爆震对豚鼠内耳损伤的研究

爆震性聋属于感音神经性聋的一种,与耳蜗损伤毛细胞有关,但不同强度噪声致聋及不同类型的神经性聋各有其特点,本研究采用爆震性聋（172dBSPL）豚鼠动物模型,检测爆震对豚鼠造成的听力损伤不同时期昕l螂卤干反应（ABR）阈值的变化,同时采用扫描电镜及光镜观察耳蜗形态学改变,探讨高强度爆震致聋豚鼠内耳损伤的特点,为探讨爆震聋发病机制提供试验依据。     

噪声对耳蜗损伤机理

噪声性耳聋是常见的后天获得性感音神经性耳聋之一，其主要损害部位是耳蜗，但噪声对耳蜗的损害机理仍不清楚。本文就目前噪声对耳蜗损伤的有关机理的研究进展作一简要阐述。     

强脉冲噪声导致的豚鼠耳蜗毛细胞凋亡及P53蛋白的表达

目的 探讨强脉冲噪声暴露引起的豚鼠毛细胞损害模式和P53凋亡蛋白在损伤毛细胞中的表达.方法 12只成年豚鼠,声压级168 dB的强脉冲噪声连续暴露80次,每次间隔时间2 s,分别在噪声暴露后3 h、6 h及12 h各处死4只动物,应用异硫氰酸荧光素(flourescein iso-thiocyanate,FITC)标记的鬼笔环肽(phalloidin)和细胞核DNA荧光染料碘化丙啶(propidium iodide,PI)来施行双重染色,在激光共聚焦显微镜下观察耳蜗毛细胞静纤毛和细胞核的形态学变化;将噪声暴露后12 h的耳蜗基底膜样品进行P53免疫荧光染色,观察受损毛细胞中是否存在P53蛋白的表达;耳蜗琥珀酸脱氢酶染色,制备基底膜铺片,400倍显微镜下行毛细胞计数并绘制耳蜗图.结果 强脉冲噪声暴露后3 h,耳蜗底回末端和第二回起始端已经发生明显的外毛细胞结构破坏,同时伴有细胞核的浓缩;暴露后6 h耳蜗毛细胞的破坏范围扩大,在毛细胞损害的中心区域可见外毛细胞的核碎片;毛细胞损害中心区域在噪声暴露后12 h,多数外毛细胞的细胞核消失,而周边区域的毛细胞可见核浓缩或核碎片.耳蜗图显示,噪声暴露后3、6及12 h,耳蜗毛细胞的破坏范围不断扩大.免疫荧光显示耳蜗第二回外毛细胞损伤的中心部位出现明显的P53阳性表达,在耳蜗底回和第三回外毛细胞中亦出现P53的阳性表达.结论 强脉冲噪声暴露引起耳蜗毛细胞死亡的主要方式是凋亡,损伤的中心部位在耳蜗第二回,并逐渐向耳蜗底回和第三回扩散;P53蛋白在此过程中可能扮演了重要角色.

Abstract：

Objective To explore the pattern of hair cell injury and expression of P53 apoptosis protein in intensive impulse noise injured cochlear hair cells in guinea pigs. Methods Twelve adult guinea pigs were exposed to a series of 40 pairs of impulse noise(2 second intervals) at the intensity of 168 dB (SPL). Animals were terminated at 3, 6 and 12 hours after noise exposure, respectively. Cochlear surface preparations were performed with a double staining of FITC-conjugated phalloidin and propidium iodide for the observations of the stereocilia and the nucleus. P53 immunochemical staining was also performed 12hours post-noise exposure to observe if there was expression of p53 protein in injured hair cells. Results Three hours after noise exposure, the outer hair cells at the end of basal turn and beginning of second turn were destroyed first with a character of nuclear condensation. Six hours post-noise exposure, many hair cells in the center of damage region had nuclear fragmentations, and the damaging area expanded towards to basal turn and apical turn. Twelve hours after noise exposure, the nucleus in most outer hair cells and inner hair cells at the region of damage center were missing. The nuclear condensation and fragmentation were appeared in hair cells in both sides of the center region of degeneration. P53 immunoreactive products were also found in damaged hair cells, not only in the central damage area, but also in the basal turn and the third turn.Conclusions Intensive impulse noise resulted in apoptosis of cochlear hair cells that initiated between the end of basal turn and the beginning of second turn. Hair cell degeneration spread to basal and third turn along the basilar membrane. P53 may play an important role in impulse noise induced-hair cell apoptosis.     

多巴胺对豚鼠噪声性听力损失的保护作用

目的观察白噪声条件下多巴胺（dopamine,DA）对耳蜗内毛细胞的保护作用,为进一步探讨多巴胺对耳蜗传入通路的负反馈保护机制奠定基础。方法健康杂色豚鼠40只,随机分4组,行活体全耳蜗灌流：①单纯给予100dB白噪声组（以下同）;②灌流人工外淋巴液组;③灌流人工外淋巴液并给予白噪声组;④灌流1mmol/L多巴胺并给予白噪声组。在灌流第0、2h记录4kHz耳蜗微音电位（cochlear mirophonics,CM）幅值,并做相对幅度输入/输出函数曲线,和不同频率复合动作电位（compound action potential,CAP）阈值。结果给予噪声暴露的3组灌流后CM输入/输出曲线非线性特点均消失,相对幅度下降,差异有统计学意义（P〈0.05）。给予噪声暴露的3组2小时后各频率CAP阈值较前均上升,差异有统计学意义（P〈0.001）。但第4组较第1组除8kHzCAP阈移差异无统计学意义外,其余各频率CAP阈移明显减小（P〈0.05）。高频的阈移相差程度均较低频明显,其中16kHz阈移相差程度最为明显。结论白噪声暴露下多巴胺对耳蜗传入通路具有保护作用,并存在频率选择性,对高频纤维保护作用较低频更强。     

西地那非对豚鼠噪声性听觉损伤阈移的影响

目的 探讨西地那非(sildenafil)对豚鼠噪声性听觉损伤阈移的影响.方法 将豚鼠按随机数字表法分为对照组、噪声暴露组和西地那非给药组,每组10只.西地那非组及噪声组豚鼠在白噪声(A计权声压级110 dB)暴露1周后分别腹腔注射西地那非10 mg/(kg·d)及生理盐水4mL/(kg·d),连续给药4周.分别测试噪声暴露前1日、噪声暴露后1、2及4周听性脑干反应(ABR)阈值,并通过扫描电镜观察噪声暴露后4周豚鼠耳蜗毛细胞的形态变化.结果 噪声暴露1后,噪声暴露组豚鼠ABR阈值(声压级)平均提高19.1 dB,随着时间推移,阈移逐渐加大,暴露后4周,阈值平均提高22.0 dB;西地那非组噪声暴露后ABR阈值提高19.8 dB,给药后阈移逐渐减小,给药后4周,阈值仅平均提高4.8 dB.西地那非组与噪声暴露组相比,除噪声暴露结束后这一时间点以外,其余给药后各时间点ABR阈值差异均具有统计学意义(P值均＜0.05).扫描电镜显示,噪声组豚鼠耳蜗内、外毛细胞均出现听毛紊乱、融合及缺失;而西地那非组耳蜗病变较轻,听毛仅有轻微倒伏、融合现象.结论 西地那非能够减轻噪声对豚鼠耳蜗毛细胞的损害,降低噪声性听觉损伤引起的ABR阈值升高.     

睫状神经营养因子防治强脉冲噪声对豚鼠内耳损伤的实验 …

目的 探索应用睫状神经营养因子防治强脉冲噪声对豚鼠内耳损伤的可能性,为强脉冲噪声致聋的防治提供新的方法。方法 制作强脉冲噪声聋豚鼠模型３６只,１８只应用ＣＮＴＦ人注射３周,１８只等量的生理盐水作对照,正常对照豚鼠１８只,进行耳蜗铺片计算机图像分析毛细胞计数,螺旋神经节细胞计数、耳蜗乙酰胆碱酯酶染色铺片观察和ＡＢＲ反应阈测定。结果 正常姐、噪声暴露后２１ｄ的生理盐水对照组和ＣＮＴＦ组耳蜗毛细胞平均总     

神经营养因子对噪声引起豚鼠耳蜗外毛细胞损伤的防护作用

目的 定量观察胶质细胞源性神经营养因子(glial cell ine-derived neurotrophic factor,GDNF)和神经营养-3(neurotrophin-3,NT-3)对噪声引起豚鼠耳蜗外毛细胞损伤的防护作用。方法 将微渗透压泵埋置于豚鼠背部,经固定于耳蜗底回鼓阶内的改良微导管将GDNF(100ng/ml)和NT-3(2.5μg/ml)的混合液缓慢注入12只豚鼠左侧内耳,以左侧内耳灌注人工外淋巴液的9只豚鼠为对照,检测噪声暴露后豚鼠听功能和耳蜗外毛细胞形态、数量的变化。结果 噪声暴露10天后,实验组手术耳和非手术耳的脑干诱发电反应阈值低于对照组(P<0.05,p<0.01)。毛细胞表皮板和纤毛肌动蛋白荧光染色计数发现,实验组手术耳和非手术耳的外毛细胞缺失率低于对照组(p<0.001,p<0.01)。毛细胞核荧光染色计数发现,实验组手术耳和非手术耳的外毛细胞核肿胀率低于对照组(p<0.01,p<0.01)。结论 GDNF和NT-3对噪声引起豚鼠耳蜗外毛细胞损伤具有较好的防护作用。     

电子自旋共振技术观察急性声损伤后耳蜗自由基改变

目的:应用电子顺磁自旋共振(ESR)技术观察急性声损伤后豚鼠耳蜗自由基的变化规律.方法:将正常白毛红目豚鼠48只分为3组:A组(对照组)取6只豚鼠不给予噪声刺激,分别在检测听功能后测定自由基和硝酸银染色.B组21只豚鼠在(125±1)dB SPL稳态噪声暴露2h后分别于即刻、2h、6h,12h、24h、48h、72h施行ABR测试和ESR检测耳蜗自由基,检测方法为断头后快速取出耳蜗,液氮速冻.样品处理后放入ESR系统谐振腔中检测自由基含量.C组21只豚鼠在噪声暴露后于上述时间点检测听觉功能并取基膜行硝酸银染色观察Corti器毛细胞形态改变.结果:①正常豚鼠耳蜗中有少量自由基存在,其相对自由基值为(21.68±1.27).噪声暴露后即刻取样组自由基值明显升高,在暴露后2h达峰值(147.01±4.95)dB SPL,此后逐渐下降,至72h恢复至接近正常水平(53.12±2.57)dB SPL;②在125dB SPL的急性噪声暴露后,豚鼠的听阈明显提高,至6h达到峰值(73.89±2.41)dB SPL,直至72h仍未恢复到暴露前正常水平(50.28±1.48)dB SPL;③急性声损伤后形态学改变表现为外毛细胞纤毛紊乱、排列不规则,部分区域可见毛细胞缺失.结论:①急性噪声暴露后,豚鼠耳蜗内自由基水平明显增高,并在2h达峰值;②应用ESR技术检测耳蜗组织中自由基含量的方法具有直接、客观和灵敏的特点,ESR技术可用于某些内耳急性损伤动物模型的实验观察.     

噪声对耳蜗损伤机理

噪声性耳聋是常见的后天获得性感音神经性耳聋之一,其主要损害部位是耳蜗,但噪声对耳蜗的损害机理仍不清楚。本文就目前噪声对耳蜗损伤的有关机理的研究进展作一简要阐述。     

高频听力损失对豚鼠低频区时间分辨率的影响

目的 构建豚鼠高频听力损失模型,通过听觉惊跳反射前抑制方法观察高频听力损失后,低频区时间分辨率的变化.方法 采用简单随机法将豚鼠分为实验组(6只)和对照组(4只),实验组暴露于声压级110 dB的12 kHz纯音下30 h,建立8 kHz以上听力损失模型,对照组未作处理.于噪声暴露前、暴露后2周、4周、6周及8周等五个时间点进行听觉惊跳反射前抑制试验和听觉脑干反应(ABR)测试.在听觉惊跳反射前抑制试验中,选用0.5～2 kHz,0.5～4 kHz和0.5～8 kHz三种背景噪音频率(均在听敏度正常的低频段)来观察低频区时间分辨率的变化.结果 噪声暴露后2周,实验组动物呈现8 kHz以上频率陡降型高频听力损失,在16 kHz、32 kHz和48 kHz频率均有平均55 dB的阈移,与声暴露前相比差异均有统计学意义(P值均＜0.05),而1 kHz、2 kHz、4 kHz和8 kHz在噪音暴露前后差异均无统计学意义(P值均＞0.05).在8周的实验期内,实验组高频听力损失仍然存在.对照组豚鼠所有频率的听敏度在实验期内各个时间点差异均无统计学意义(P值均＞0.05).实验组在8 kHz的背景噪音下,声暴露后第2周抑制能力减弱,与暴露前相比差异具有统计学意义(P=0.036),其余时间点和暴露前相比差异无统计学意义(P值均＞0.05);在4 kHz的背景噪音下,声暴露后所有时间点的抑制能力与暴露前相比差异均无统计学意义(P值均＞0.05);在2 kHz的背景噪音下,声暴露后第6周、第8周抑制能力降低,与暴露前相比差异具有统计学意义(P值均＜0.05).结论 高频听力损失可降低正常低频区的时间分辨率.

Abstract：

Objective To constitute the animal model of high frequency hearing loss and observer the temporal processing abilities of low frequency regions using prepulse inhibition of auditory startle response (gap-PPI).Methods Ten guinea pigs were randomly grouped into two groups: the high frequency hearing loss group with six guinea pigs and the control group with four guinea pigs.The former group was exposed to 12 kHz tone at 110 dB SPL for 30 hours to establish the high frequency hearing loss above 8 kHz and the latter group received no stimuations.Before and two,four,six and eight weeks after noise exposure,gapPPI and auditory brainstem response (ABR) were recorded in both groups.In the gap-PPI experiment,three different background noises as 0.5-2 kHz,0.5-4 kHz and 0.5-8 kHz were applied to test the temporal gap.Results High frequency hearing loss above 8 kHz was shown two weeks after noise exposure.The averaged ABR thresholds of 16 kHz,32 kHz and 48 kHz were elevated about 55 dB and shown statistical significance compared to those before exposure (P ＜ 0.05＝.No significant difference of ABR thresholds were shown between 1 kHz,2 kHz,4 kHz and 8kHz before and after noise exposure(P ＞0.05).In the control group,the ABR thresholds remained stable during experiment.In the gap-PPI test,two weeks after noise exposure of 8 kHz,the experiment group showed attenuated inhibition ability and recovered gradually four weeks after noise exposure.No statistical differences of inhibition ability at time points of two,four,six and eight weeks after noise exposure of 4 kHz were detected when compared with that of pre-exposure.Under the background noise of 2 kHz,the inhibition ability attenuated and reached statistical significance at 6-8 weeks after noise exposure.Conclusion The high frequency hearing loss might induce an impairment of the temporal processing in the low frequency region.     

Development of cis-platinum-induced hearing loss in guinea pigs

Summary The resultant hearing loss can be variable if cis-dichlorodiammineplatinum is given to guinea pigs. In order to find out more about these differences, we used brainstem audiometry to study the start and development of hearing loss over time for several frequencies. Our results confirmed previous observations that hearing loss starts at the higher frequencies but can also occur at lower frequencies. Furthermore, there were great differences in the start and the rate of the increase of hearing loss found in the individual animals. The individual differences in susceptibility can not be explained by one factor alone, but by the combination of three factors, namely deviation point, slope and length of survival.This study was supported by the Heinsius Houbolt Foundation     

Hyperbaric oxygenation with corticoid in experimental acoustic trauma

Among possible therapies after acute acoustic trauma, hyperbaric oxygenation (HBO) combined with corticoid was found effective in several animal studies. Such evidence was obtained for moderate 20–25 dB losses. The aim of this study was to further assess this therapy for noise-induced hearing losses greater than previously examined. Sixty-five ears from thirty-six adult guinea pigs were used. Acoustically evoked responses from intracranial electrodes chronically implanted bilaterally into the ventral cochlear nucleus were used to assess acoustic sensitivity alterations. Trauma sound was a third-octave noise-band around 8 kHz presented bilaterally at 115 dB SPL for 45 min. One control group received no treatment, one group was treated with HBO only and another with corticoid only both starting within one day post-trauma, two groups were treated with both HBO and corticoid starting for one group within one day post-trauma, and for the second group at 6 days post-trauma. Acoustic thresholds were measured between the 6th and the 16th days after acoustic trauma. Animals treated with HBO alone or corticoid alone did not differ from controls. Combined HBO and corticoid therapy provided significant protection from noise-induced loss of auditory thresholds, especially when started one day post-exposure. Hearing loss reduction induced by HBO combined with corticoid was of similar magnitude (about 10–15 dB) as in previous studies although the induced hearing loss was considerably greater (about 40 dB instead of 20–25 dB).     

卡那霉素联合速尿致聋豚鼠的Math1基因治疗

目的 观察卡那霉素和速尿联合致聋豚鼠耳蜗鼓阶导入Math1基因后的形态学及功能改变,探讨Mathl基因治疗药物中毒性耳聋的可行性.方法 健康成年豚鼠经硫酸卡那霉素(500 mg/kg)和速尿(50 mg/kg)联合致聋,将听性脑干反应(ABR)反应阈＞95 dB SPL的豚鼠按随机数字表法分为空白对照组(不做任何处置,3只),手术对照组(右耳单纯鼓阶钻孔,3只),人工外淋巴液组(右耳鼓阶钻孔导入人工外淋巴液,3只),单纯病毒载体组[右耳鼓阶钻孔导入携带增强型绿色荧光蛋白基因(enhanced green fluorescent protein,EGFP)的重组腺病毒(Ad.EGFP),4只]、Math1基因治疗组[右耳鼓阶钻孔导入携带Math1及EGFP基因的重组腺病毒(Ad.Math1-EGFP),6只].各组动物分别于鼓阶注射前及注射后8周时行ABR测试,结束测试后处死动物,取出耳蜗组织行扫描电镜观察.结果 各组豚鼠不同频率(4、8、16、20 kHz)短纯音ABR阈值在不同检测时间段差异均无统计学意义,组间比较差异亦无统计学意义(P值均＞0.05).除Math1基因治疗组外,其余各组右耳耳蜗各回毛细胞形态和数目与左耳(自身对照)比较无明显差别.4只Math1基因治疗组豚鼠中,有2只右耳耳蜗第三回内、外毛细胞数量明显比左耳多,其中内毛细胞排列形态较外毛细胞整齐.结论 鼓阶显微注射导入Math1基因能使部分卡那霉素和速尿联合致聋豚鼠的耳蜗毛细胞修复或再生,但其听觉功能没有改善.     

一氧化氮合酶抑制剂和神经营养因子3对噪声暴露下豚鼠耳蜗的保护作用

目的观察一氧化氮合酶抑制剂——N-硝基左旋精氨酸甲酯（N^G-nitro-L-arginine methyl ester，L-NAME）和神经营养因子3（neurotrophin 3，NT3）对噪声性听力损失的保护作用。方法80只雄性杂色豚鼠按区组随机分为非噪声组（n=20）和噪声暴露组（n=60），噪声暴露组又分为生理盐水组（n=20）、L-NAME组（n=20）、L-NAME＋NT3组（n=20）。L-NAME组和L-NAME＋NT3组动物在噪声暴露（4kHz倍频程、声压级115dB，5h）之前2d和噪声暴露前30min给予L-NAME 10mg／kg（腹腔注射），生理盐水组动物给予等体积的生理盐水。NT3（10μg／ml）在噪声暴露前4d经微量渗透泵（200μl，0．5μl／h）输入到L-NAME＋NT3组动物的右侧耳蜗鼓阶，持续到噪声暴露后10d。噪声暴露前和暴露后10d测试听性脑干反应（auditory brainstem response，ABR），暴露后3d测试耳蜗组织一氧化氮（nitric oxide，NO）水平，最后一次ABR测试后计数耳蜗毛细胞的存活率。结果无噪声暴露组动物无明显的听力改变和毛细胞缺失；生理盐水组动物的ABR阈移、毛细胞缺失率及耳蜗组织NO水平均高于L-NAME组和L-NAME＋NT3组，差异有统计学意义（P值均〈0．01）；与L-NAME组相比，L-NAME＋NT3组豚鼠的ABR阈移减小，差异有统计学意义（P〈0．01），而耳蜗组织NO水平和毛细胞缺失率差异则没有统计学意义（P=0．197及P=0．095）。结论与单独给予L-NAME相比，联合使用NT3可以更大程度减轻噪声对豚鼠耳蜗的损伤。     

Maturation of auditory evoked potentials in young guinea pigs with binaural conductive hearing loss

Summary A reversible conductive hearing loss produced during the first 4 weeks post partum caused marked alterations in the development of click-evoked auditory brainstem (ABR) and middle latency (MLR) responses in guinea pigs. The early component PI in the ABR in controls showed adult-like latencies at the time of birth, while the later ABR components P_III and P_V and all components investigated in the MLR showed postnatal development characterized by a shortened latency that persisted for the whole period of investigation. The course of the ABR latencies showed the sharpest decrease during the first 2–3 weeks of life, while that for the MLR took place during the first 5 weeks. In addition to the increased ABR thresholds and lengthened ABR latencies due to the conductive hearing loss, development of the ABR inter-peak latencies (IPL) and MLR latencies was retarded. The IPL reached control values 1 week after the end of the deprivation phase, while the delay in MLR started later (4th week) and lasted longer than did that in the ABR. These findings showed that a long-lasting postnatal conductive hearing loss does not generate sustained impairment at the level of the brainstem but can evoke longer-lasting deficiencies at higher stations of the auditory pathway.     

紫杉醇对豚鼠听阈的影响

目的：探讨紫杉醇对豚鼠听阈的影响。方法：将30只雌性豚鼠随机分为6组（1个对照组和5个实验组,每组5只）,实验组分别予以不同剂量紫杉醇腹腔内注射,检测用药前、后各组动物ABR阈值。结果：实验组听力水平用药后均显著减低,相关分析显示,总药量与听力损失程度之间无显著相关性。结论：紫杉醇可导致轻到中度听力损失。     

Long-term administration of magnesium after acoustic trauma caused by gunshot noise in guinea pigs

In a previous study we observed that a 7-day post-trauma magnesium treatment significantly reduced auditory threshold shifts measured 7 days after gunshot noise exposure. However this improvement was only temporary, suggesting that it could be potentially beneficial to prolong this treatment. The aim of the present study was to evaluate the efficacy of a long-term (1 month) magnesium treatment after an impulse noise trauma, in comparison with either a 7-day magnesium treatment, an administration of methylprednisolone (conventional treatment), or a placebo (NaCl). Guinea pigs were exposed to impulse noise (three blank gunshots, 170 dB SPL peak). They received one of the four treatments, 1 h after the noise exposure. Auditory function was explored by recording the auditory brainstem response (ABR) and measuring the distortion product otoacoustic emissions (DPOAE) over a 3-month recovery period after the gunshot exposure. The functional hearing study was supplemented by a histological analysis. The results showed that a 1-month treatment with magnesium was the most effective treatment in terms of hair cell preservation. The DPOAE confirmed this effectiveness. Methylprednisolone accelerated recovery but its final efficacy remained moderate. It is probable that magnesium acts on the later metabolic processes that occur after noise exposure. Multiple mechanisms could be involved: calcium antagonism, anti-ischaemic effect or NMDA channel blockage. Regardless of the specific mechanism, a 1-month treatment with magnesium clearly attenuates NIHL, and presents the advantage of being safe for use in humans.