急性錳中毒對小鼠的耳毒性

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(1)Acute Manganism Induces Ototoxicity of Mice. Yu-Pu Han. Chung-Hsin Wu, Ph.D..

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(5) ..…………………………………………………………..…...4 ……………………………………………………..………….5 1 (1.1) (1.2) (1.3). ………..………………...……….........7 ……………….………...………………......9 …...………...………………....9 ………………………………………………………………12. 2 3 (3.1) (3.2) (3.3) (3.4) (3.5) (3.6) (3.7) (3.8) (3.9) (3.10). ……………………………………………...………….13 ……………………………………………...………….13 ………………………………………………...……….13 ……………………………………………...………….14 …………………………………………...…………….14 ……………….…………...……15 (ROS) …………………….…..……………...16 ………………………………………...……….16 ……...……………………………………...……..17 ………………………………………...………...18. 4 (4.1) (4.2) (4.3) (4.4) 5 6 7 8. !. ……………….…...…19 ………………….......….20 ………………….…...20 prestin c-caspase 3 ……………..…20 ……………………………………………………………………22 ……………………………………………………………………27 ………………………………………………………………28 ………………………………………………………………39. 3!.

(6) 2011. Ding. ICR (ABR ) (10 /. /. /. /. 5 ). (50. ) ICR. ICR prestin. cleaved caspase-3 ICR. ABR. ICR prestin. ICR ICR cleaved caspase-3. !. 4!.

(7) Abstract In vitro, overdose of manganese is toxic to auditory nerve fibers as well as spiral ganglion neurons and cochlear hair cells. In 2011, Ding and his colleagues published a study in Neurotoxicology showing for the first time that low levels of manganese initially damage neurons and sensory hair cells in the inner ears of rats. To understand the mechanism for manganese-induced auditory impairment, we assessed the degree of hearing impairment and the cochlear histopathological damage in ICR mouse model resulting from various levels of manganese. The auditory brainstem response (ABR) recording was used to determine and compare the auditory threshold in ICR mice after 5 times subcutaneous injection of saline, low (10 mg/kg/day) or high dose (50 mg/kg/day) of manganese chloride. Immunochemistry staining techniques were used to compare expression of prestin, a cochlear motor protein, and cleaved caspase-3, a apoptosis protein, in the cochlea of ICR mice. Our results show that the ABR thresholds of ICR mice were significantly elevated with low and high dose of manganese treatments. Although the difference of prestin expression in outer hair cells was not observed among ICR mice with subcutaneous injection of saline, low and high dose of manganese. The apoptosis-related cleaved caspase-3 was obviously observed in outer hair cells, spiral ganglion neurons, and stria vascularis cells in the cochlea of ICR mice with subcutaneous injection low and high dose of manganese. !. 5!.

(8) Thus we suggest that the cochlear apoptosis may be involved in manganese-induced auditory impairment.. Keywords: manganism, neurotoxicity, cochlea, auditory impairment, hair cells, spiral ganglion, stria vascularis.. !. 6!.

(9) 1 (1.1) (manganese, Mn). glutamine synthetase (GS). mitochondrial superoxide dismutase. pyruvate carboxylase. (McCord, 1976; Wedler et al., 1984; Gonzalez-Zulueta et al., 1998; Keen et al., 2000). 3~5%. (Von, 1907; Cotzias, 1958). (Keen et al., 2000; Myers et al., 2003; Myers et al, 2009; Park et al., 2009) (Yang et al., 2012) methylcyclopentadienyl manganese tricarbonyl (MMT) !. 7!.

(10) 18 mg/L 0.05-0.5 µg/m3. (. MMT)(Yin et al., 2010). (Rose et al., 1999; Katsoyiannis et al., 2006; Bouchard et al., 2007) (Manganism). (Von, 1907; Cotzias, 1958; Barbeau, 1984) 1837. Glascow. Couper (Couper, 1837) (Gupta et al., 1980) (Yamada et al., 1986; Pal et al.,. 1999). (Olanow et al.,. 1996). (dopamine) (norepinephrine). !. 8!.

(11) (Ding et al., 2011; Ma et al., 2008). (1.2). (Ding et al., 2011). (1.3) (hearing loss). (basilar membrane) (tectorial membrane). (outer hair cells, OHCs) (electromotility) !. 9!.

(12) prestin. (Zheng et. al., 2000) prestin. prestin (Peter et al., 2008). (Uy et al., 2013). prestin. prestin. (Liberman et al., 2002; Dallos et al., 2008) (Rajagopalan et al., 2007; Rajagopalan et al., 2009; Sfondouris et al., 2008). (Oliver et. al., 2001; Santos-Sacchi et al., 2006; Schaechinger et al., 2007) prestin prestin. prestin (Cheatham et al.,. 2009). (mitochondria). (Gold et al., 1994) !. 10!.

(13) (Hutchin et al., 2000). (Degoul. et al., 1991; Goto et al., 1992) ATP (Seidman et al., 1999). !. 11!.

(14) 2 (MnCl2). (Ding et al., 2011) MnCl2. !. 12!.

(15) 3 (3.1) ICR (. ). 25 C. ICR. 12 h/12 h. (3.2). (Subcutaneous injection, SC). ABR. (3.3) (3.3.1)0.9%. (Isotonic Sodium Chloride Solution, Saline) 4DH2433. (3.3.2). .4H2O (Manganese(II) Chloride Tetrahydrate). Sigma-Aldrich CAS No. : 13446-34-9 !. 13!. SI-M3635-100G.

(16) 0.5%. (3.4) (3.4.1). ICR 5 ml/kg. (Subcutaneous. injection, SC) (3.4.2). ICR 10 mg/kg. (3.4.3). :. ICR 50 mg/kg. (3.5) ABR St. Louis, Mo, U.S.A.). Atropine (Sigma, 0.5 mg/kg. (Intramuscular injection, IM). 20. Urethane(Sigma, St. Louis, Mo, U.S.A.) (intraperitoneal injection, IP). !. 14!. 1.2 g/kg.

(17) (3.6). (auditory brainstem response, ABR) ABR (Moller, 2006) (. ABR. 1). (msec). (cochlear) (cochlear nucleus) (superior olivary complex) (nucleus of the lateral lamniscus) (inferior colliculus). (medial. geniculate). (auditory. radiation)(Ruebhausen et al., 2012) ABR. (threshold). (Ruebhausen et al., 2012; Willott, 2006) ABR. Tucker-Davis Technologies BioSigRP – Stimulate/Record System RZ5. 10 8k. 100~20 dB. !. RP Filter. 12k. 24k. 300~3k Hz. 15!. 10 32k Hz NT. 50 Hz. 4.4.1 701.

(18) Tucker-Davis Technologies(ES1 SN:3532) 15. (3.7). (ROS) (saline, MnCl2 10mg/kg, MnCl2 50mg/kg). 40. l. 10. l. heparin. CL. CL. Chemiluminescence Analyzing System CLD-110. Tohoku Electronic Industrial Co.,. Sendai, Japan 25 l. l. 60. 25. luminol/lucigenin. CL. 180. CL count. (3.8) ABR 10% !. 16!.

(19) 5. Leica RM 2125. 3-5. m. (3.9) Novolink polymer diction system (Leica) buffered saline(PBS). 0.1 M phosphate. tween-20. 60 C. 95%. ddH2O. epitope retrieval buffer peroxidase block (3% H2O2) antigoat polymer. protein block (goat antibody. ). DAB chromogen(substrate buffer hematoxylin solution(. Novelinktm 40. PBS. ) ) ddH2O. 95%. (1) Prestin Antibody (Santa Cruz. SC-22694. goat. polyclonal Ab) (2)Cleaved Caspase-3 (Asp175) Antibody (Cell Signaling Technology. 9661). Cleaved form. caspase. !. caspase-3. caspase. 17!. caspase.

(20) (3.10) Prism. GraphPad. Software, Inc., La Jolla, CA 92037 USA one-way ANOVA. Dunnett. (mean) (95% confidence intervals). !. Alpha = 0.05 * p-values. 18!. 0.05. ** p-values. 0.01.

(21) 4 (4.1) (saline) (10mg/kg). (50mg/kg) 12k Hz 50. 2a&b). 80. 32k Hz. (. 50 80. (. (MnCl2 10mg/kg). 3a&b). (MnCl2 50mg/kg) (saline). ABR 12k Hz. (. 8k Hz 4a&b). 44. (MnCl2 10mg/kg) (MnCl2 50mg/kg). 24k Hz. 32k Hz(. 72.5. 76.67. 80. 85. 4c&d). 62. 58. 93.33 87.5. !. 47.5. 19!. 82.5. 86.67.

(22) (4.2) CL count luminol MnCl2 10mg/kg. (. 5a). saline. 35382). MnCl2 50mg/kg. 27318 49161. luminol lucigenin. (. 5b). CL count. 26082.67. 24982.33. 27228.67. (4.3). (. 6). (4.4). prestin cleaved caspase-3. prestin. (. 7). cleaved caspase-3(Asp175). cleaved caspase-3 cleaved caspase-3 (spiral ganglion !. 20!.

(23) neurons, SGN). (. 9). cleaved caspase-3. SGN. SGN. cleaved caspase-3. !. 21!.

(24) 5. (extra pyramidal symptoms, EPS). (Bowler et al., 2007; Antonini et al., 2009; Antonini et al., 2009). (PEL-TWA). 1 mg/m3. 5 mg/m3 (Occupational Safety and Health. Administration, OSHA) (. ). 5 mg/m3. (auditory nerve fibers, ANF) (Ding et al., 2011). (Kalliomaki. et al., 1983; Josephs et al., 2005; Bouchard et al., 2008) (lung) !. (inner ear)(Kalliomaki et al., 1983; 22!.

(25) Park et al., 2007; Ma et al., 2008; Park et al., 2009) 2011 Ding. Prof. MnCl2. (reactive oxygen species, ROS). (Ingersoll et al., 1995). MnCl2 ABR. (. urethane. pure tone. 4) ABR. ABR. Prof. Ding prestin (Peter et al., 2008; Xia et al., 2013). !. 23!.

(26) (Zhang et al., 2008). (Zhang et al., 2004). al., 2006). (Kwik-Uribe et. (Milatovic et al., 2009; Zhao et al., 2009). dopamine. glutamate. (Desole et al., 1995; Normandin et al., 2002;. Gwiazda et al., 2007; Sistrunk et al., 2007; Erikson et al., 2008). luminol. lucigenin. (O2-). H2O2. O2-. luminol. endoperoxide. lucigenin. O2H2O2. SOD(superoxide dismutase) H2O2. H2O2. catalase. H2O2. O2-. SOD H2O2. catalase. catalase. H2O2. luminol. lucigenin (MnCl2 50mg/kg). 14. luminol. 14. lucigenin. ( (O2-). !. 24!. ).

(27) (Hazell et al., 2006; Milatovic et al., 2009). (Mn2+). (Ca2+) (Gavin et al., 1990;. Gunter et al., 2004). (oxidative. phosphorylation). (Gunter et al., 2004) (Yin et al., 2008). (Rama et al., 2007; Yin et al., 2008). (stria vascularis). (Wangemann et al., 1995). cleaved caspase-3 (Wangemann et al., 2012). SGN. ABR. SGN !. 25!.

(28) ABR glutamate. glutamate. Wolfram. (endoplasmic reticulum stresss) (Inoue et al., 1998; Bespalova et al., 2001). !. 26!.

(29) 6. !. 27!.

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(40) outer hair cells. Nature. 2000;405:149–155.. 2001;20:10.. !. 38!.

(41) 8. 1. ABR. (Willott JF, 2006). !. 39!.

(42) 2. 12k Hz (MnCl2). !. (saline). 10mg/kg. 50mg/kg. 40!. 50 70.

(43) 3. 32k Hz (MnCl2). !. (saline). 10mg/kg. 50mg/kg. 41!. 50 80.

(44) 4. Hz(c) 32k Hz(d). 8k Hz(a). (saline) (* p 0.05. !. 24k. ABR (MnCl2 10mg/kg). 50mg/kg)都. 16k Hz(b). ** p 0.01). 42!. (MnCl2.

(45) 5.. (saline) total CL count. luminol. CL count (* p 0.05. !. ** p 0.01). 43!. lucigenin. (a) (b).

(46) 6.. (a) ( ). !. 44!. (b)(c).

(47) 7.. (a). (b)(c). prestin (. /. !. 45!. prestin positive area).

(48) 8.. (b)(c). c-caspase-3. ( c-caspase-3 positive. area) !. 46!.

(49) 9. SGN c-caspase-3. c-caspase-3. (a). SGN. (c). (. !. c-caspase-3 positive area). 47!.

(50)