Effects of Hyperbaric Therapy on Function and Morphology of Guinea Pig Cochlea with Endolymphatic Hydrops

Transcription

1 Otology & Neurotology 25: , Otology & Neurotology, Inc. Effects of Hyperbaric Therapy on Function and Morphology of Guinea Pig Cochlea with Endolymphatic Hydrops Fang-Lu Chi, Qin Liang, and Zheng-Min Wang Department of Otolaryngology, Eye Ear Nose and Throat Hospital, Fudan University, Shanghai, P.R. China. Objective: The objective of this study was to investigate the effect on experimental endolymphatic hydrops in guinea pigs after hyperbaric therapy. Background: The histopathologic character of Ménière s disease is the presence of endolymphatic hydrops. Endolymphatic hypertension could be one of the factors resulting from endolymphatic hydrops. Some treatments of Ménière s disease are aimed toward preventing the endolymphatic hypertension. Exposure to pressure change has risen in recent years. Methods: Thirty-two guinea pigs were operated on the right ears to induce endolymphatic hydrops by obliterating the endolymphatic sac through an extradural posterior cranial fossa approach. After 5 weeks survival, 12 guinea pigs were put into a chamber with an absolute atmospheric pressure of 2.2 for 3 weeks (90 minutes once a day 5 times a week). We observed the morphologic and functional changes in guinea pig cochleae of the pressure group, 4-week hydrops group (n 10), 8-week hydrops group (n 10), and the normal group (n 10). We measured the hearing threshold of the auditory brainstem response, the 70-dB SPL action potential (AP) latency, the ratio of 70-dB SPL summating potential magnitude to action potential magnitude (-SP/AP) of the electrocochleogram, and the maximum scala media area (SMA) ratio, respectively. Results: The average 70-dB SPL SP/AP magnitude of right ears (0.29 ± 0.09) and the average maximum SMA ratio (2.23 ± 0.20) in the pressure group were significantly less than that in the 8-week hydrops group (0.69 ± 0.15 and 4.04 ± 0.52, respectively) with the same survival time (p < 0.05). The results in the pressure group were almost as similar as that in the 4-week hydrops group (0.29 ± 0.13 and 2.22 ± 0.20, respectively) (p > 0.05). The average hearing threshold of ABR of right ears in the pressure group (36.67 ± dB SPL) was lower than that of the 8-week hydrops group (44 ± dB SPL), but the difference was insignificant (p > 0.05). The average 70-dB SPL AP latency of right ears in the pressure group was not significantly different from those of the 8-week hydrops group, the 4-week hydrops group, or the normal group (p > 0.05). Conclusions: Our findings suggest hyperbaric therapy can significantly suppress the development of endolymphatic hydrops and improve cochlear function in guinea pigs. This study provided strong evidence for the development of pressure treatment of Ménière s disease without destroying the inner ear. Key Words: Endolymphatic hydrops Therapy pressure Guinea pig Histology Auditory brainstem response Electrocochleogram. Otol Neurotol 25: , The etiology and pathogenesis of Ménière s disease are still unknown. Since Hallpike and Cairns (1938) discovered the presence of an endolymphatic hydrops in the temporal bones of patients with Ménière s disease, endolymphatic hydrops has generally been considered to be the histopathologic basis of Ménière s disease. This finding has led to the assumption that increased endolymphatic pressure could be one of the reasons for inner ear dysfunction in these patients. This hypothesis has been confirmed by previous experimental studies (1 6). Address correspondence and reprint requests to Dr. Fang-Lu Chi, Department of Otolaryngology, Eye Ear Nose and Throat Hospital, Fudan University, 83 Fenyang Road, Shanghai, , P.R. China. chifanglu@yahoo.com.cn In some extension, management of Ménière s disease is aimed at reducing endolymphatic pressure. Until now, there are three different methods of reducing a high endolymph pressure: application of diuretics, decompression of the endolymphatic sac, and exposure to pressure change. Since 1975, middle ear overpressure has been introduced to treat Ménière s disease (7). Previous studies showed that relief of acute attacks of Ménière s disease was achieved by placing the patients in an under-pressure chamber and instructing them not to perform active equilibration of middle ear pressure while pressure is reducing (7 11). Subsequently, it has been demonstrated that direct application of overpressure to the external auditory canal improves hearing in patients with Ménière s disease (12,13) and inhibits the development of endolymphatic hydrops in guinea pigs (14). Re- 553

2 554 F.-L. CHI ET AL. cently, the new, nondestructive device called Meniett has been used in patients with Ménière s disease and showed significant improvement of symptoms, primarily of vertigo, but also in terms of tinnitus and hearing (15 20). Fattori et al. (21,22) found that patients treated with alternobaric oxygen therapy (ABOT), at the end of the 2-year follow-up period, had significantly fewer vertiginous episodes and improved pure-tone average (PTA) and tinnitus were noted, compared with the controls. In recent years, Feijen et al. (23,25), Thalen et al. (24,26), and Wit et al. (27) have studied the relationship between inner ear pressure change and intracranial or middle ear pressure manipulation. In this study, to develop a nondestructive treatment of Ménière s disease, and to clarify the efficacy and mechanism of hyperbaric therapy for patients with Ménière s disease, we established an animal model of experimental endolymphatic hydrops by blockage of the endolymphatic sac and duct, then conducted electrophysiological measurements of the cochlea and quantified the degree of hydrops. As a result, we determined whether application of hyperbaric therapy could prevent the development of the endolymphatic hydrops. MATERIALS AND METHODS Forty-two healthy albino male guinea pigs weighing 300 to 450 g were used in this experiment. These animals were divided randomly into four groups. Ten normal animals without operation on both ears served as control (group 1). Thirty-two animals were operated on the right ear to induce endolymphatic hydrops by obliteration of the endolymphatic sac and duct through an extradural posterior cranial fossa approach (16). Ten animals survived for 4 weeks (group 2, n 10), and another 10 animals survived for 8 weeks (group 3, n 10) and the left 12 animals also survived for 8 weeks (group 4, n 12), respectively. After 5 weeks survival, the animals in group 4 underwent hyperbaric treatment in a pressure chamber for 3 weeks (90 min once a day 5 times a wk), including 17 minutes of compression time, 60 minutes of stable compression time with 2.2 absolute atmospheric pressure, and 13 minutes of decompression time. Surgical Procedure The operation was performed under sterile conditions with a Zeiss operating microscope. Anesthesia was induced with a combination of 10 mg/kg ketamine hydrochloride intramuscularly and 30 mg/kg 1% pentobarbital sodium subcutaneously. During the course of the operation, the temperature was stabilized using an electric heating pad. Through an extradural posterior cranial fossa approach, the endolymphatic sac was identified after exposing the sigmoid sinus through the occipital bone and moving it slightly medial. The endolymphatic sac was obliterated using a 0.5-mm diamond burr, and aseptic bone wax was interposed between the sigmoid sinus and the endolymphatic duct using a small otologic elevator (28). Electrophysiological Measurements The electrophysiological measurements were conducted on both ears of each animal preoperatively and 8 and/or 4 weeks postoperatively using a brain stem analyzer (DANPLEX BRA 2, Siemens) in a sound electric screen room, which included an auditory brainstem response and an electrocochleogram. The animals were anesthetized with 10 mg/kg ketamine hydrochloride intramuscularly and 30 mg/kg 1% pentobarbital sodium subcutaneously and placed in a box with two holes where earphones were put on. Auditory Brainstem Response A brainstem analyzer was used to obtain the auditory brainstem response (ABR) threshold. Electrodes were placed on both mastoids (reference electrode and ground electrode) and vertex (recording electrode). The animals were given 0.1-ms alterative clicks sound with a 10-ms time window value and 10 to 80-dB SPL stimulus intensity. There hundred click stimuli are applied. The ABR threshold of the guinea pigs was given by the computer automatically. Electrocochleogram The electrodes were placed on both mastoids (reference electrode and ground electrode) and either right or left posterior superior par of external ear canal clung to the bone (recording electrode). The animals were given 0.1-ms alterative clicks sound with a 10-ms time window value and 10 to 80-dB SPL stimulus intensity and -10-dB SPL masking. Three hundred click stimuli were given. We measured the 70-dB SPL action potential (AP) latency, the ratio of 70-dB SPL summating potential magnitude to action potential magnitude (-SP/AP) of electrocochleogram (EcochG). Fixation Procedure The animals were killed by intracardiac perfusion and the temporal bones were removed and fixed with 4% formaldehydum polymerisatum (ph 7.4). The temporal bones were decalcified in 5% trichloroacetic acid for approximately 5 days and dehydrated in a graded ethanol series. Specimens were processed in celloidin and serially sectioned at 12 m. Cochlear midmodiolar sections were stained with hematoxylin and eosin, and then observed under light microscope. Measurement of Hydrops Two midmodiolar sections per ear were selected at random and photographs were taken with a digital camera (Nikon Cool PIX990) under a light microscope (4 10). This procedure was performed on both ears of each animal. The figures were input to a computer for quantitative analysis using auto-computedaided design software (AutoCAD R14). According to the method described by Klis et al. (29), we measured the area of scala media (SM) and scala vestibuli (SV) in each turn of the cochlea. Thus, we obtained four measurements per turn, and calculated the ratio of SM area to SM + SV area for each turn in both ears of each animal, so-called the proportional SM area. Then we divided the proportional SM area from the right ear by the proportional SM area from the left ear and obtained the SMA ratio. We used the maximum of the SMA ratio as the index of endolymphatic hydrops degree. In formula: SM area SM area + SV area right SMA ratio = SM area SM area + SV area left Statistical Analysis Statistical analysis of the data of four groups was conducted with Student-Newman-Keuls test using the Statistical Analysis System.

3 FUNCTION AND MORPHOLOGY OF GUINEA PIG COCHLEA 555 RESULTS Electrophysiological Measurements ABR Threshold of Right Ears The average ABR threshold of right ears in the 4-week hydrops group (25 ± 5.27-dB SPL) was greater than that in the normal group (19 ± 3.16-dB SPL). The threshold of the 8-week hydrops group (44 ± dB SPL) was greater than that of the 4-week hydrops group. Both were significantly different (p < 0.05). After hyperbaric treatment, the average threshold of ABR of right ears in group 4 (36.67 ± dB SPL) was less than that of the 8-week hydrops group and greater than that of the 4-week hydrops group; however, the difference had no significance (p > 0.05). 70-dB SPL AP Latency of Right Ears The results of the average 70-dB SPL AP latency of right ears were not significantly different among the normal group (1.74 ± 0.13 ms), the 4-week hydrops group (1.76 ± 0.15 ms), the 8-week hydrops group (1.86 ± 0.16 ms), and the pressure group (1.77 ± 0.18 ms) (p > 0.05). 70-dB SPL SP/AP Magnitude of Right Ears The average 70-dB SPL SP/AP magnitude of right ears in the 4-week hydrops group (0.29 ± 0.13) was more than that in the normal group (0.16 ± 0.10). The magnitude of the 8-week hydrops group (0.69 ± 0.15) was greater than that of the 4-week hydrops group. These was significantly different (p < 0.05). After pressure treatment, the average 70-dB SPL SP/AP magnitude of right ears (0.29 ± 0.09) in the pressure group was significantly less than that in the 8-week hydrops group (p < 0.05) but similar to the result in the 4-week hydrops group (p > 0.05). Degree of Endolymphatic Hydrops There was no endolymphatic hydrops in both ears of the control group (group 1) and the left ears of the experimental groups (group nos. 2, 3, and 4). The angle between Reissner s membrane and basilar membrane was approximately 45 (Fig. 1). Obvious morphologic changes in right ears of the 4-week hydrops group were noted, that different degrees of endolymphatic hydrops in each turn was observed, endolymphatic hydrops in the apical turn was greater than that in the basal turn. Reissner s membrane bulged to scala vestibuli (Fig. 2). The distension of Reissner s membrane in right ears of the 8-week hydrops group was more obvious than that in the 4-week hydrops group (Fig. 3). The average maximum SMA ratio in the 4-week hydrops group (2.22 ± 0.20) was greater than that in the control group (1.10 ± 0.07). The average maximum SMA ratio of the 8-week hydrops group (4.04 ± 0.52) was greater than that of the 4-week hydrops group. There was a significant difference (p < 0.05). After 3 weeks of pressure treatment, the development of endolymphatic hydrops in group 4 was inhibited, especially in the first and second turns. The average FIG. 1. Midmodiolar section of cochlea in both ears of the control group (group 1) and the left ears of the experimental groups (groups 2, 3, and 4), showing that the angle between Reissner s membrane and basilar membrane was approximately 45, implying no endolymphatic hydrops. This also shows the way we drew the outline of scala media (SM) and scala vestibuli (SV) on each turn of cochlea for quantitative analysis using auto-computedaided design software (AutoCAD R14). The average maximum SMA ratio in the control group was 1.10 ± maximum SMA ratio (2.23 ± 0.20) in group 4 was significantly less than that in the 8-week hydrops group (p < 0.05) (Fig. 4). It was similar to that in the 4-week hydrops group (p > 0.05) (Fig. 5). DISCUSSION The histopathologic character of Ménière s disease is the presence of endolymphatic hydrops. Endolymphatic hypertension could be one of the factors resulting in FIG. 2. Midmodiolar section of cochlea in right ears of the 4-week hydrops group showing the main morphologic changes with the different degrees of endolymphatic hydrops in each turn. Reissner s membrane bulged to scala vestibuli. The average maximum SMA ratio in the 4-week hydrops group (2.22 ± 0.20) was greater than that in the control group (1.10 ± 0.07) (p < 0.05).

4 556 F.-L. CHI ET AL. FIG. 3. Midmodiolar section of cochlea in right ears of the 8-week hydrops group showing the distension of Reissner s membrane was more significant than that in the 4-week hydrops group. The average maximum SMA ratio of the 8-week hydrops group (4.04 ± 0.52) was greater than those of the 4-week hydrops group (2.22 ± 0.20) (p < 0.05). endolymphatic hydrops (1 6). In this case, several treatments for Ménière s disease were developed that aimed toward preventing the endolymphatic hypertension. For more than 100 years, the influence of middle ear pressure on inner ear fluids has been investigated (10). Vertigo induced by changes in atmospheric pressure has been known in divers and caisson workers, resulting from decompression sickness with bubble formation in the inner or hemorrhages in the labyrinths. Since 1975, middle ear overpressure has been introduced to treat Ménière s disease (7). Pressure treatments include general treatment and local treatment. In some studies, relief of acute attacks of Ménière s disease was achieved by placing the patients in an underpressure FIG. 4. After 3 weeks of pressure treatment, the degrees of endolymphatic hydrops of group 4 were inhibited, especially in the first and second turns. The average maximum SMA ratio (2.23 ± 0.20) in group 4 was significantly less than that in the 8-week hydrops group (p < 0.05). chamber and instructing them not to perform active middle ear pressure equilibration while pressure reducing (7 11). Subsequently, it has been demonstrated that direct application of overpressure to the external auditory canal improves hearing in patients with Ménière s disease (12,13) or prevents the development of experimental endolymphatic hydrops in guinea pigs (14). Recently, a great progress was made in pressure treatment of Ménière s disease; a new, nondestructive device called Meniett has been used in patients with Ménière s disease and showed significant improvement of symptoms, primarily of vertigo, but also in terms of tinnitus and hearing (15 20). Fattori et al. (21,22) found that hyperbaric (2.2 ATA) and, in particular, alternobaric treatment ( ATA) permitted a significant control of the principal attacks of vertigo during a follow up of 2 years. Hearing loss also showed a more significant and more persistent improvement in the patients treated with alternobaric oxygenation. Therefore, the change of atmospheric pressure could have some effect on body, including the inner ear. The present study demonstrates that 3 weeks of hyperbaric treatment can significantly inhibit the development of endolymphatic hydrops and improve cochlear function in guinea pigs. This study provided an experimental base for the development of pressure treatment without trauma for Ménière s disease. However, the mechanism of pressure treatment of Ménière s disease is unknown. Several hypotheses that could contribute to this inhibition of hydrops are proposed. For the sake of explaining the relief of endolymphatic hydrops by hyperbaric treatment, we consider both mechanical and physiological factors. Mechanical Effects The main pressure transduction channel between endolymph fluid and intracranial fluid is the cochlear aqueduct. Experimental observations of inner ear fluid dynamics studied by Salt and DeMott (30) have indicated that induction of changes in the perilymphatic pressure could produce longitudinal movement of the endolymph. Recently, Feijen et al. (23,25), Thalen et al. (24,26), and Wit et al. (27) have studied the relationship between inner ear pressure change and intracranial or middle ear pressure manipulation, showing that the pressure equalization process is nonlinear. This nonlinearity could be a consequence of the dependence of the compliance and/or flow resistance on pressure. According to the theory mentioned here, we think mechanical effects play an important role. First, increased middle ear pressure could facilitate endolymph drainage through the cochlear aqueduct as a result of increasing perilymph fluid pressure. In our study, guinea pigs in group 4 underwent hyperbaric treatment. Increased pressure was mainly transmitted to the middle ear through eustachian tube and then to perilymph fluid through the round window. High pressure could also be partly transmitted to the tympanic membrane, ossicular chain, and finally to the perilymph through the vestibular window.

5 FUNCTION AND MORPHOLOGY OF GUINEA PIG COCHLEA 557 FIG. 5. Endolymphatic hydrops developed progressively after obliterating the endolymphatic sac, and hyperbaric treatment in guinea pigs can prevent the development of the endolymphatic hydrops. As a result, perilymph fluid pressure increases, pushing the Reissner s membrane back and discharging excessive endolymph fluid through the endolymphatic duct and sac. Finally, it inhibits the development of endolymphatic hydrops and improves cochlear function in guinea pigs. Second, increased middle ear pressure could reduce endolymphatic hydrops. When the pressure in the middle ear cavity is relatively lower, blood vessels of the middle ear mucosa are congested with blood to compensate for the relative lower pressure in the middle ear. In contrast, high pressure in the middle ear causes constriction of the middle ear vascular bed. It was suggested that a pressure transmission from the middle ear to the inner ear might induce constriction of the vascular bed in the inner ear similarly after reduction of endolymphatic hydrops (7,8). Physiological Effects The cochlear microcirculation is impaired in the hydropic ear. Increased endolymphatic pressure inevitably causes increasing mechanical pressure on the stria vascularize and the blood vessels of the spiral ligament in the cochlea and interferes with inner ear function (6). The diameters and velocities of stria vascular vessels and spiral ligament vessels decreased in experimental endolymphatic hydrops (31,32). Milier et al. (33) used laser Doppler flowmetry to assess hydrops-induced change in cochlear blood flow (CBF). They found that the magnitude of the change of CBF elicited by local electrical stimulation of the cochlea and direct stimulation of the superior cervical ganglion was reduced by approximately 30% and one third in the hydropic ear compared with a normal ear. Rhythmic (flux motion or vasomotion) variations and the autoregulatory response to a decreased perfusion pressure in CBF were reduced in the hydropic ear. These experiments suggested that the cochlear microcirculation in the hydropic ear is impaired, leading to inner ear anoxia. There is a decreased endolymphatic potential and an increased endolymphatic Ca 2+ concentration in experimental hydropic endolymph compared with the normal ear (14,34). These changes have also been observed in the anoxemic ear (14). Hence, increased oxygen tension could improve the situation of inner ear anoxia and the cochlear microcirculation disorder, which lead to decreased endolymphatic hydrops, thereby suppressing the development of endolymphatic hydrops and improving cochlear function. In our pressure group, the level of hyperbaric treatment was 2.2 absolute atmospheric (ATA) pressure. This pressure is 2.2 times higher than that of the standard atmospheric pressure. The oxygen tension in the middle ear was increased through the eustachian tube in the hyperbaric chamber, and subsequently the oxygen tension in the inner ear could have also increased. Increased oxygen tension in the inner ear could improve the electrogenic activity of the stria vascularize related to Na + - K + -ATPase, the energy-yielding enzyme, and the cytochrome oxidase. These changes result in a decrease in endolymphatic Ca 2+ concentration and a reduction of the osmotic pressure, thereby minimizing hydropic changes (14). In addition, hyperbaric treatment could decrease the permeability of blood and lymphatic vessels in the inner ear and then reduce its exudation, thereby improving the microcirculation of the inner ear and suppress endolymphatic hypertension, which lead to the remission of its ill effect on the cochlear receptor. Our findings suggest hyperbaric therapy can significantly prevent the development of endolymphatic hydrops and improve cochlear function in guinea pigs. It could provide strong evidence for the development of hyperbaric treatment without trauma for Ménière s disease. REFERENCES 1. Bohmer A. Hydrostatic pressure in the inner ear fluid compartments and its effects on inner ear function. Acta Otolaryngol (Stockh) 1993;Suppl 507:3 24.

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