untitled

Size: px

Start display at page:

Download "untitled"

歪悟
5 years ago
Views:

1 Acta Physiologica Sinica, February 25, 2007, 59 (1): * (1) (NS, 10 µl) NS (10 µl) + (kynurenic acid, KYNA, 5 mmol/l, 10 µl) + 15 NS KYNA 110 db SPL 1 h (auditory brainstem response, ABR) (compound action potential, CAP) N1 (2) NS + ABR CAP KYNA + N1 NS + KYNA + / KYNA R764 Role of glutamate receptors in the spiral ganglion neuron damage induced by acoustic noise ZHANG Yan-Min, MA Bei *, GAO Wen-Yuan, WEN Wen, LIU Hai-Ying Department of Physiology, the Second Military Medical University, Shanghai , China Abstract: The aim of the present study was to investigate the role of glutamate receptors in the damage of spiral ganglion neurons (SGNs) induced by acute acoustic noise. This investigation included in vivo and in vitro studies. In vivo, kynurenic acid (KYNA), a broad-spectrum antagonist of glutamate receptors, was applied to the round window of guinea pigs, and its protective effect was observed. The animals were divided into three groups: control (saline, 0.9%, 10 µl), saline (0.9%, 10 µl) + noise and KYNA (5 mmol/l, 10 µl) + noise. Saline and KYNA were applied to the round window membrane with a microsyringe. The animals were exposed to 110 db SPL of white noise for 1 h. Hearing thresholds for auditory brainstem responses (ABRs) and compound action potentials (CAPs) in all animals were measured before and after treatment. The amplitudes of III waveform of ABR and N1 waveform of CAP and the latency of N1 waveform at different stimulation levels (intensity-amplitude and intensity-latency functions) were also measured. The cochleas were then dissected for transmission electron microscopy (TEM) after final electrophysiological measurement. In vitro, the SGNs of the normal guinea pigs were isolated and glutamate (100 µmol/l or µmol/l) was added into the medium. The morphology of the SGNs was examined by light microscopy. In vivo results showed that the hearing function and morphology of the inner ear including hair cells and SGNs in the control group were normal. Compared with that in the control group the thresholds for ABR and CAP (click and tone burst) in saline + noise group were elevated significantly. The input-output functions showed that the amplitudes of III waveform of ABR and N1 waveform of CAP decreased and the latency of N1 waveform increased obviously. There was significant difference in the amplitude and latency between saline + noise group and KYNA + noise group (P<0.05). TEM indicated obvious swelling and vacuoles Received Accepted This work was supported by the National Natural Science Foundation of China (No ). * Corresponding author. Tel: ; Fax: ; mabei2004@yahoo.com.cn

2 104 Acta Physiologica Sinica, February 25, 2007, 59(1): at the terminate of dendrites of SGNs in NS + noise group. On the contrary, the afferent dendrites in KYNA + noise group showed normal appearance without swelling and vacuoles. In vitro experiment showed that the isolated SGNs of guinea pigs obviously swelled and even died after application of 100 µmol/l or µmol/l glutamate. These results suggest that noise exposure causes hearing impairment, damage of hair cells and hair cell/afferent synapse and death of SGNs. The antagonist of glutamate receptors provides protective effects against hearing loss and SGN damage. It is inferred that excessive release of glutamate from the inner hair cells induced by noise may be responsible for these damages. Glutamate receptors are involved in the degeneration and death of SGNs. Key words: spiral ganglion neuron; hearing loss; glutamate; glutamate receptors; kynurenic acid (noise-induced hearing loss, NIHL) - (spiral ganglion neurons, SGNs) [1,2] SGNs SGNs ( / ) 40~60 nm [3-5] AMPA kainate NMDA [6-9] AMPA Na + Cl AMPA NMDA Ca 2+ Ca 2+ [10-13] 135 db 30 min 1.5 [14] 115 db 60 min 47% SGNs ( ) [15] ; SGNs SGNs SGNs [16,17] ( ) [18] SGNs [19] (115 db, 5 h) NMDA SGNs (auditory brainstem response, ABR) [20] (kynurenic acid, KYNA) NMDA AMPA KYNA [3] KYNA [ABR (compound action potential, CAP) - ] SGNs SGNs KYNA A Sigma

3 105 Hanks Gibco ~300 g (NS, 0.9%, 10 µl) NS (0.9%, 10 µl) + KYNA (5 mmol/l, 10 µl) Khan [21] 2% (40 mg/kg, i.p.) (38±0.5) 10 min (120 cm 60 cm 60 cm) 40 db SPL (B&K 1405) (FJG600-1) 4145 B&K db ±1 db 4 khz 110 db SPL 1 h ABR Spirit 2000 (Nicolet ) ABR 2% (40 mg/ kg ) (click) (tone burst)( khz) THD-39P Click 0.1 ms 11.1 /s 0.1 ms 10 ms 100~3 000 Hz 128 ABR ABR CAP click ( khz) CAP N1 CAP N1 CAP CAP (N1 latency) 1.5 1% 2% 1:1 4 Epon Å 1.6 SGNs 20% Hanks 0.1 mg/ml 10 min Hanks A 1.7 means±sem EXCEL t P< KYNA ABR CAP NS NS + click ( khz) ABR 30~39.5 db CAP 29~36.5 db KYNA + ABR 7.5~11 db CAP 6.5~13.5 db NS + ABR CAP NS KYNA + (P<0.05)( 1) NS NS ABR CAP / SGNs 2.2 KYNA ABR CAP NS click ABR CAP N1 NS db click ABR CAP N1 - KYNA + click ABR CAP N1

4 106 Acta Physiologica Sinica, February 25, 2007, 59(1): KYNA Fig. 1. Effect of KYNA on noise-induced threshold shifts of the auditory brainstem response (A) and compound action potential (B). * P<0.05 vs NS group and KYNA + noise group. 2. KYNA Fig. 2. Effect of KYNA on noise-induced amplitudes of the auditory brainstem response (A) and compound action potential (B). * P<0.05 vs KYNA + noise group. NS + (P< 0.05)( 2) 2.3 KYNA CAP N1 NS CAP N1 0.8~1.6 ms NS db click N1 - KYNA + click CAP N1 NS + (P<0.05) (P>0.05)( 3) 2.4 KYNA / / NS (outer hair cells, OHCs) / 3. KYNA N1 Fig. 3. Effect of KYNA on noise-induced N1 latency of compound action potential. * P<0.05 vs KYNA + noise group. ( 4) NS + OHCs /

5 / Fig. 4. Transmission electron microscopy pictures of one outer hair cell and the hair cell/afferent synapse in the saline group. A: The outer hair cell displayed normal appearance and structure. Scale bar, 5 µm. B: The hair cell/afferent synapse showed normal structure without swelling and intracellular vacuoles. Scale bar, 2.5 µm. 5. NS + / Fig. 5. Transmission electron microscopy pictures of the outer hair cell and the hair cell/afferent synapse in NS + noise group. A: The outer hair cell showed swelling with small vacuoles beneath the membrane (arrows). Scale bar, 2.5 µm. B: A large vacuole appeared in the cytoplasm of one outer hair cell. Scale bar, 2 µm. C: Some relative large vacuoles in the afferent dendrites. Scale bar, 2.5 µm. D: The afferent dendrites had a severe swelling appearance. Scale bar, 2.5 µm. E: The mitochondria in the afferent dendrites showed edema with vacuoles inside. Scale bar, 0.5 µm. ( 5) KYNA + OHCs / NS ( 6) 2.5 SGNs SGNs 5~6 h 100 µmol/l µmol/l 1 SGNs SGNs ( 7)

6 108 Acta Physiologica Sinica, February 25, 2007, 59(1): KYNA + / Fig. 6. Transmission electron microscopy pictures of the outer hair cells and the hair cell/afferent synapse in KYNA + noise group. A, B: The outer hair cells displayed normal appearance without swelling and vacuoles inside. The hair cell/afferent synapse showed normal structure. Scale bar, 5 µm and 2.5 µm, respectively. C: There was no swelling and vacuoles in the dendrites and the mitochondria had normal structure. Scale bar, 0.4 µm. 7. Fig.7. The toxic effect of glutamate on the isolated spiral ganglion neurons of guinea pigs. A: The isolated spiral ganglion neurons in the normal medium showed normal appearance. Scale bar, 25 µm. B: Spiral ganglion neurons showed swelling and vacuoles 1 h after application of glutamate in the medium. Scale bar, 25 µm. C: Degeneration and death of spiral ganglion neurons after application of glutamate. Scale bar, 25 µm. 3 SGNs [22] SGNs (1) SGNs - SGNs [2] (2) SGNs [23]

7 109 [24,25] SGNs NS + click ( khz) ABR 30~39.5 db CAP 29~36.5 db ABR CAP KYNA ABR 7.5~ 11 db CAP 6.5~13.5 db NS + (P<0.05) KYNA NIHL NIHL CAP CAP N1 / N1 (0.5 ms) N1 0.8~1.6 ms NS + click N1 / AMPA AMPA Na + Cl KYNA + click N1 / KYNA SGNs CAP - SGNs [26] NS db click CAP N1 ABR SGNs SGNs KYNA + click CAP N1 NS + KYNA SGNs SGNs NS + OHCs KYNA KYNA + OHCs OHCs OHCs OHCs KYNA SGNs SGNs 20 h 100 µmol/l µmol/l SGNs SGNs SGNs (1 µmol/l, 10 µmol/l) ( )

8 110 Acta Physiologica Sinica, February 25, 2007, 59(1): ABR CAP / SGNs SGNs KYNA SGNs 1 Kotarbinska E. Hearing protectors in prevention of occupational hearing loss. Med Pr 2000; 51(2): Chertoff ME, Yi X, Lichtenhan JT. Influence of hearing sensitivity on mechano-electric transduction. J Acoust Soc Am 2003; 114 (6 Pt 1): Eybalin M. Neurotransmitters and neuromodulators of the mammalian cochlea. Physiol Rev 1993; 73(2): Puel JL. Chemical synaptic transmission in the cochlea. Prog Neurobiol 1995; 47(6): Glowatzki E, Fuchs PA. Transmitter release at the hair cell ribbon synapse. Nat Neurosci 2002; 5(2): Ryan AF, Brumm D, Kraft M. Occurrence and distribution of non-nmda glutamate receptor mrnas in the cochlea. Neuroreport 1991; 2(11): Niedzielski AS, Wenthold RJ. Expression of AMPA, kainate, and NMDA receptor subunits in cochlear and vestibular ganglia. J Neurosci 1995; 15(3 Pt 2): Usami S, Matsubara A, Fujita S, Shinkawa H, Hayashi M. NMDA (NMDAR1) and AMPA-type (GluR2/3) receptor subunits are expressed in the inner ear. Neuroreport 1995; 6 (8): Matsubara A, Laake JH, Davanger S, Usami S, Ottersen OP. Organization of AMPA receptor subunits at a glutamate synapse: a quantitative immunogold analysis of hair cell synapses in the rat organ of Corti. J Neurosci 1996; 15; 16(14): Koch RA, Barish ME. Perturbation of intracellular calcium and hydrogen ion regulation in cultured mouse hippocampal neurons by reduction of the sodium ion concentration gradient. J Neurosci 1994; 14(5 Pt 1): Doble A. The role of excitotoxicity in neurodegenerative disease: implications for therapy. Pharmacol Ther 1999; 81 (3): Diao MF ( ), Gao WY. The effect of NO in noiseinduced hearing loss: research progress. Chin J Ophthalmol Otolaryngol ( ) 2004; 4(3): (Chinese, English abstract). 13 Diao MF ( ), Liu HY, Zhang YM, Gao WY. Changes in antioxidant capacity of the guinea pig exposed to noise and the protective effect of α-lipoic acid against acoustic trauma. Acta Physiol Sin ( ) 2003; 55(6): (Chinese, English abstract). 14 Jager W, Goiny M, Herrera-Marschitz M, Brundin L, Fransson A. Noise-induced aspartate and glutamate efflux in the guinea pig cochlea and hearing loss. Exp Brain Res 2000; 134(4): Gao WY, Jiang YP. Effect of noise on concentration of amino acid in perilymph of guinea pig cochlea. Acad J Sec Mil Med Univ ( ) 2002; 23(1): Zhang L ( ), Wang XP, Song CR, Song MH, Jiang J. Effect of adenosine on perilymph glutamate concentration and compound action potentials in guinea pigs exposed to noise. Acad J Sec Mil Med Univ ( ) 2005, 26(2): (Chinese, English abstract). 17 Zhang L, Gao WY. Protective effect of adenosine on temporary threshold shift of audition caused by intensive white noise in guinea pigs. Acad J Sec Mil Med Univ ( ) 2001; 22(7): Hakuba N, Koga K, Gyo K, Usami SI, Tanaka K. Exacerbation of noise-induced hearing loss in mice lacking the glutamate transporter GLAST. J Neurosci 2000; 20(23): Chen GD, Kong J, Reinhard K, Fechter LD. NMDA receptor blockage protects against permanent noise-induced hearing loss but not its potentiation by carbon monoxide. Hear Res 2001; 154(1-2): Diao MF ( ), Zhang YM,Liu HY, Han H,Gao WY. Observation on the protective effect of MK-801 against hearing loss in acoustic trauma. J Clin Otorhinolaryngol ( ) 2005; 19(1): (Chinese, English abstract). 21 Khan MJ, Seidman MD, Quirk WS, Shivapuja BG. Effects of kynurenic acid as a glutamate receptor antagonist in the guinea pig. Eur Arch Otorhinolaryngol 2000; 257(4): Beutner D, Moser T. The presynaptic function of mouse cochlear inner hair cells during development of hearing. J Neurosci 2001; 21(13): Seidman MD, Quirk WS, Shirwany NA. Mechanism of alteration in the microcirculation of the cochlea. Ann NY Acad Sci 1999; 884: Jager W, Goiny M, Herrera-Marschitz M, Brundin L, Fransson A, Canlon B. Noise-induced aspartate and glutamate efflux in the guinea pig cochlea and hearing loss. Exp Brain Res 2000; 134(4): Pujol R, Puel JL. Excitotoxicity, synaptic repair, and functional recovery in the mammalian cochlea: a review of recent findings. Ann NY Acad Sci 1999; 884: Gao WY ( ), Chi FL, He BK. Clinical Auditory Physiology. Beijing: People s Military Medical Press, 2004, (Chinese).

21 13 D / 2 10 ig ig 10 d 1. 0 g kg /2 2 β- 5 g kg g kg mg kg - 1 ig ig 24 h 5% 1 2% CMC-Na 2. 5 ml 30

21 13 D / 2 10 ig ig 10 d 1. 0 g kg /2 2 β- 5 g kg g kg mg kg - 1 ig ig 24 h 5% 1 2% CMC-Na 2. 5 ml 30 21 13 1 2 1* 2 1. 430065 2. 445000 20 10 5 g kg - 1 5 mg kg - 1 ig 10 d ig HE 1 TFF1 TFF1 P < 0. 01 P < 0. 01 P < 0. 01 P < 0. 05 R285. 5 doi 10. 13422 /j. cnki. syfjx. 2015130100 A 1005-9903 2015 13-0100-05