内皮舒张因子对豚鼠耳蜗血管纹血管的保护作用

本实验利用活体显微镜摄像技术、透射电镜技术,观察了内皮舒张因子（EDRF）对豚鼠耳蜗微循环的保护作用。结果提示：①速尿组（F）动物,经静脉注射药10min后,耳蜗微动脉缺血,血管内皮细胞损伤;②对速尿／L-精氨酸组（F／L－Arginine）动物,EDRF能使速尿引起的微动脉缺血明显改善,血管纹血管超微结构的损伤程度较单纯F组减轻;③速尿／L-硝基-精氨酸组（F／L－NNA）动物,耳蜗的缺血程度较F组加重。结论提示;EDRF能通过增加局部血流的灌注而改善和保护耳蜗微循环。本研究提供的实验结果,于临床开展微循环致聋疾病的治疗有所启迪。     

Cochlear microcirculation in living guinea pigs following explosion

The cochlear microcirculation in living guinea pigs was observed, explosion was then imposed on the left ear and the velocity of blood cell flow and vessel diameters were measured within one hour. The results showed that: 1. there was an orderly stable movement of blood cells in all vascular beds under normal conditions; 2. the flow rate in both spiral ligament and stria vascularis increased promptly after explosion, that in the spiral ligament might return to normal within 30 minutes with vasodilatation, indicating increased blood supply early after explosion; 3. there was no relationship between changes in microcirculation and hearing loss and hair cell damage. The cause of hair cell damage and hearing loss were discussed and the techniques of vital microscopic studies of cochlear microcirculation assessed.     

A surgical approach appropriate for targeted cochlear gene therapy in the mouse

Therapeutic manipulations of the mammalian cochlea, including cochlear gene transfer, have been predominantly studied using the guinea pig as the experimental model. With the significant developments in mouse genomics and the availability of mutant strains of mice with well-characterized hearing loss, the mouse justifiably will be the preferred animal model for therapeutic manipulations. However, the potential advantages of the mouse model have not been fully realized due to the surgical difficulty of accessing its small cochlea. This study describes a ventral approach, instead of the routinely used postauricular approach in other rodents, for accessing the mouse middle and inner ear, and its application in cochlear gene transfer. This ventral approach enabled rapid and direct delivery of liposome-transgene complex to the mouse inner ear while avoiding blood loss, facial nerve morbidity, and mortality. Transgene expression at 3 days was detected in Reissner's membrane, spiral limbus, spiral ligament, and spiral ganglion cells, in a pattern similar to that previously described in the guinea pig. The successful access and delivery of material to the mouse cochlea and the replication of gene expression seen in the guinea pig demonstrated in this study should promote the use of the mouse in future studies investigating targeted cochlear therapy.     

Recent advances in cochlear blood flow measurements

Changes in blood flow to the inner ear have been thought to influence or underlie a number of cochlear diseases, including some forms of noise-induced hearing loss, sudden hearing loss, and Meniere's disease. Recently, important advances have been made in two technologies for the study of cochlear blood flow. The first is in the area of vital microscopic studies of cochlear microcirculation, and the second is based on the introduction of laser technology in the form of laser Doppler flowmetry. In this report, measurements are given of changes in cochlear circulation caused by carbon dioxide breathing, intravenous phenylephrine injection, systemic hemodilution, positive end expiratory pressure, and direct electrical stimulation of the cochlea. From these changes, we observe that cochlear blood circulation responds to systemic blood pressure alterations and is subject to local flow control mechanisms. Linearity and speed of response of the laser Doppler instrumentation also are shown. These advances show promise for contributing to our knowledge of control mechanisms of inner ear blood flow and for revealing the influence of various pharmacologic agents of potential clinical value.     

Relationship between three inner ear antigens with different molecular weights and autoimmune inner ear disease

Crude inner ear antigen (CIEAg) can induce autoimmune inner ear disease (AIED) although it is not known which subcomponent of CIEAg is involved. In this study, we investigated the relationship between 3 purified inner ear antigens (31, 42-45 and 60 kD proteins) and AIED, and determined their distribution in normal guinea pig cochlea. Three groups of guinea pigs were immunized with the three inner ear antigens and one group served as a control. The hearing thresholds, serum IgG level and morphological changes in the inner ear were observed. The expression of the three antigens in the cochlea was detected using immunohistochemical techniques. No obvious changes in hearing thresholds or inner ear morphology were observed between the control and 42-45 kD groups. Animals immunized with the 31 or 60 kD proteins showed a significant increase in hearing thresholds (p < 0.05 vs control), accompanied by morphological changes in the inner ear. The serum IgG level was increased significantly (p < 0.05) in all immunized animals. The 31 kD protein was distributed in the cochlear nerve and spiral ganglion, while the 42-45 and 60 kD proteins were distributed widely, being found in the spiral ganglion, organ of Corti, stria vascularis and spiral ligament. These results suggest that two subcomponents of CIEAg (the 31 and 60 kD proteins) may induce AIED independently, that several inner ear antigens may contribute to the pathogenesis of AIED and that the 31 kD protein is of high tissue specificity and may be used as a marker protein for the clinical diagnosis of AIED.     

Experimental studies of ischemia of the cochlea. Part 3: Pathophysiology]

Selective interruption of cochlear blood flow is possible without operative manipulations in the middle or inner ear. By fitting a magnet into the external auditory canal and injecting iron filings into the cephalic vein is it possible to obtain an impaired inner ear in otherwise healthy experimental animals that can be kept alive for any length of time. The hearing loss induced by the ischemia normally remains unchanged over a period of weeks. The highly vascularised areas of the cochlea, namely the spiral ligament, the vascular stria, the spiral prominence and the root cells in the external spiral sulcus, as well as the spiral limbus were all clearly degenerated, but to different degrees. Pronounced vacuolisation of cells, degeneration of tissue, reduction of cellular elements, stasis in large and small blood vessels and morphological disorganisation were observed. The organ of Corti showed no changes worthy of note. On revascularisation of the spiral ligament and the spiral limbus with resumption of function, the organ of Corti can return to normal activity again. Repeat blood flow disturbances can also lead to recurrent sudden hearing loss and intermittent loss of hearing.     

Probational treatment of sudden deafness with prostacyclin: a pilot study.

Sudden idiopathic hearing loss has occasionally been supposed to be caused by a disturbed microcirculation in the inner ear of unknown origin. Little is known about the regulation of cochlear blood flow and the effectiveness of drugs in cochlear microcirculation. Because animal experiments gave evidence that prostacyclin (PGI2) might be one biochemical substratum of local regulators in the flow of blood in the stria vascularis, 11 patients with sudden idiopathic hearing loss were treated once for 6 h with prostacyclin (10 ng/kg body weight/min) in a first open clinical trial. In most cases prostacyclin increased hearing level (mean value: 7.4 dB/frequency/day) more than a standard therapy with pentoxifylline. The substitution of PGI2 could be another indication of a rheologic disorder--whether per se or within a larger context of inflammation-like interaction--in the inner ear of patients with sudden hearing impairment.     

Die Rolle der Spiralganglienneurone im Kochleaimplantat

Sensorineural hearing impairment is caused by pathologies within the cochlear portion of the inner ear or the central auditory pathway. Within the last decade, tremendous progress has been made in inner ear biology, thus greatly increasing our understanding of congenital and acquired inner ear pathologies. Moreover, the discovery of hair cell regeneration and the presence of neuronal stem cells in the cochlea has raised hopes of being able to treat the causes of sensorineual hearing impairment in the mid-term future. To do so, the regenerated cells will have to be reinnervated through the peripheral axons of the spiral ganglion neurons (SGNs). So far, most factors with the potential to guide peripheral axons of SGNs have been investigated in the developing cochlea of rodent models but not in humans. Remaining SGNs can already be directly stimulated electrically by cochlear implants, electrode arrays surgically inserted into the cochlea, providing effective treatment for severe cochlear hearing impairment.     

重组腺病毒构建及介导报告基因在豚鼠耳蜗中的表达

目的：带绿色荧光蛋白（GFP）基因的重组腺病毒,为利用重组腺病毒介导外源基因转导内耳提供依据。方法：通过细菌内同源重组方法构建携带有报告基因的重组腺病毒（Ad-GFP）,将重组腺病毒经耳蜗底回途径导入豚鼠耳蜗鼓阶外淋巴后,观察手术后不同时间GFP基因在豚鼠耳蜗内的表达、分布情况;检测手术前后听觉脑干诱发电位,观察手术和病毒对豚鼠听觉功能的影响。结果：构建的重组腺病毒经酶切电泳鉴定正确;豚鼠耳蜗底回途径导入腺病毒24 h后即有报告基因表达,3 d后最高,表达时间可持续2周以上;报告基因表达部位主要分布在血管纹、鼓阶外淋巴面的间皮细胞和耳蜗Corti器等部位;听觉脑干诱发电位（ABR）在各组动物手术前后无明显改变。结论：成功构建了携带GFP基因的重组腺病毒,通过该方法构建的腺病毒可以介导报告基因在豚鼠耳蜗中表达,并且对豚鼠听觉功能无明显影响,为内耳基因治疗提供了理论依据。     

Expression of heat shock protein 70 in immune response of the guinea pig inner ear]

OBJECTIVE: To explore whether the immune response of the inner ear could induce heat shock protein (hsp) 70 in guinea pig cochlea. METHODS: A model of autoimmune inner ear disease (AIED) was established by systemically immunizing the guinea pig with the homologous crude inner ear antigen (CIEAg). The immunized cochleae and normal control cochleae were examined for the expression of hsp70 with techniques of immunohistochemistry and in situ hybridization. RESULTS: In the control animals, the expression of the hsp70-like protein appeared only in the spiral ganglion, whereas in the cochleae with CIEAg immunization, strong expression of the hsp70-like protein and its mRNA appeared in the spiral ganglion as well as in the stria vascularis and the spiral ligament. The hearing thresholds were significantly increased in 10 out of 28 cochleae (35.7%) with CIEAg immunization. CONCLUSION: The results suggest that the immune response of the inner ear can induce the expression of hsp70 in the guinea pig cochlea.     

Feasibility of pulse oxymetry to measure arterial O2 saturation in studies on cochlear blood circulation.

To understand the characteristics of oxygen transport to the inner ear, the relationship between arterial O2 saturation and cochlear microcirculation was investigated under different respiratory condition in guinea pigs. To monitor arterial O2 saturation a pulse oxymeter instead of an arterial blood gas analyzer was used. When the arterial O2 saturation was measured in the foot pad by a pulse oxymeter under different respiratory conditions, the data showed a close correlation with the results of blood gas analysis. For the measurement of cochlear microcirculation, a pulse oxymeter was found to be a feasible respiratory monitor for animal experiments. With this apparatus our study demonstrated a slower reaction in the decrease of perilymphatic oxygen tension than of cochlear blood flow during stepwise induction of hypoventilation monitored by a pulse oxymeter. Under certain conditions of hyperventilation in which arterial O2 saturation and perilymphatic oxygen tension increased gradually, cochlear blood flow was found to decrease. This decrease of cochlear blood flow could be attributed to chemical controls which are regulated, as in the cerebral blood circulation, by the content of CO2 and H+ in the vascular bed in the cochlea.     

Tissue resistivities determine the current flow in the cochlea

PURPOSE OF REVIEW: In individuals with severe to profound hearing loss, cochlear implants bypass normal inner ear function by applying electrical current directly into the cochlea, thereby stimulating cochlear nerve fibers. Stimulating discrete populations of spiral ganglion cells in cochlear implant users' ears is similar to the encoding of small acoustic frequency bands in a normal-hearing person's ear. Thus, spiral ganglion cells stimulated by an electrode convey the information contained by a small acoustic frequency band. Problems that refer to the current spread and subsequent nonselective stimulation of spiral ganglion cells in the cochlea are reviewed. RECENT FINDINGS: Cochlear anatomy and tissue properties determine the current path in the cochlea. Current spreads largely via scala tympani and across turns. While most of the current leaves the cochlea via the modiolus, the facial canal and the round window constitute additional natural escape paths for the current from the cochlea. Moreover, degenerative processes change tissue resistivities and thus may affect current spread in the cochlea. SUMMARY: Electrode design and coding strategies may result in more spatial stimulation of spiral ganglion cells, resulting in a better performance of the electrode-tissue interface.     

Distribution of type IV collagen in the cochlea in Alport syndrome

OBJECTIVE: To determine the distribution of alpha1, alpha3, and alpha5 chains of type IV collagen in the cochlea in Alport syndrome. DESIGN: Case-control study. PATIENTS: Two patients with sensorineural hearing loss due to Alport syndrome. Both patients had known mutations in the COL4A5 gene. MAIN OUTCOME MEASURES: Immunostaining was used to study the distribution of type IV collagen (alpha1, alpha3, and alpha5 chains) within the cochlea. Immunostaining was also performed in the cochlear tissues of an unaffected individual used as a control. RESULTS: In the control ear, alpha1 staining was observed in the basement membrane overlying the basilar membrane, in the basement membrane of cochlear blood vessels and Schwann cells, and within the spiral limbus. In the control ear, we also observed strong staining for alpha3 and alpha5 chains in the basement membrane overlying the basilar membrane and within the spiral ligament. In both cases with Alport syndrome, no immunostaining was observed for alpha3 or alpha5 chains within the cochlea, whereas alpha1 staining was present in locations similar to that seen in the control ear. CONCLUSIONS: The results indicate that isotype switching does not occur within the cochlea in Alport syndrome. The results are also consistent with the hypothesis that the sensorineural hearing loss in Alport syndrome may be due to alterations in cochlear micromechanics and/or dysfunction of the spiral ligament.     

Inner ear blood flow measured with a laser Doppler system

Cochlear blood flow was studied in guinea pigs, using a laser Doppler system during exposure to methoxamine hydrochloride and phentolamine hydrochloride, local cooling, or terminal asphyxia. Measurements were made through the intact lateral wall of the first turn of the cochlea. Observed changes in relationship to simultaneous measures of skin blood flow and BP indicated that this system provides a dynamic measure of inner ear blood flow. This technique may provide a strategy to evaluate the role of blood flow in normal and pathologic cochlear function and the utility of presumed cochlear vasoeffective agents proposed for treatment of certain inner ear disorders.     

实验性自身免疫性内耳病耳蜗热休克蛋白70的表达

目的　探讨自身免疫性内耳病模型动物耳蜗热休克蛋白的表达。方法　利用同种异体内耳抗原免疫豚鼠 ,以建立自身免疫性内耳病 (autoimmuneinnereardisease ,AIED)动物模型 ,并应用免疫组织化学及原位杂位技术 ,研究热休克蛋白 (heatshockprotein ,hsp) 70在正常对照组及AIED模型动物实验组耳蜗中的表达情况。结果　对照组豚鼠螺旋神经节细胞中存在hsp70样蛋白基础表达 ;实验组 2 8耳中 10耳 (35 7% )听阈提高≥ 10dB ;此组动物螺旋神经节细胞和血管纹、螺旋韧带hsp70及其mRNA表达明显增强。结论　以粗制膜迷路抗原免疫豚鼠 ,听阈提高动物hsp70在螺旋神经节细胞和血管纹、螺旋韧带合成增加。     

降钙素基因相关肽在豚鼠耳蜗中的分布

目的：观察降钙素基因相关肽(CGRP)在成年豚鼠耳蜗中的分布。方法：采用免疫组织化学，光镜观察的方法，对CGRP在豚鼠耳蜗中的分布进行研究。结果：CGRP阳性反应物分布于螺旋神经元．骨螺旋板神经孔，内毛细胞、外毛细胞与外支持细胞交界区，螺旋韧带血管纹。结论：CGRP广泛分布于豚鼠耳蜗，是耳蜗传出神经系统重要的神经递质或神经凋质，CGRP可能通过调节螺旋神经元、毛细胞和支持细胞等影响听功能．也可能影响耳蜗的血流。     

纯化内耳P0蛋白诱发实验性自身免疫性内耳病动物模型

目的：用从内耳组织中纯化的P0蛋白免疫豚鼠,建立P0蛋白诱发的自身免疫性内耳病动物模型,研究其在自身免疫性内耳病中的作用。方法：采用制备性SDS-PAGE从内耳组织中分离、纯化P0蛋白。以纯化的豚鼠内耳P0蛋白作为抗原免疫豚鼠,观察其听性脑干反应阈,血清中抗体水平和内耳形态学的改变,并用免疫组织化学法确定P0蛋白在耳蜗的分布情况。结果：SDS-PAGE结果显示纯化的蛋白质只在分子量为30 000的位置上出现单一的蛋白染色带,Western blot结果示该蛋白即为P0蛋白。免疫后有22%豚鼠的听性脑干反应阈升高,对照组动物无变化。实验组血清IgG显著升高（F=6.48,P〈0.01）,反应阈提高豚鼠的螺旋神经节细胞均有不同程度的数目减少,蜗轴小血管周围有炎性细胞浸润。P0蛋白在耳蜗仅分布于螺旋神经节、蜗轴神经纤维的髓鞘上。结论：用制备性SDS-PAGE能够成功地从内耳纯化P0蛋白,用于自身免疫性内耳病的研究。P0蛋白在部分豚鼠能够诱发自身免疫性内耳病,可能是自身免疫性内耳病的自身抗原之一。     

不同内耳组织抗原免疫致自身免疫性感音神经性聋的研究

目的：探讨不同内耳组织抗原免疫所致内耳主要病理损伤部位和听力障碍类型。方法：采用同种螺旋韧带（ＳＬ）、基底膜（ＢＭ）、螺旋神经节（ＳＧ）组织抗原免疫豚鼠，观察内耳组织病理改变和听觉功能变化。结果：ＳＬＡｇ和ＢＭＡｇ免疫组主要表现耳蜗微音器电位阈值升高和复聪现象，以及蜗管内和血管纹的免疫炎性病理改变；ＳＧＡｇ免疫组主要表现听神经复合动作电位阈值升高和幅值降低，内耳病理变化主要位于蜗轴血管及周围和ＳＧ     

Cochlear blood circulation studied with microspheres

The cochlear blood flow was studied with the microsphere method in guinea pigs, rats and cats. Results from experiments using different sizes of microspheres were compared and the effect of middle ear infection or perforation of the otic capsule on the cochlear microcirculation was investigated. Neither the opening of the cochlear bony wall nor the middle ear infection influenced the blood flow in the cochlea. 9- and 15-micrometer spheres were given simultaneously and the results were equivalent, indicating no more shunting of the smaller ones than of the bigger ones. Thus, the smaller spheres are to be preferred, since the smaller ones can be used in a larger number without negative effects on the general circulation. In cats, the cochlear blood circulation was determined before and during cervical sympathetic stimulation. Sympathetic stimulation caused a reduction of 25% of the cochlear blood flow.