Bax在D-半乳糖诱导的老化大鼠耳蜗中的作用

目的研究Bax介导的细胞凋亡在D-半乳糖诱导的老化大鼠耳蜗中的作用,探讨老年性耳聋外周听觉系统细胞凋亡的发生机制。方法 36只1月龄雄性Spragua-Dawley大鼠随机分成2组(各18只):1 D-半乳糖组:每日颈背部皮下注射D-半乳糖(500mg/kg),连续8周;2对照组:每日颈背部皮下注射同体积的生理盐水,连续8周。造模完成后,取两组大鼠耳蜗,利用实时定量PCR检测线粒体DNA(mitochondrial DNA,mt DNA)普遍缺失(common deletion,CD)的累积;利用western blot检测Bax蛋白的表达;利用免疫组织化学检测Cleaved caspase-3蛋白的表达;利用原位末端转移酶标记(terminal deoxynucleotidyl transferase(Td T)-mediated deoxyuridine triphosphate(d UTP)nick-endlabelling,TUNEL)染色检测大鼠耳蜗细胞凋亡发生的情况。所有实验数据采用两样本的t检验进行分析。结果和对照组的大鼠相比较,D-半乳糖诱导的老化大鼠mt DNA CD的累积在耳蜗组织中明显增多,差异有统计学意义(t值为6.631,P值<0.01);Bax和Cleaved caspase-3蛋白的表达在耳蜗组织中明显增强,差异有统计学意义(t值分别为20.914、20.043,P值均<0.01)。对照组的大鼠耳蜗没有检测到凋亡细胞,而D-半乳糖诱导的老化大鼠耳蜗底回血管纹检测到少量凋亡细胞。结论 Bax介导的细胞凋亡参与了D-半乳糖诱导的耳蜗老化过程,可能是导致老年性耳聋外周听觉系统细胞凋亡发生的重要原因。     

罗布麻宁减轻D-半乳糖诱导的老化大鼠听皮层线粒体氧化损伤

目的研究NADPH氧化酶抑制剂罗布麻宁(Apocynin, APO)对D-半乳糖诱导的老化大鼠听皮层线粒体氧化损伤的作用。方法 36只1月龄雄性Sprague-Dawley大鼠适应性喂养1周后随机分为3组(每组12只):①对照组:大鼠颈背部皮下注射生理盐水,每日1次,连续8周;②D-半乳糖组:大鼠颈背部皮下注射500mg/kg D-半乳糖,每日1次,连续8周;③D-半乳糖+罗布麻宁组:大鼠颈背部皮下注射500mg/kg D-半乳糖+腹膜内注射50mg/kg罗布麻宁,每日1次,连续8周。我们利用酶化学法检测H2O2、总超氧化物歧化酶(Total superoxide dismutase, T-SOD)活性、ATP和线粒体膜电位水平,利用免疫组织化学法检测DNA氧化损伤标记物8-羟基-2-脱氧鸟苷(8-hy?droxy-2-deoxyguanosine, 8-OHdG),利用透射电子显微镜观察听皮层线粒体超微结构。结果和对照组相比较,D-半乳糖组大鼠听皮层H2O2和8-OHdG水平显著增加,而T-SOD活性、ATP和线粒体膜电位水平则显著降低。和D-半乳糖组相比较,D-半乳糖+罗布麻宁组大鼠听皮层H2O2和8-OHdG水平显著降低,而T-SOD活性、ATP和线粒体膜电位水平则显著增加。同时,我们也观察到罗布麻宁可有效保护D-半乳糖诱导的老化大鼠听皮层线粒体超微结构。结论罗布麻宁可有效保护中枢听觉系统老化过程中线粒体氧化损伤。     

NADPH氧化酶在鼓室硬化症大鼠模型中的表达

 目的研究NADPH氧化酶（NOX, NOX2及NOX3）在鼓室硬化症大鼠模型的中耳组织中的表达,探讨鼓室硬化症氧化性损伤的发生机制。方法20只雄性、体重250~300 g的Spragua Dawley大鼠随机分成两组（实验组15鼠30耳,对照组5鼠10耳）：①实验组：鼓膜后上象限人工制造2 mm大小穿孔,连续饲养18 d;②对照组：不做任何处理,连续饲养18 d。造模完成后,利用内镜检查两组大鼠鼓膜形态;利用RT PCR法检测NOX基因的表达;采用列联表二项分类逻辑回归分析,分析鼓膜穿孔和NOX基因表达的相关性以及鼓室硬化和两种NOX基因表达的相关性。结果实验组15只大鼠全部造模成功,对照组大鼠无一出现鼓膜钙化及浑浊;RT PCR检验大部分实验组大鼠发现NOX2及NOX3基因同步表达;而对照组大部分大鼠NOX2及NOX3基因表达均为阴性,通过列联表二项分类逻辑回归分析,提示鼓膜穿孔可致NOX2及NOX3基因的表达增强（P＜0.01）,而NOX基因的激活与鼓室硬化症的发生相关,并具有统计学意义（P＜0.01）。结论在穿孔导致的鼓室硬化症发病过程中,NOX是活性氧的来源之一,可能是导致鼓室硬化氧化性损伤发生的重要原因。     

D-半乳糖诱导的小鼠耳蜗带状突触损伤

目的研究D-半乳糖诱导的衰老小鼠耳蜗带状突触数量的变化。方法 1)D-半乳糖诱导的衰老小鼠模型的构建:24只5周龄雄性C57BL/6J小鼠适应性喂养1周后随机分为2组(每组12只):①对照组:小鼠颈背部皮下注射同体积的生理盐水,每日1次,连续6周;②D-半乳糖组:小鼠颈背部皮下注射1000mg/kg D-半乳糖,每日1次,连续6周;2)ABR检测听小鼠听功能;3)免疫组织化学染色检测小鼠耳蜗线粒体DNA氧化损伤产物8-羟基-2-脱氧鸟苷(8-hydroxy-2-deoxyguanosine, 8-OHdG)的表达;4)免疫组织化学染色检测小鼠耳蜗带状突触突触前标记物CtBP2的数量;5)Western blot检测小鼠耳蜗CtBP2蛋白的表达。结果 1)D-半乳糖诱导的衰老小鼠听功能表现为ABR I波幅值的降低,而无阈值的改变;2)8-OHdG在D-半乳糖诱导的衰老小鼠耳蜗表达明显增加;3)D-半乳糖耳蜗带状突触数量明显减少;4)CtBP2蛋白在D-半乳糖诱导的衰老小鼠耳蜗表达明显减少。结论耳蜗带状突触是D-半乳糖诱导的衰老小鼠耳蜗早期出现的病变部位,D-半乳糖诱导的衰老小鼠可作为研究老年性聋耳蜗带状突触损伤的动物模型。     

石杉碱甲对D-半乳糖诱导老年性聋大鼠听觉功能的影响评估

目的通过测试听性脑干反应和复合动作电位评估石杉碱甲对D-半乳糖致老年性聋大鼠听觉功能的改变。方法出生后3～4 W的Sprague-Dawley大鼠随机分为3组,模型组用5%D-半乳糖(200 mg/kg)行颈背部皮下连续注射8 W制备大鼠老年性聋模型;干预组用5%D-半乳糖(200 mg/kg)和石杉碱甲(0.1 mg/kg)行大鼠颈背部皮下注射;空白组作为对照。用药前、后分别检测大鼠听性脑干反应(auditory brainstem response,ABR)以检测听觉敏感性;复合动作电位(compound actionpotential,CAP)测试耳蜗输出幅度;以成对短声(pair clicks)为时间紧张性刺激,通过各CAP2/CAP2(20 ms)比值与短声间隔的关系函数反映耳蜗时间分辨能力。β-半乳糖苷酶染色下丘衰老细胞。结果 3组动物用药前后ABR阈值无明显改变(P>0.05);和模型组相比,干预组8kHz下80 dBSPL时ABRⅢ波潜伏期、I～V波间期缩短,提示听觉信号从外周到中枢的传入时间减少;以成对短声CAP响应所代表的耳蜗时间分辨力干预组高于模型组,但仍低于对照组;模型组下丘衰老细胞密度最高,干预组次之,对照组最少。结论石杉碱甲可以提高D-半乳糖致老年性聋大鼠与言语识别相关的听觉处理能力,为临床寻找改善老年患者听觉功能药物提供实验支持。     

蛋白磷酸酶1在D-半乳糖诱导老化小鼠耳蜗中的表达

目的 观察D-半乳糖诱导老化小鼠耳蜗中蛋白磷酸酶1(protein phosphatase 1,PP1)的定位表达及作用.方法 40只昆明小鼠随机分为对照组和D-半乳糖组,每组20只.D-半乳糖组颈背部皮下注射D-半乳糖(800mg·kg-1·d-1),对照组每日皮下注射等量生理盐水,连续给药8w;停药后检测血浆总超氧化物歧化酶(superoxide dismutase,SOD)活力和丙二醛(Malondialdehyde,MDA)水平;免疫荧光法定位PP1在耳蜗的表达,re-al-time PCR法检测耳蜗中PP1 mRNA水平,Western blot法检测小鼠蛋白磷酸酶1核目标亚基(protein phos-phates 1 nuclear targeting subunit,PNUTS)、PP1及caspase-3的表达.结果 与对照组比较,D-半乳糖组小鼠总SOD活力降低而MDA水平升高(均为P<0.01);PP1主要定位表达于小鼠耳蜗毛细胞、螺旋神经节细胞及血管纹细胞的胞核和胞浆中,且给予D-半乳糖后,小鼠耳蜗中PP1 mRNA及其蛋白表达水平显著升高,而PNUTS表达减少,caspase-3表达增加(均为P<0.05).结论 PP1在D-半乳糖诱导老化小鼠耳蜗毛细胞、螺旋神经节及血管纹细胞中表达,且参与了其耳蜗老化过程.     

左旋肉碱对大鼠线粒体DNA 4834bp缺失突变的影响及与老年性聋的关系

目的　研究探讨左旋肉碱（L-carnitine）对D-半乳糖诱导的模拟衰老大鼠mtDNA 4834bp缺失突变的影响。方法　实验分为4组：NaCl组（对照组）、D-gal+NaCl组（模型组）、D-gal+LC组、NaCl+LC组，每组12只。听性脑干反应（ABR）评估确定用药前后大鼠的听阈。比色法检测血清与耳蜗腹侧核中丙二醛（malondialdehyde，MDA）含量、H2O2浓度和超氧化物歧化酶（superoxide dismutase，SOD）活性的变化情况；Western blot检测耳蜗腹侧核中NOX2蛋白水平的变化；实时荧光定量PCR法检测mtDNA 4834bp缺失突变率。结果　在血清和耳蜗腹侧核中，D-gal+NaCl组中H2O2浓度和MDA含量显著增加，SOD活性显著降低（P<0.05），NOX2表达水平显著增加（P<0.05）；D-gal+NaCl组、D-gal+LC组、NaCl+LC组和NaCl组mtDNA4834bp的缺失率分别为12.54%±2.23%、8.13%±1.29%、2.31%±0.76%、2.12%±0.27%（P<0.05）。结论　L-carnitine可以一定程度上改善由D-半乳糖诱导的大鼠体内的氧化损伤。     

氧化还原因子1对体外培养大鼠耳蜗螺旋神经节氧化损伤的保护作用

目的 观察表达无嘌呤无嘧啶核酸内切酶/氧化还原因子1(apurinic/apyrimidimicendonuclase/redox factor 1,APE/Ref-1)的重组腺病毒感染对H2O2所致体外培养大鼠耳蜗螺旋神经节细胞氧化损伤的保护作用.方法 体外培养大鼠耳蜗螺旋神经节细胞,APE/Ref-1腺病毒表达载体感染48 h后,加入不同浓度H2O2(0、10、25、50、100及300 μmol/L)干预1 h,更换正常培养液后继续培养24 h,通过蛋白免疫印迹分析、四甲基偶氮唑蓝(MTT)法、原位缺口末端标记法(TUNEL)分别检测感染后螺旋神经节细胞APE/Ref-1蛋白表达、细胞活力以及凋亡情况.结果 通过腺病毒感染实现了APE/Ref-1基因在体外培养耳蜗螺旋神经节细胞的过表达,H2O2浓度为50～300 μmol/L时,APE/Ref-1组同对照组比较,细胞活力提高、凋亡率降低(P值均<0.01).结论 腺病毒介导的APE/Ref-1过表达对H2O2所致螺旋神经节细胞氧化损伤具有保护作用.     

NKCC1和Na-K-ATPase通道蛋白与老年性耳聋的关系及其在老年性耳聋不同阶段中的作用

目的：通过对C57BL/6J小鼠耳蜗中NKCC1和Na-K-ATPase的年龄相关性表达的研究,分析其与老年性耳聋的关系并进一步探讨其在老年性耳聋发生发展不同阶段中的作用。方法通过听性脑干反应（ABR）分别检测C57BL/6J小鼠在4、24和48周年龄段的听力水平。采用实时免疫荧光定量逆转录聚合酶链反应（RT-PCR）法分别检测NKCC1和Na-K-ATPase mRNA在各年龄段小鼠耳蜗中的表达水平。结果随着C57BL/6J鼠龄的增加,其ABR反应阈值逐渐升高（P〈0.05）。RT-PCR显示NKCC1和Na-K-ATPase mRNA在耳蜗的表达水平存在随鼠龄增加逐渐下降的趋势（P＜0.01）。结论 C57BL/6J小鼠的ABR反应阈值随鼠龄增加逐渐增高,具有老年性耳聋的特征;NKCC1和Na-K-ATPase两种通道蛋白随鼠龄的增加逐渐下降,与C57BL/6J小鼠的年龄相关性听力下降密切相关,可能与老年性耳聋后期发展及恶化有关。     

大鼠内耳拟老化伴线粒体突变模型的建立

目的:探讨建立大鼠内耳拟老化伴线粒体突变模型,为进一步揭示老年性聋发病机制及其防治策略奠定基础。方法:Wistar大鼠24只,随机分为2组:A组14只,5%D-半乳糖颈部皮下注射(150mg·kg-1·d-1),共8周,继以生理盐水腹腔注射10d;B组10只,仅给予生理盐水注射。听性脑干反应(ABR)检测2组大鼠听阈,比色法检测2组大鼠膜迷路谷胱甘肽过氧化物酶(GSH-PX)活力。利用巢氏聚合酶链反应技术检测内耳组织线粒体DNA4834bp缺失突变的发生情况。结果:用药后A组大鼠线粒体DNA4834bp缺失突变发生率为100%(28/28),B组无线粒体DNA4834bp缺失突变检出。A组GSH-PX活力[(59.07±8.70)U]明显低于B组[(142.10±7.02)U],其差异具有统计学意义(P<0.01)。而A组ABR听阈平均提高(5.36±3.08)dBpeSPL,B组为(6.50±3.37)dBpeSPL,经t检验,A组和B组之间差异无统计学意义(P>0.05)。结论:D-半乳糖可以诱导大鼠内耳组织拟老化,此模型大鼠内耳组织线粒体DNA4834bp缺失突变率极高,但听阈无明显提高。     

Effect of auditory stress agents on heterozygous German waltzing guinea pigs

The German waltzing guinea pig is a strain of animals expressing deafness and severe balance disorders at birth. The mutation arose spontaneously in a breeding facility in Germany and as the affected animals show a characteristic waltzing behavior, the strain is named the German waltzing guinea pig. The strain is presently bred only at Karolinska Institutet.The hereditary inner ear impairment has a recessive mode of inheritance and the strain thus produces not only affected homozygotes but also symptom-free heterozygotes and fully normal offspring. The outcome depends solely on the genotype of the parents. The heterozygotes, which have obtained the “waltzing” gene from one parent only, have normal hearing and no balance dysfunction. The heterozygous animals appear normal but will, in turn, carry the genetic defect to the next generation. The present thesis is focused on these animals.Noise and ototoxic drugs are well known stress factors that interfere negatively with the hearing organ in both humans and animals, causing hearing impairment. However, the inter-individual variability in susceptibility to auditory stress factors is surprisingly large, most likely due to different genetic predisposition. In this study, heterozygous animals of the German waltzing guinea pig, animals carrying a genetic defect known to cause severe hearing impairment, were used to study how an unexplored gene for deafness interacts with auditory stress agents, i.e. noise exposure and the ototoxic drugs gentamicin and cisplatin.Animals were exposed to both narrowband as well as broadband noise at different ages and hearing thresholds were measured using ABRs. Heterozygotes of the German waltzing guinea pig showed less threshold shifts compared to control strains. Older animals were less affected by the noise trauma than younger animals.To test the hypothesis that the efferent system contributes to protection of the inner ear against noise trauma, measurements using a new method of post onset adaptation of DPOAEs and maximum adaptation magnitude were conducted. The post onset adaptation of DPOAEs detected a strain difference at the higher frequency region while in the maximum adaptation magnitude method showed no difference between the strains.The heterozygous German waltzing guinea pigs displayed a distinctly increased resistance to noise exposures, manifested as reduced threshold shifts and faster recovery following acoustic overstimulation. However, when exposed to ototoxic drugs, the heterozygous carriers suffered from a more pronounced hearing loss.It is concluded that endogenous resistance to noise in the heterozygotes does not offer any protection against ototoxic drugs. The detailed mechanisms remain to be explored.     

Effect of perinatal biotin deficiency on auditory pathway of the Wistar-Albino rats

Aim: Severe biotin deficiency associated with biotinidase enzyme deficiency in newborns is seen as severe neurological problems and hearing loss. However, the effect on the infant of deficiencies in the maternal diet during pregnancy are not clear.

Material and methods: The study included 16 female Wistar albino rats and 4 male Wistar albino rats, that were mated and then the females were separated into 4 groups. At 40 days after the birth, 3 pups were selected from each group, and these 12 pups were evaluated with DPOAE and ABR electrophysiologically and the cochlea was examined ultrastructurally with electron microscopy.

Results: In the DPOAE evaluation, At 8000 and 11,000?Hz, the signal-noise ratios in the B-N and B-B groups were statistically significantly higher (p?<?.05). In ABR, lengthening of the latency periods was determined in all the waves at both 8 and 16?kHz in the B-B group. When the IPL periods were examined, lengthening in IPL 1–5 was statistically significant in the B-B group only at 8?kHz.

Conclusions: Biotin can be said to have an effect on hearing pathways. However, specifically where on the hearing pathways that biotin is involved has not been clarified.     

The Effect of Obstructive Sleep Apnea on DNA Damage and Oxidative Stress

Objectives

Obstructive sleep apnea syndrome (OSAS) is associated with repeated hypoxia and re-oxygenation. This characteristic of OSAS may cause oxidative stress and DNA damage. However, the link of OSAS with oxidative stress and DNA damage is still controversial. In the current study, we investigated whether OSAS causes DNA damage using alkaline single-cell gel electrophoresis (comet assay) and measuring oxidative stress by monitoring serum malondialdehyde (MDA) levels.

Methods

From March 2009 to August 2010, 51 patients who underwent polysomnography (PSG) during the night were enrolled in this study. We obtained serum from the patients at 6 AM. DNA damage and oxidative stress were evaluated using a comet assay and measuring serum MDA, respectively. We divided the patients into two groups according to the existence of comets appearing in the comet assay. Group 1 included 44 patients with negative assay results and group 2 consisted of seven patients with positive comet assay findings. We compared the age, gender proportion, PSG data (respiratory disturbance index [RDI], lowest O₂ saturation level, and arousal index [AI]), time of disease onset, smoking habits, and serum MDA levels between the two groups.

Results

The average age and gender proportion of the two groups were not statistically different (P>0.05). The average of RDI for group 1 was 30.4±18.4 and 8.0±7.7 (P<0.01) for group 2. The average of lowest O₂ saturation level for group 1 was 81.2±7.2 and 87.4±6.5 (P<0.05) for group 2. The average AI for group 1 was 32.8±15.1 and 20.8±7.7 (P<0.05) for group 2. Similarly, serum MDA levels of the two groups were not statistically different (P>0.05). No relationship between positive comet assay results and OSAS severity was identified.

Conclusion

Results of the current study showed that OSAS was not associated with DNA damage as measured by comet assays or oxidative stress according to serum MDA levels.     

用MP3播放器通过耳机播放音乐对豚鼠听觉系统的影响

目的研究用MP3播放器通过耳机播放音乐对豚鼠的听性脑干反应(auditory brainstem response,ABR)阈值及耳蜗形态学的影响,探讨其对听觉系统的损伤作用。方法实验动物分为对照组及实验组,每组动物12只。对照组不给予音乐暴露,其他条件与实验组相同。实验组每天用MP3播放器播放流行音乐5 h,连续14 d。观察对照组、实验组ABR反应阈及耳蜗形态学的变化。结果实验组动物在音乐暴露过程中及暴露后数天均呈现明显ABR反应阈上移,而对照组ABR反应阈无明显变化,两组间差异有统计学意义;基底膜铺片示实验组耳蜗各转外毛细胞均有不同程度的缺失,以耳蜗底转近钩端最为严重,3排外毛细胞以第二、三排损伤较重。结论用MP3播放器通过耳机播放音乐能引起豚鼠听觉系统的损害,表现为ABR反应阈升高及耳蜗外毛细胞形态的改变。     

Effect of minocycline and its nano-formulation on central auditory system in blast-induced hearing loss rat model

Blast injuries are common among the military service members and veterans. One of the devastating effects of blast wave induced TBI is either temporary or permanent hearing loss. Treating hearing loss using minocycline is restricted by optimal drug concentration, route of administration, and its half-life.Therefore, therapeutic approach using novel therapeutic delivery method is in great need. Among the different delivery methods, nanotechnology-based drug delivery is desirable, which can achiev...