive days. All animals underwent measurements for auditory brainstem response after the noise exposure; the results revealed that there was a better improvement in the threshold shift for the 1.0% and 1.5% H2 -treated groups than the non-treated group. Furthermore, outer hair cell (OHC) loss was examined 7 days after noise exposure. A significantly higher survival rate of OHCs was observed in the 1.0% and 1.5% H2 -treated group as compared to that of the non-treated group in the basal turn. Immunohistochemical analyses for 8-hydroxy-2 -deoxyguanosine (8-OHdG) were performed to examine the amount of oxidative DNA damage. While strong immunoreactivities against 8-OHdG were observed of the non-treated group, the H2 -treated group showed decreased immunoreactivity for 8-OHdG. These findings strongly suggest that inhaled hydrogen gas protects against NIHL. © 2014 Published by Elsevier Ireland Ltd.

Recent studies have revealed that molecular hydrogen (H2 ) mediates beneficial effects in different systems as an optimal antioxidant agent by selectively scavenging free hydroxyl radicals (OH) (Ohsawa et al., 2007). Hydrogen gas reduces OH to H2 O, thereby preventing lipid peroxidation, DNA oxidation, thiyl radical formation, and mitochondrial depolarization that contribute to cellular apoptosis and necrosis (Ohsawa et al., 2007). In some fields, clinical application of hydrogen is already carried out and has been shown to improve lipid and glucose metabolism in type 2 diabetes patients (Kajiyama et al., 2008). Hydrogen gas is permeable to cell membranes and can target organelles, including mitochondria and nuclei. This is especially favorable in otolaryngology, because many

Abbreviations: ABR, auditory brainstem response; NIHL, noise-induced hearing loss; OHC, outer hair cell; ROS, reactive oxygen species; SE, standard error; SPL, sound pressure level; OH, hydroxyl radical; H2 , molecular hydrogen; PBS, phosphate-buffered saline; 8-OHdG, 8-hydroxy-2 -deoxyguanosine. ∗ Corresponding author at: Department of Otolaryngology – Head and Neck Surgery, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 3598513, Japan. Tel.: +81 4 2995 1686; fax: +81 4 2996 5212. E-mail address: [email protected] (T. Matsunobu).

therapeutic compounds are blocked by the blood-labyrinth barrier and cannot gain access to the inner ear (Coimbra et al., 2007; Laurell et al., 2008). There are several methods of hydrogen intake or consumption, including inhaling hydrogen gas or drinking water dissolved with hydrogen (hydrogen water). However, the amount of orally administered H2 may not be enough to scavenge hydroxyl radicals (Le Prell et al., 2007). Considering the safety and easy accessibility of hydrogen gas to the inner ear cells, hydrogen gas seems a promising agent to investigate for the potential prevention of oxidative stress. Hearing loss due to noise exposure is a common form of sensorineural hearing loss in industrialized countries, and noiseinduced hearing loss (NIHL) caused by excessive exposure to either acute or chronic acoustic trauma is preventable. Although the mechanisms underlying the phenomenon are not fully understood, it is widely accepted that acoustic over-stimulation of the cochlea generates the production or formation of free radicals, including ROS, which may induce outer hair cell (OHC) death (Coleman et al., 2010). ROS are short-lived, unstable, highly reactive clusters of atoms. They are essential for cellular life processes; however, when in excess, they damage cellular lipids, proteins, and DNA. In this study, we tested the ability of inhaled hydrogen gas to attenuate NIHL and decrease oxidative stress by scavenging ROS,

http://dx.doi.org/10.1016/j.neures.2014.08.009 0168-0102/© 2014 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Kurioka, T., et al., Inhaled hydrogen gas therapy for prevention of noise-induced hearing loss through reducing reactive oxygen species. Neurosci. Res. (2014), http://dx.doi.org/10.1016/j.neures.2014.08.009

34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

G Model

ARTICLE IN PRESS

NSR 3723 1–6

T. Kurioka et al. / Neuroscience Research xxx (2014) xxx–xxx

2

Day0

Day1 Day2 Day3

Day7

Day4

Inhaled Hydrogen Gas , 5h 1.5 % , 1.0 % , 0.5 % Cell count

ABR

ABR

ABR

ABR

ABR

ABR Noise Exposure 121dB , 5h Fig. 1. Schedule of the experiment procedures.

57 58 59

as well as protect cochlear cells and tissues against oxidative stress as assessed by fluorescent immunohistochemistry for free radical formation.

60

2. Materials and methods

61

2.1. Animals

69

Female Hartley guinea pigs weighing 300–350 g (Japan SLC, Inc., Shizuoka) with normal Preyer’s reflex were used in this study. In addition to free access to water, the animals were given a regular diet. All experimental protocols were performed in accordance with the guidelines for animal experiments of the National Defense Medical College and the law and notification of the government of Japan. All animals were randomly divided into five groups: normal, non-treated, 0.5% H2 -treated, 1.0% H2 -treated and 1.5% H2 -treated.

70

2.2. Noise exposure

62 63 64 65 66 67 68

80

Guinea pigs were anesthetized by an intraperitoneal (i.p.) injection of ketamine (50 mg/kg) and medetomidine (1.0 mg/kg). They were exposed to one octave band noise centered at 4 kHz at 121 dB SPL for 5 h in a ventilated sound exposure chamber. The sound chamber was fitted with speakers (Model 2380A; JBL, Northridge, CA, USA) driven by a noise generator (DANAC-31; Dana Japan, Tokyo, Japan) and power amplifier (D-45; Crown International, Elkhart, IN, USA). Sound levels were calibrated (Type 6224 precision sound level meter; Aco Instruments, Japan) at multiple locations within the sound chamber to ensure uniformity of the stimulus.

81

2.3. Auditory brainstem response

71 72 73 74 75 76 77 78 79

produced a reliable peak III or IV. Peak III is the manifestation of neural activity in the cochlear nucleus, and the anatomical location of the neural generator of peak IV is probably the superior olivary complex. Thresholds obtained immediately before noise exposure were used as the baseline thresholds for estimating noise-induced threshold shifts. All data values for the ABR threshold shift in the text and in the figures are presented as mean + standard error (SE). 2.4. Inhaled hydrogen gas treatment After noise exposure, animals in the H2 -treated group were put in a sealed Plexiglas chamber with inflow and outflow outlets for 5 h a day over five consecutive days (Fig. 1). In the chamber, H2 (0.5%, 1.0%, 1.5%) in air (PLM-30493; Saisan, Saitama, Japan) was supplied through a gas flowmeter at total rate of 2 L/min. The concentration of H2 in the chamber was monitored with the Breath Gas Analyzer (BGA-1000D; Breath Lab, Nara, Japan) and maintained during the treatment. The animals without H2 treatment were only exposed to room air in the chamber. The room and chamber temperature was maintained at 20 ◦ C–24 ◦ C. 2.5. Quantitative assessment of hair cell loss Thirty guinea pigs were divided into either the 0.5% H2 -treated group (n = 6), 1.0% H2 -treated group (n = 6), 1.5% H2 -treated group (n = 6) or non-treated control group (n = 6) were decapitated 1 week after noise exposure (Fig. 1). The normal group (n = 6) that was neither exposed to noise nor inhaled hydrogen gas treatment was also examined. The bone near the apex was removed and the round and oval windows of the inner ear were opened, followed by a gentle local perfusion with 2× 1 mL 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4); the tissues were preserved overnight in the fixative. The cochleae were dissected by removing the lateral wall bones, lateral wall tissues, and tectorial membranes. After several washings with PBS, the remaining parts of the cochleae were incubated in 0.3% Triton X-100 in PBS for 5 min and washed three times with PBS. The organ of Corti was stained for Factin with 1% rhodamine-phalloidin (Invitrogen, Carlsbad, CA, USA) for 60 min to outline hair cells and their stereocilia for quantitative assessment. After several washings with PBS, the organ of Corti was dissected and mounted for surface preparation. The tissues were observed under confocal fluorescence microscopy using the Nikon C1 system (Nikon, Tokyo, Japan) and fluorescence microscope (Axio Imager A1; Carl Zeiss MicroImaging Gmbh, Jena, Germany), and the numbers of missing OHCs and inner hair cells (IHCs) in the basal, middle, and apical turns of the cochleae were counted. The ratio of missing-to-whole OHCs and IHCs were expressed as a percentage. 2.6. Immunohistochemistry for 8-OHdG

82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98

ABR was measured using a single recorder (Synax 1200; NEC, Tokyo, Japan) before and immediately after noise exposure, and then 2, 4, and 7 days after the noise exposure (Fig. 1). Following anesthesia, stainless steel needle electrodes were placed subcutaneously at the vertex and ventrolateral to the left and right ears for recording. Tone burst stimuli of 1 ms rise/fall time at frequencies of 4, 8, 12, 16, and 20 kHz were generated; the amplitudes were specified by the sound generator and attenuated by a real-time processor and programmable attenuator (RP2.1 and PA5; TuckerDavis Technologies, Alachua, FL, USA). The sound stimuli were produced by a coupler-type speaker (ES1spc; Bio Research Center, Nagoya). ABR waveforms were recorded for 12.8 ms at a sampling rate of 40,000 Hz using 50–5000 Hz bandpass filter settings; waveforms from 256 stimuli at a frequency of 9 Hz were averaged. ABR waveforms were recorded in 10 dB SPL intervals down from the maximum amplitude until no waveform could be visualized. ABR threshold was defined as the lowest stimulus intensity that

The expression of 8-hydroxy-2 -deoxyguanosine (8-OHdG) was observed in the cochlea of twelve guinea pigs. 8-OHdG was one of the predominant forms of free radical-induced lesions of DNA and used as an indication of free-radical formation in the inner ear. Ten animals received noise exposure, and immediately after the noise exposure, five animals received inhaled hydrogen gas therapy for 5 h (1.5% H2 -treated group), while the other five animals were not given any treatment (non-treated group). ABR thresholds were measured before noise exposure, and the auditory thresholds were equivalent in all groups. All animals were deeply anesthetized and killed immediately after the 5-h of noise exposure. The bone near the apex was removed and both the round and oval windows were opened, followed by gentle local perfusion with 2× 1 mL of 4% paraformaldehyde. The tissues were preserved overnight in the fixative. The cochleae were dissected by removing the lateral wall bone, lateral wall tissues, and the tectorial membranes.

Please cite this article in press as: Kurioka, T., et al., Inhaled hydrogen gas therapy for prevention of noise-induced hearing loss through reducing reactive oxygen species. Neurosci. Res. (2014), http://dx.doi.org/10.1016/j.neures.2014.08.009

99 100 101 102 103 104 105

106

107 108 109 110 111 112 113 114 115 116

117

118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141

142

143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158

G Model NSR 3723 1–6

ARTICLE IN PRESS

172 173 174 175 176 177 178 179

184

All data are presented as means + SE. ABR threshold shifts and missing OHC rates were evaluated using the Mann–Whitney U-test. A p value

Inhaled hydrogen gas therapy for prevention of noise-induced hearing loss through reducing reactive oxygen species.

Reactive oxygen species (ROS) that form in the inner ear play an important role in noise-induced hearing loss (NIHL). Recent studies have revealed tha...
1MB Sizes 2 Downloads 4 Views