Ethanol infiltration into the stapedio-vestibular joint reduces low-frequency vibration of the ossicular chain and round window membrane in the guinea pig

Background: The synovial stapedio-vestibular joint (SVJ), which serves as a bridge between the stape and oval window, can be found in guinea pigs and most human adults. Unlike the fibrous SVJs in other animals, the contribution of the synovial SVJ to middle ear sound transmission remains unknown.

Aims/objectives: In this study, we investigate whether sclerosis of the synovial SVJ contributes to frequency-dependent vibration of the ossicular chain and round window membrane (RWM).

Materials and methods: A model of SVJ sclerosis model was established in the guinea pig using 75% ethanol. A laser Doppler vibrometer was then used to measure vibrations of the RWM and the long process of the incus (LPI) under pure tone sound stimulations of 0.25–16?kHz. The influence of SVJ sclerosis was analysed by comparing structural vibration displacement between the normal and sclerosis groups.

Results: Both LPI and RWM vibrations significantly decreased at low frequencies after infiltration of ethanol, which caused SVJ sclerosis.

Conclusions: SVJ sclerosis reduces low-frequency vibration of the ossicular chain and RWM in the guinea pig, which indicates that the synovial SVJ is vital to low-frequency sound transmission in the middle ear.

Significance: Providing useful data for further research regarding middle ear biomechanics.     

Hyaluronan applied to lesioned round window membrane is free from cochlear ototoxicity

Hyaluronan (HYA) in 1% solution was instilled into the round window (RW) niche of rats (n = 6) prior to perforating the round window membrane (RWM). Cochlear functioning and structure were then monitored by recording auditory brainstem responses (ABRs) at 2-31.5 kHz and by scanning electron microscopy. Perforation of the RWM alone (n = 6) resulted in immediate loss of ABR thresholds between 6 and 31.5 kHz in 2 of 6 animals. Similar results were obtained after instilling HYA into the RW niche and subsequent RWM perforation (n = 6). After 2 months, ABR thresholds were recorded at all frequencies in the HYA-treated animals, whereas in 2 of the controls no ABR thresholds could be elicited at 20 and 31.5 kHz. However, in both treatment groups the mean ABR thresholds and mean latencies for wave II at the ABR threshold returned to the pre-surgical (normal) range after 2 months. With respect to the cochlear morphology the results in both treatment groups were also alike including minor structural changes in hair cell stereociliae but no loss of hair cells. It is concluded that HYA, when instilled into the middle ear with the inner ear opened, is free from cochlear otoxicity.     

Measurement of Basilar Membrane Motion During Round Window Stimulation in Guinea Pigs

Driving the cochlea in reverse via the round window membrane (RWM) is an alternative treatment option for the hearing rehabilitation of a nonfunctional or malformed middle ear. However, cochlear stimulation from the RWM side is not a normal sound transmission pathway. The basilar membrane (BM) motion elicited by mechanical stimulation of the RWM is unknown. In this study, the BM movement at the basal turn was investigated in both reverse via RWM drive and acoustic stimulation in the ear canal or forward drive in postmortem isolated temporal bone preparations of guinea pigs. During reverse drive, a magnet-coil was coupled on RWM, and the BM vibration at the basal turn and the movement of the incus tip were measured with laser Doppler vibrometry. During forward drive, the vibration of the incus tip induced by sound pressure in the ear canal resulted in BM vibration and the BM movement at the same location as that in the reverse stimulation was measured. The displacement ratio of the BM to RWM in reverse drive and the ratio of the BM to incus in forward drive were compared. The results demonstrated that the BM response measured in both situations was similar in nature between forward and reverse drives. This study provides new knowledge for an understanding of BM movement induced by reverse drive via the RWM stimulation.     

Influence of liquid volume in the middle ear on tympanic membrane vibration (experimental study by holographic interferometry)

The influence of middle ear liquid on the tympanic membrane (TM) vibration was studied using the holographic interferometry. The physiological saline solution was injected into the middle ear cavity of the canine temporal bone. The liquid volume in the middle ear cavity was changed into 3 grades in order to observe the difference of the vibration pattern between them. The vibratory patterns of a normal canine TM remained unchanged in their first mode up to a frequency of approximately 2 kHz. In this mode, there were peak displacement regions in the posterior and anterior quadrants. The TM vibrations showed sectional patterns, above 3 kHz in the posterior and above 4 kHz in the anterior. After liquid retention, the following were observed. 1. The frequency at which sectional patterns occurred shifted to a lower frequency in the immerged portion, but showed no definite tendency in the unimmerged portion. 2. Maximum amplitudes of the anterior and posterior quadrants of the TM and the mallar tip shifted to somewhat higher frequencies for the liquid volume below the level of the tip portion, but contrary, shifted to lower frequencies for the liquid volume there above. 3. The ossicular chain tended not to make normal function for the TM vibration with an increase of liquid volume.     

Experimental and Modeling Study of Human Tympanic Membrane Motion in the Presence of Middle Ear Liquid

Vibration of the tympanic membrane (TM) has been measured at the umbo using laser Doppler vibrometry and analyzed with finite element (FE) models of the human ear. Recently, full-field TM surface motion has been reported using scanning laser Doppler vibrometry, holographic interferometry, and optical coherence tomography. Technologies for imaging human TM motion have the potential to lead to using a dedicated clinical diagnosis tool for identification of middle ear diseases. However, the effect of middle ear fluid (liquid) on TM surface motion is still not clear. In this study, a scanning laser Doppler vibrometer was used to measure the full-field surface motion of the TM from four human temporal bones. TM displacements were measured under normal and disease-mimicking conditions with different middle ear liquid levels over frequencies ranging from 0.2 to 8 kHz. An FE model of the human ear, including the ear canal, middle ear, and spiral cochlea was used to simulate the motion of the TM in normal and disease-mimicking conditions. The results from both experiments and FE model show that a simple deflection shape with one or two major displacement peak regions of the TM in normal ear was observed at low frequencies (1 kHz and below) while complicated ring-like pattern of the deflection shapes appeared at higher frequencies (4 kHz and above). The liquid in middle ear mainly affected TM deflection shapes at the frequencies higher than 1 kHz.     

Math1基因内耳导入径路的探索研究

目的研究腺病毒携带Math1-EGFP基因经完整圆窗膜途径及鼓阶打孔途径导入耳蜗后对听功能和转导效率的影响,为内耳基因治疗提供实验基础和理论依据。方法健康成年白色红目豚鼠40只,雌雄不限,体重250—300g。随机分成四组,完整圆窗膜组12只,鼓阶打孔组12只,各组分别设对照8只。实验组（24只）导入重组腺病毒携带的Math1基因及增强型绿色荧光蛋白基因（enhanced green fluorescent protein,EGFP）,对照组（16只）导入人工外淋巴液,所有动物均以左耳作为导入耳。术前及术后分别行听性脑干反应（ABR）检查。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片观察基因表达情况。结果完整圆窗膜组导入耳ABR阈值,术后5天各频率与术前比较无显著性差异（P〉0．05）;鼓阶打孔组导入耳ABR阈值,术后5天在2kHz、4kHz与术前比较无差异（P〉0．05）,8kHz较术前增高（P〈0．05）,16kHz、20kHz较术前明显增高（P〈0．01）,术后14天在16kHz、20kHz较术后5天时明显好转（P〈0．01）,但较术前仍有增高（P〈0．05）。转导成功率鼓阶打孔组为91．6％,优于完整圆窗膜组的50％。两种转导途径对目的基因在耳蜗内的表达部位和表达时间没有显著影响。结论完整圆窗膜途径及鼓阶打孔途径在转导成功率及听功能保护方面各有优劣。完整圆窗膜途径因其对耳蜗的损伤极小,在临床应用方面具有更好的发展前景。     

Effect of round window membrane application of nitric oxide on hearing and nitric oxide concentration in perilymph

Nitric oxide (NO), a free radical, has been found to be important in the development of middle ear effusions. However, the effect of NO in the middle ear effusion on cochlear function and on perilymph concentrations of NO has not been reported. We placed S-nitroso-N-acetylpenicillamine (SNAP), a NO donor compound, on the round window membrane (RWM) of adult chinchillas. Auditory brainstem response (ABR) thresholds were measured before and after the placement of SNAP on the RWM and hourly for 8 h after SNAP placement. Samples of perilymph were collected 2 h after application of SNAP and were assayed for total nitrate and nitrite, the end products of NO. Experimental ears demonstrated significant ABR threshold elevations after 5 h and elevated nitrate/nitrite in the perilymph. These findings suggest that NO present in the middle ear passes through the RWM into the inner ear and can cause significant hearing loss.     

Outer- and middle-ear contributions to presbycusis in the Brown Norway rat

This paper examines the contribution of the outer and middle ears to the hearing loss associated with presbycusis in Brown Norway rats. Animals were formed into two groups; young adults (2-3 months old) and aged animals (approximately 34 months old). Auditory brainstem response (ABR) thresholds were obtained with the outer ear intact or surgically removed. Tympanic membrane (TM) velocity transfer functions were measured from the umbo with the outer ear removed. The length of the auditory meatus, TM surface area, and TM thickness were quantified. The ABR thresholds were 17-26 dB less sensitive in the aged animals between 8.0 and 40.0 kHz when the outer ear was intact. A significant and reliable reduction in the aged rat velocity transfer function of 5-8 dB occurred between 10.0 and 32.0 kHz, while the low frequency velocity response was only a few decibels greater in the younger animals. The ABR threshold differences between young adult and aged ears were compensated by removing the outer/middle ear effects of aging to reveal a purely sensorineural component of presbycusis. The outer and middle ear effects were calculated directly when the ABR and TM velocity data were obtained with the outer ear removed. The outer ear intact condition was modeled in order to compare the ABR data obtained with the outer ear intact with the TM velocity data obtained with the outer removed. With either procedure, removal of the age-related contributions of the outer and middle ear to the ABR threshold resulted in similar age-related ABR threshold shifts between the two age groups. The pure sensorineural threshold shift component of the ABR response was restricted to frequencies between 5.0 and 20.0 kHz and reached a maximum of approximately 15 dB. These results support the conclusion that there is an outer- and middle-ear contribution to the threshold loss defining presbycusis.     

Conductive Hearing Loss Induced by Experimental Middle-Ear Effusion in a Chinchilla Model Reveals Impaired Tympanic Membrane-Coupled Ossicular Chain Movement

Otitis media with effusion (OME) occurs when fluid collects in the middle-ear space behind the tympanic membrane (TM). As a result of this effusion, sounds can become attenuated by as much as 30–40 dB, causing a conductive hearing loss (CHL). However, the exact mechanical cause of the hearing loss remains unclear. Possible causes can include altered compliance of the TM, inefficient movement of the ossicular chain, decreased compliance of the oval window-stapes footplate complex, or altered input to the oval and round window due to conduction of sound energy through middle-ear fluid. Here, we studied the contribution of TM motion and umbo velocity to a CHL caused by middle-ear effusion. Using the chinchilla as an animal model, umbo velocity (V _U) and cochlear microphonic (CM) responses were measured simultaneously using sinusoidal tone pip stimuli (125 Hz–12 kHz) before and after filling the middle ear with different volumes (0.5–2.0 mL) of silicone oil (viscosity, 3.5 Poise). Concurrent increases in CM thresholds and decreases in umbo velocity were noted after the middle ear was filled with 1.0 mL or more of fluid. Across animals, completely filling the middle ear with fluid caused 20–40-dB increases in CM thresholds and 15–35-dB attenuations in umbo velocity. Clinic-standard 226-Hz tympanometry was insensitive to fluid-associated changes in CM thresholds until virtually the entire middle-ear cavity had been filled (approximately >1.5 mL). The changes in umbo velocity, CM thresholds, and tympanometry due to experimentally induced OME suggest CHL arises primarily as a result of impaired TM mobility and TM-coupled umbo motion plus additional mechanisms within the middle ear.     

Mechanisms of Tympanic Membrane and Incus Mobility Loss in Acute Otitis Media Model of Guinea Pig

Acute otitis media (AOM) is a rapid infection of middle ear due to bacterial or viral invasion. The infection commonly leads to negative pressure and purulent effusion in the middle ear. To identify how these changes affect tympanic membrane (TM) mobility or sound transmission through the middle ear, we hypothesize that pressure, effusion, and structural changes of the middle ear are the main mechanisms of conductive hearing loss in AOM. To test the hypothesis, a guinea pig AOM model was created by injection of Streptococcus pneumoniae. Three days post inoculation, vibration of the TM at umbo in response to input sound in the ear canal was measured at three experimental stages: intact, pressure-released, and effusion-drained AOM ears. The vibration of the incus tip was also measured after the effusion was removed. Results demonstrate that displacement of the TM increased mainly at low frequencies when pressure was released. As the effusion was removed, the TM mobility increased further but did not reach the level of the normal ear at low frequencies. This was caused by middle ear structural changes or adhesions on ossicles in AOM. The structural changes also affected movement of the incus at low and high frequencies. The results provide new evidence for understanding the mechanism of conductive hearing loss in AOM.     

Electrophysiological effects of the clinically used local anesthetics lidocaine,lidocaine-prilocaine and phenol on the rat's inner ear

Summary Local anesthetics, even if applied to the outer ear canal, may still enter the middle ear, running the risk of penetrating the round window. To elucidate the effect of certain topical anesthetics on the inner ear, the round window niche in the laboratory rat's middle ear was exposed separately to lidocaine, lidocaine-prilocaine and phenol. Auditory brain-stem responses (ABR) were recorded at 2, 4, 6, 8, 12, 16, 20, and 31.5 kHz before the application, and 24 h, 3 weeks, 2 months and 6 months after exposure. After terminating the 6-month ABR measurements, the animals were sacrificed and the temporal bones fixed and decalcified for light microscopic analysis. All three drugs affected the ABR thresholds and the cochlear morphology with a pattern characteristic for each drug. At 24 h, all three substances caused severe impairment of ABR thresholds, followed by a period of restitution lasting up to 2 months. Even 6 months after exposure, the ABR thresholds at and above 12 kHz were impaired, as compared with the pre-treatment level, for all substances tested. In the lower frequencies the original ABR threshold was reached in the order: (1) lidocaine, (2) lidocaine-prilocaine, (3) phenol. The cochlear structures were unaffected by lidocaine, whereas lidocaine-prilocaine and phenol caused morphological damage which was most pronounced after exposure to phenol. The heterogeneity of the changes in the ABR thresholds suggests differences in the mechanism of action of each type of local anesthetic investigated. The effects following lidocaine were transient. However, clinicians must be aware of the ototoxic potential of both lidocaine and phenol.     

Change of middle ear transfer function in otitis media with effusion model of guinea pigs

Otitis media with effusion (OME) is an inflammatory disease of the middle ear that causes most cases of conductive hearing loss observed in the pediatric population. With the long term goal of evaluating middle ear function with OME, the aim of the current study was to create an animal model of OME in which middle ear transfer functions could be measured. In guinea pigs, OME was created by injecting lipopolysaccharide (LPS) into the middle ear. Evidence of OME was assessed by otoscopy, tympanometry, histology, and by measuring the volume of fluid in the middle ear. Vibrations of the umbo and round window membrane were measured with a laser Doppler vibrometer at frequency range of 200-40kHz in three groups of 3, 7, and 14 days after injection of LPS. Changes in displacement of the umbo and round window membrane in response to 80dB SPL sound in the ear canal were measured across the frequency range. Displacement of both the umbo and round window membrane was reduced at all time points following LPS injections. Further, the change of the displacement transmission ratio (DTR) from the tympanic membrane to the round window occurred mainly in chronic (e.g. 14 days post-LPS injection) OME ears. This study provides useful data for analyzing the change of middle ear transfer function in OME ears.     

虚拟耳镜对鼓室成形术前后鼓室结构的观察

目的 探讨虚拟耳镜在鼓室成形术中的临床评估作用.方法 应用虚拟耳镜观察耳病患者102例(204耳),72例(75耳)完成鼓室成形术和外耳道鼓室成形术,其中慢性中耳炎53例(55耳),先天性外耳道闭锁1 9例(20耳).结果 23耳慢性化脓性中耳炎胆脂瘤型,虚拟耳镜观察19耳听骨链破坏,手术证实23耳均有听骨腐蚀;32慢性化脓性中耳炎骨疡型,虚拟耳镜观察29耳听骨链不同程度破坏,手术证实23耳锤骨和砧骨侵蚀,11耳镫骨头或镫上结构缺失;20耳先天性外耳道闭锁,中耳畸形,术前提示18耳听骨畸形,2耳小鼓室无听骨链,手术证实17耳外耳道闭锁,听骨严重畸形,2耳镫骨缺失,1耳前庭窗闭锁.2耳术后突然听力下降,复查虚拟耳镜发现移植听骨与鼓膜脱离.虚拟耳镜与手术探查符合率,中耳炎为92%,先天性外耳道闭锁、中耳畸形为100%.结论 虚拟耳镜为鼓室成形术术前病变程度及术后疗效评估提供可靠的影像学依据.     

听骨链CT扫描并三维重建的临床应用

目的:探讨CT仿真内镜(CTVE)显示中耳正常结构的显示方法,评价中耳病变特别是听骨链连接状态的价值及意义,确立显示听骨链及其他中耳结构的位点和方法。方法:选择40例无耳科疾病的志愿者(正常组)和30例疑有中耳疾病的患者(病例组),分别采用德国西门子(Siemens SOMATOM Sensation 16)螺旋CT机Inner Ear扫描程序对患者进行轴位扫描,重建原始图像,用Fly-through软件经选择的A、B、C点入路进行CTVE成像研究。重点显示听骨链连接状态,并对慢性中耳炎的显示结果与手术中探查图像进行对照。结果:正常组CTVE中锤骨、砧骨、鼓岬、面神经管水平段、外半规管隆凸显示率均为100%;而镫骨头、两足弓的显示率在3个显示层面则分别为57.5%、70.0%,97.5%;圆窗、卵圆窗为90.0%、93.0%、97.5%。在中耳炎、外伤、颞骨畸形等情况下可显示听小骨损伤、移位、中断、缺失和畸形。结论:CTVE在显示3个听小骨之间的链接关系(如中断等方面)有一定优势。通过选择恰当的入路,CTVE在显示听小骨及其链接关系以及病理状态方面有相当优势。通过比较CTVE在3个不同显示层面观察镫骨的能力,明确CTVE是断层影像的有利补充。     

Transmission of changes in the external ear atmospheric pressure to the perilymph

Using guinea pig, pressures in the external ear canal, in the middle ear and in the perilymph were registered simultaneously, while pressure was applied to the external ear canal using an impedance audiometer. In the first experiment, applied pressure was changed in the range from 200 mmH2O to -200 mmH2O with and without the opening of the otic bulla. The change in the perilymphatic pressure with the opening was smaller than that without the opening. The result indicates that the external ear pressure is transmitted to the perilymph not only via the ossicular chain but also via the middle ear cavity without the opening, while it is exclusively transmitted via the ossicular chain with the opening. Pressure transmission to the perilymph was significantly impaired either by disrupting the ossicular chain or by closing the round window niche, especially by the latter. Thus the middle ear cavity itself plays an important role in pressure transmission from the external ear canal to the perilymph mainly via the round window. In the second experiment, applied pressure to the external ear canal was changed in the range from 1000 mmH2O to -1000 mmH2O after the Eustachian tube being closed. Between 400 mmH2O and -200 mmH2O, the middle ear and perilymphatic pressures paralleled well with the applied pressure. Beyond these levels, the middle ear pressure increased or decreased in response to the applied pressure but the perilymphatic pressure reversed against the middle ear pressure. Communication between perilymph and cerebrospinal fluid via the cochlear aqueduct is thought to be a major factor causing this reversal which, in turn, aggravates pressure gradient between the middle ear and the perilymph.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ossicular vibration in human temporal bones

Vibration mode of the ossicles was investigated in twelve fresh human temporal bones using a video measuring system (VMS, Technical Instrument). This system allows one to observe the ossicular vibration and to measure its vibration amplitude (up to 0.2 micron) and phase angle. In this study the inner and middle ear was kept intact except for two small holes in the tympanic tegmen. These holes were for the observation of ossicular movement and were covered with a thin cover glass during the experiment. The vibration amplitude and phase angle of the umbo, malleus head, lenticular process and stapes head were measured at 19 frequencies between 0.1 kHz and 4.5 kHz. The umbo moved piston-like at 0.1-0.8 kHz and 2.6-4.5 kHz but in an ellipse at 1.0-2.4 kHz. The malleus head showed elliptical movement with its long axis anteriorly tilted around 45 degrees from the direction of the umbo vibration at 0.1 kHz. Both the lenticular process and stapes head showed similar movement; piston-like in lower frequencies and elliptical in higher frequencies. The umbo, lenticular process and stapes head vibrated parallel at lower frequencies. The position, displacement and phase angle of the rotation axis of the ossicles was calculated based on the displacement and phase angle of the umbo, malleus head and lenticular process. The axis was around the level of the neck of the malleus in frequencies lower than the resonant frequency, beneath the level of the short process near the resonant frequency and at the top of the malleus head in higher frequencies. The average axis displacement was 0.9 microns at 1 kHz but much smaller at frequencies lower than 0.8 kHz or higher than 2 kHz. This suggests that such axis movement may reduce the efficiency of the middle ear sound transmission.     

腺病毒携带EGFP基因经完整圆窗膜转导豚鼠耳蜗的实验研究

目的研究腺病毒携带目的基因经完整圆窗膜途径耳蜗转导的可行性及安全性，为内耳基因治疗提供实验基础和理论依据。方法20只白色红目豚鼠，术前及术后分别行听性脑干反应（ABR）检查。实验组（12只）以重组腺病毒携带的增强型绿色荧光蛋白基因（enhancedgreenfluorescentprotein，EGFP），对照组（8只）以人工外淋巴液注入豚鼠圆窗龛内。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片，耳蜗冰冻切片观察。结果于圆窗龛内注入腺病毒携带目的基因的转导方法对听力无明显影响。转染耳蜗及对侧耳蜗内目的基因呈广泛表达。5天组表达产物最高，14天组逐渐降低。对照组耳蜗未见EPFP表达。结论于圆窗龛内注入腺病毒携带目的基因转导的方法对耳蜗无明显毒害作用，且能够将目的基因成功转导至双侧耳蜗组织并广泛表达。     

自体砧骨听骨链成形术的听力重建效果

目的探讨以自体砧骨重建听骨链的开放式IIIa型鼓室成形术的听力改善疗效。方法回顾性收集16例(16耳)因慢性化脓性中耳炎或胆脂瘤中耳炎接受开放式Ⅲa型鼓室成形术患者的临床资料,所有病例均采用自体砧骨进行听骨链重建。术后随访3-6个月,评估患者手术后听力改善情况,分析指标为手术前后言语频率的纯音听阈及气骨导差。结果所有均达到干耳。纯音气导听阈从术前的45.2到干耳。降至术后的30.6的干耳。纯音气导听阈(P<0.05)。术前、术后气骨导差分别为33.1、术后7dB及19.8、术后7dB分别为从术前的析,其中气骨导差小于20dB者占62.5%(10/16)。结论在本组病例中,以自体砧骨行听骨链重建开放式Ⅲa型鼓室成形术,获得了较好的近期听力改善效果,其远期疗效有待于进一步观察。     

Experimental study of an adjustable-length prosthesis in a temporal bone model

CONCLUSIONS: This prosthesis has the advantage of rapid adjustment at the time of insertion in order to achieve optimal tension and, as a result, optimal sound transmission. OBJECTIVE: To test the acoustic performance of a new, adjustable incus replacement prosthesis in a human temporal bone model. MATERIAL AND METHODS: Experiments were performed in seven human temporal bones, before and after removal of the incus and insertion of the prosthesis. The input comprised 406 pure tones ranging in frequency between 0.1 and 10 kHz at an intensity of 80 dB SPL at the tympanic membrane. The output measurement was stapes footplate displacement, determined by means of a laser Doppler vibrometer. Three lengths of the prosthesis were investigated: optimal, optimal +0.2 mm and optimal +0.4 mm. RESULTS: The optimal-length prosthesis produced similar results to those of an intact middle ear. The slightly longer prostheses decreased middle ear sound transmission at all test frequencies, except those near 1.5 kHz.     

Hearing loss in relation to round window membrane morphology in experimental chronic otitis media.

The present study was performed to test the effect of single and repeated Pseudomonas aeruginosa exotoxin A (PaExoA) instillations in the middle ear of the rat. The hearing level was examined by the ABR technique, round window membrane (RWM) thickness was measured and morphology was studied by light microscopy. The results showed both reversible and permanent hearing loss (HL). In animals that received a single dose of PaExoA, the RWM thickness doubled initially and remained thickened during the observation period. When PaExoA was instilled on several occasions, RWM thickness doubled, before decreasing to near-control levels. This study confirms the toxicity of PaExoA and the partially reversible HL occurring after a single application of the toxin. The diminished effect of repeated toxin instillations--despite the decreasing thickness of the RWM--is discussed.