Ann. N.Y. Acad. Sci. ISSN 0077-8923
A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S Issue: Dizziness and Balance Disorders
Meniere’s disease: histopathology, cytochemistry, and imaging Gail Ishiyama,1 Ivan A. Lopez,2 Ali R. Sepahdari,3 and Akira Ishiyama2 1
Department of Neurology, Reed Neurological Research Center, David Geffen School of Medicine at UCLA, Los Angeles, California. 2 Department of Head and Neck Surgery, Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, California. 3 Department of Radiology, David Geffen School of Medicine at UCLA, Los Angeles, California Address for correspondence: Gail Ishiyama, M.D., Department of Neurology Reed Neurological Research Center, UCLA David Geffen School of Medicine, 710 Westwood Blvd., Box 951769, Los Angeles, CA 90095. [email protected]
Meniere’s disease is a poorly understood, disabling syndrome causing spells of vertigo, hearing fluctuation, tinnitus, and aural fullness. In this paper, we present a review of the histopathology, cytochemistry, and imaging of M´eni`ere’s disease. Histopathology is significant for neuroepithelial damage with hair cell loss, basement membrane thickening, and perivascular microvascular damage. Cytochemical alterations are significant for altered AQP4 and AQP6 expression in the supporting cell, and altered cochlin and mitochondrial protein expression. Current developments include imaging techniques to determine the degree and presence of endolymphatic hydrops, and future studies will endeavor to correlate the observance of hydrops with clinical findings. Keywords: endolymphatic hydrops; inner ear; Meniere’s disease; vestibular; MRI imaging; aquaporins
Introduction Meniere’s disease (MD) is a disabling syndrome characterized by fluctuating hearing loss, episodic vertigo, ear fullness, and tinnitus. In 1861, Prosper M´eni`ere was accredited with describing this clinical syndrome as an inner ear labyrinthine dysfunction, rather than a central neurological disorder.1 It was not until almost 80 years later that the most prominent and consistent pathological correlate of MD was revealed in postmortem human temporal bone studies: endolymphatic hydrops (EH), the dilation of the membranous labyrinth of the inner ear. In two simultaneous publications on temporal bone histopathology by Yamakawa2 and Hallpike and Cairns,3 these independent findings of EH were noted in subjects that had MD. Schuknecht conducted detailed postmortem temporal bone histopathology on subjects with a history of MD and reported various abnormal pathologies, including distension of Reissner’s membrane, a ballooning out of the saccular wall, ruptures of the membranous labyrinth, and
sometimes fistulae. However, he reported the relative sparing of the neuroepithelia, the region of the vestibular end organ containing the receptors for balance, the vestibular hair cells.4 It had been proposed that EH leads to the symptoms of MD—vertigo spells and hearing loss—because of potassium intoxication of the hair cells during membrane ruptures, allowing the potassium-rich endolymph to paralyze the sensory hair cells that are located within the perilymph. Temporal bone studies demonstrated that nearly all cases of MD are associated with EH;4 however, not all EH cases are associated with the symptoms of MD.5 In a study using an archival human temporal bone bank, all 28 cases with classical symptoms of MD demonstrated hydrops. However, there were nine cases with idiopathic hydrops and 10 cases with secondary hydrops that did not exhibit MD during their lifetimes. Of note, in all these cases, there was ipsilateral sensorineural hearing loss, sometimes fluctuating, indicating that they may have had what was once called “cochlear Meniere’s disease.” In that study, it was proposed that EH is an
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epiphenomenon of MD, rather than the etiology of symptoms.5 The currently accepted American Academy of Otolaryngology–Head and Neck Surgery (AAO– HNS; 1995) criteria for definite MD include two or more spontaneous episodes of vertigo lasting 20 min or longer, audiometrically documented hearing loss on at least one occasion, tinnitus or aural fullness in the treated ear, and other causes excluded. Probable MD is similar, except there is only one episode of vertigo, whereas possible MD includes episodic vertigo of MD type without hearing loss or hearing loss without definitive vertigo episodes (such as disequilibrium spells).6 One of the difficulties in the previous archival temporal bone studies is that, in many cases, the prior history may not have always been detailed as in prospective or concurrent histopathological studies. Second, postmortem histopathological analyses, by necessity, occur many years and sometimes several decades after the symptoms of MD. Many theories have been proposed regarding the pathophysiology of MD, including anatomic abnormalities affecting endolymph resorption, vascular abnormalities, postviral autoimmune mechanisms, and factors relating to water homeostasis.7,8 Although EH is nearly universally present in postmortem human temporal bones of subjects with a history of MD, the relationship between EH and MD symptoms remains unclear. The study of the vestibular end organs from subjects with a history of intractable MD may reveal clues to an underlying pathophysiology that causes both the symptoms of MD and EH. Previous studies had reported that surgically obtained end organs from MD patients at the light microscopic level exhibited a relative preservation of the vestibular neuroepithelium.4,9,10 Rivzi9 reported that there was severe dilatation of the ampullary walls and proposed that this interfered with cupular movement, accounting for caloric paresis in the early stages. This mechanism was proposed because of the relatively spared neuroepithelia in the histopathological studies. Similarly, Ylikoski et al.10 reported the relative preservation of the vestibular neuroepithelium under light microscopy in intractable MD. Previous studies had systematically and predominantly studied the utricular maculae. Therefore, we carried out a study
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of the horizontal cristae ampullares and otolithic organs from subjects with intractable end-stage MD. Histopathology and electron microscopy findings were reported from 17 patients (10 male, 7 female) with an average age of 62 years (range: 29–83 years) and an average duration of MD of 7 years, and with intractable MD with profound ipsilateral hearing loss. All patients, except one, had varying degrees of ipsilateral caloric paresis, ranging from 25% to 90% ipsilateral caloric paresis. McCall et al.11 demonstrated varied degrees of degeneration of the neuroepithelium, the region of the vestibular end organ that contains the vestibular hair cells. Often, there was partial or complete replacement of the normal epithelial cytoarchitecture by a monolayer of cells: 92% of the horizontal cristae (n = 13) and 75% of the saccular maculae (n = 4), contrasting with only 24% of the utricular maculae (n = 17). In the majority of the cristae, there was severe hair cell loss, basement membrane thickening, and fibrosis (Fig. 1A). There were also nonspecific changes, including the loss of hair cell stereocilia and cellular vacuolization, which were equally present in the utricular maculae and the cristae ampullaris. Electron microscopy demonstrated perivascular basement membrane thickening, an increased number of fibrocytes, and endothelial cell cytoplasm vacuolization in both cristae and utricular maculae (Fig. 1B). The finding of severe neurodegeneration and loss of hair cells is at odds with previous studies that reported relatively normal cytoarchitecture under light microscopy.9,10,12,13 The neuroepithelial degeneration was associated with the presence of thickening of the underlying basement membrane. Furthermore, neuroepithelial degeneration of the horizontal crista was associated with a caloric paresis. Of note, most prior studies had systematically analyzed only the utricular maculae, and thus, the relative sparing of the utricular maculae from neuroepithelial degeneration may explain why previous studies reported relatively normal neuroepithelium in MD. Alternatively, the thickness of the temporal bone specimens and processing of celloidin-embedded specimens may affect histopathological analysis. The relative resistance of the utricular maculae to degenerative damage has been noted additionally in both age-related neurodegeneration and
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Figure 1. Histopathology of the vestibular end organs in Meniere’s disease (courtesy of McCall et al.11 ). (A) The vestibular end organ (superior crista) from a patient with Meniere’s disease demonstrates degeneration and disorganization. The sensory epithelia demonstrated disorganization of the cytoarchitecture. The epithelium demonstrated significant vestibular hair cell (HC) loss and degenerative changes. Supporting cell (SC) cytoplasm was expanded into areas of HC loss, and the stroma nerve fibers (NF) appeared normal. Thickening of the BM underneath the subepithelium (SE) was prominent (asterisk). Bar in A = 50 m. (B) Transmission electron micrograph of the basement membrane (BM) and stroma beneath the sensory epithelia from patients with Meniere’s disease (BM from a Meniere’s utricular macula). The thickening of the BM appeared to be a disarray of collagen-like fibrils (arrows). The stromal fibroblast (FB) cytoplasm demonstrated mild vacuolization. The stromal perivascular BMs surrounding the blood vessels (BV) was also thickened ({}), and endothelial cell cytoplasm was vacuolated (arrowheads). Bar in B = 5 m.
intratympanic gentamicin ototoxicity. In our case, a report on the histopathology of the vestibular end organs after intratympanic gentamicin failure in intractable MD, a relative sparing of the utricular macula was noted. The utricle exhibited 7.3% of type I hair cells and 4.9% of type II hair cells remaining, when compared with age-matched normative controls. In contrast, there was a near complete loss
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of hair cells in the cristae ampullaris, which correlated with a 100% caloric paresis.14 Human temporal bone studies have indicated that, in the early stages of MD, the hydrops is predominantly in the cochlear duct and the saccule, and later can involve the entire endolymphatic system.4 Correspondingly, there was nearly always severe degeneration of the saccule, often rendering the small organ atrophied in appearance at surgery. Although the utricular macula is relatively spared, intraoperatively the otolithic membrane overlying the utricle appeared to be friable and degenerating. A related phenomenon, the vestibular drop attack, also known as a Tumarkin fall, occurs in a small subset of patients with MD. In 1936, Tumarkin described the sudden fall, without loss of consciousness, in patients and proposed that the otolithic organs were to blame, calling the falls “an otolithic catastrophe.”15 Patients describe being pushed down to the ground, often with tilting of the surrounding environment, and the multiple falls tend to occur in the same direction.15–17 These falls are referred to as an otolithic crisis because they are attributed to the sudden mechanical stimulation of the saccule or utricle, triggering the vestibulospinal reflex. Another characteristic that is evidence for otolithic organ involvement is that a subset of patients experience an ocular tilt reaction at the same time as the fall.17 In a study of otolithic membrane histopathology in MD, there was evidence that the instability of the utricular otolithic membrane may be the cause of Tumarkin falls, causing sudden otolithic stimulation, and that MD is associated with otolithic membrane damage.18 Figure 2 is reproduced from Calzada et al.,18 demonstrating otolithic atrophy and degeneration in MD and vestibular drop attacks. This comparative study examined the degenerative changes and thickness of the otolithic membrane in intractable MD; delayed EH, drop attacks, or acoustic neuroma (AN) were studied.18 Using archival temporal bones, the thickness of the membrane from subjects with MD was compared with age-matched normative controls. In MD, there was significant atrophy of the otolithic membrane (average thickness = 38 m) compared with normative controls (average thickness = 11.45 m), and the otolithic membrane in MD appeared to be degenerated and disorganized. Surgical observations had noted that 100% of those with drop attacks had
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Figure 2. Thickness of the utricular otolithic membrane in archival temporal bone specimens of Meniere’s disease compared with age-matched normative controls (courtesy of Calzada et al.18 ). The utricular otolithic membrane thickness was evaluated in five temporal bone specimens from patients with documented normal audiovestibular function and in five specimens from patients with documented MD. The mean thickness of the otolithic membrane in the five archival temporal bone MD specimens was 11.45 m, versus 38 m in healthy specimens (P = 0.001).
disrupted otoconial membranes. Six of seven patients had disruptions, and one otolithic membrane could not be identified owing to severe atrophy and damage. Approximately 50% of the patients with delayed EH and 56% of those with MD had disrupted otoconial membranes, whereas no otolithic membranes from patients with AN were damaged. These data support the hypothesis that damage to the otolithic organs occurs as part of the underlying pathophysiology of MD and may also play a role in otolithic or vestibular drop attacks18 (Fig. 2). Given the nearly universal finding of EH in MD, the overproduction of endolymph secondary to a disruption of fluid homeostasis has been proposed, because of an alteration in the expression of water and ion channels.19,20 Aquaporins (AQPs) play a fundamental role in mediating bidirectional water transport across membranes, regulating the volume and internal osmotic pressure of cells, and
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are found in all living organisms, including bacteria, plants, and animals.21 The distribution of AQPs 1, 4, and 6 in the human inner ear revealed a tightly conserved expression of AQPs across mammalian animal models.22 AQP1 is an osmotic water channel in the kidney, brain, and vascular system and has been proposed to function as a cGMP-gated anion channel.23 AQP1 is localized to nonsensory epithelial cells in the spiral ligament and the subbasilar tympanic cells in humans. In the macula utricle and crista ampullaris, AQP1 is localized to the fibrocytes beneath the sensory epithelium. AQP4 is critical for the movement of water from the blood or cerebrospinal fluid into and out of the brain, and is abundantly expressed in astrocyte foot processes, with the highest water permeability.24 The AQP4 knockout mouse exhibits significant hearing loss with hair cell dysfunction.25 In the normal human inner ear and in rodents, AQP4 is expressed
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in cochlear supporting cells and the basal region of vestibular supporting cells.22 Finally, AQP6 is a water channel of unknown physiological significance and is expressed in renal cells. We discovered the expression of AQP6 in apical supporting cells of the vestibular neuroepithelium in humans and rodents.22 MD has been proposed to be secondary to an alteration of AQP expression in the inner ear, causing an accumulation of endolymphatic fluid, or EH. Surgically acquired utricular maculae from subjects with intractable MD and ipsilateral profound hearing loss were evaluated for alterations in AQP1, AQP4, and AQP6 expression. As a normative control, age-matched postmortem normal human temporal bone specimens were studied, and as a surgical normative control, surgical specimens from AN were examined. The expression of AQP1 in subepithelial fibrocytes of the vestibular end organs was unchanged in MD. However, within supporting cells of the vestibular end organ in MD, AQP4 expression was significantly decreased and AQP6 expression was significantly increased compared with both AN and postmortem normative. The AQP6 expression exhibited a loss of polarity, being spread throughout the cell, rather than being polarized to the apical supporting cell. The effect of a loss of polarization likely has physiological consequences26 (Fig. 3). Although the alteration in AQP expression is likely most relevant to the final outcome of increased endolymphatic fluid and EH, we have noted other related alterations in cytochemical expression in the inner ear from subjects with MD. Using archival human temporal bones, diminished expression of the translocase of the outer mitochondrial membrane (Tom20) in human cochlea from MD patients has been reported,27 as well as upregulated expression of mRNA and cochlin protein28 and altered regional expression of glutamate aspartate transporter (GLAST) within human cochlea from MD patients.29 The stria vascularis and spiral ligament demonstrate significant atrophy compared with age-matched controls.30 Part of the difficulty in establishing causative factors for MD and in determining its relationship to EH is that most of the studies of MD inner ear have been conducted using postmortem human temporal bone. Many of the cytochemical findings may be part of the universal pathway that occurs in
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neurodegeneration of the vestibular epithelium. For example, the diminished expression of Tom2027 and GLAST28 appear to occur with aging as well. However, some cytochemical changes appear to be specific to MD and do not occur with aging or with other otological diseases. An example of an apparent MD-specific cytochemical alteration is that of AQP4 and AQP6 expression. Understanding the role of altered AQP expression in other systems is noteworthy. After cerebral injury, the astrocytic expression of AQP4, normally highly enriched at perivascular endfeet (the outermost layer of the blood–brain barrier), becomes dysregulated and spreads throughout the astrocytic plasmalemma, and is associated with cerebral edema.24 Thus, the loss of polarized AQP4 expression appears to be a principal component of pathological cerebral edema. The ability to detect inner ear structural changes, such as the degree of EH, would be an invaluable tool to begin to understand the relationship between EH and MD. Only since 2007 has the technology been developed for the visualization of EH using magnetic resonance imaging (MRI) techniques, and these studies demonstrate EH in clinically defined MD, originally using intratympanic (IT) gadolinium by researchers in Japan.31 Other researchers have used IT gadolinium and MRI to demonstrate a lack of correlation between EH and caloric paresis.32,33 In some of the prior studies, a significant minority of subjects with MD did not demonstrate EH of the vestibular space.34 In the study performed by Barath et al.,35 90% of the subjects with MD had ipsilateral EH. Gurkov et al.36 demonstrated that the degree of hearing loss correlated with cochlear hydrops, using an observerbased grading of EH on a Likert scale (0–3). Recently, objective quantitative criteria for EH using delayed intravenous gadolinium 3D fluid attenuation inversion recovery (3D-FLAIR) MRI have been developed, and the degree of EH was significantly correlated with the degree of hearing loss. In Sepahdari et al., vestibular endolymphatic space (VES)/vestibule ratio is used as a quantitative indicator of the degree of EH. There was a strong positive correlation with PTA in subjects with MD. Threedimensional analysis was able to evaluate for EH in the anterior/inferior vestibule. It was found that the degree of EH strongly correlated with auditory hearing loss based on the pure tone average in the setting of MD (Spearman = 0.89, P = 0.0003).37
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Figure 3. Aquaporin (AQP) 1, 4, and 6 expression in the vestibular end organs of Meniere’s disease (MD) compared with acoustic neuroma (AN) and normative (N1) (courtesy of Ishiyama et al.26 ) AQP1 expression was not different in MD compared with N1. AQP1 (red) is localized to the fibrocytes beneath the overlying sensory epithelium (se, arrow) and to the fibrocytes in the underlying stroma (st, double arrows) in the utricular maculae (lu, luminal region) in MD (A), AN (a ), and Nl (a ). The AQP4 expression in the utricular macula from subjects with MD (B) was significantly diminished in the sensory epithelium (arrows) when compared with that in AN (b , arrows) and in Nl (b , arrows). Quantitative immunoreactivity analysis (b ) shows a statistically significant decrease in AQP4 immunoreactivity in MD when compared with AN and N1. The AQP6 (red) expression was altered in MD (C) and diffusely distributed throughout the supporting cells (arrows), compared with a subapical polarized expression in the supporting cell in AN (c ) and Nl (c ). Quantitative immunoreactivity analysis (c ) shows a statistically significant increase in AQP6 immunoreactivity in Meniere’s disease specimens when compared with acoustic neuroma and autopsy specimens. Phalloidin Oregon green staining (green) was used to identify actin at the apical portion of the sensory epithelium. No colocalization was observed between AQP6 (red) and phalloidin (green), corroborating a subapical distribution in the supporting cell of utricular maculae derived from acoustic neuroma (B) and normative (C) samples. Bars 50 m (a–a , b–b , c–c ). 6
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Figure 4. 3D maximum-intensity projection (MIP) image in unilateral Meniere’s disease (methodology as described in Ref. 37). Shown are results from a 60-year-old man with right-sided definite Meniere’s disease. A 3D MIP image from a heavily T2-weighted 3D-FLAIR sequence (TR = 9000 ms, TE = 534 ms, TI = 2350 ms) was obtained at a 4-h delay after double-dose intravenous contrast administration. The cochlear basal turn (BT), lateral semicircular canal (LSC), posterior semicircular canal (PSC), and medulla are labeled. The superior semicircular canals are en face in this projection and not well seen. The vestibular endolymphatic structures (VES) are outlined in blue, and the vestibules are outlined in yellow, with respective cross-sectional areas noted on the image. The right inner ear shows a dilated utricle and saccule, effacing the vestibular perilymph, with a VES/vestibule ratio of 67%. The normal left side shows a small utricle, with a VES/vestibule ratio of 25%. Increased signal is also seen in the perilymph of the cochlear basal turn on the right side. (Image courtesy of Ali Sepahdari, M.D., David Geffen School of Medicine at UCLA, Department of Radiology.)
Figure 4 illustrates imaging methodology to quantify the degree of EH in patients. The image uses 3D maximum-intensity projection images (MIPs) from a heavily T2-weighted 3D-FLAIR sequence obtained at a 4-h delay after double-dose intravenous gadolinium administration. Illustrated is imaging of the internal auditory canals of a 60-year-old man with unilateral right-sided definite MD. The technique allows for visualization of the VES, outlined in blue, and of the vestibule, outlined in yellow. By evaluating the fraction of the vestibule occupied by the VES, a quantitative indication of EH was obtained. A high VES/vestibule ratio is indicative of EH. In this case, the normal left side shows a VES/vestibule ratio of 25%, whereas the right side, with MD, demonstrates a dilated utricle and saccule, effacing the vestibular perilymph, with a VES/vestibule ratio of 67%. Also of note is the increased contrast signal intensity seen in the perilymph of the cochlear basal turn on the MD side. The development of a quantitative method using MRI during life will allow for the study of other related phenomena, including vestibular migraine, migraine in MD, delayed EH, and secondary
hydrops. There is the possibility that other otopathological entities exhibit hydrops during the active phase of the illness and then the hydrops dissipate with time. It is known that a multitude of insults can trigger the development of EH: postinfectious,38 autoimmune,38 and acoustic trauma. Microvascular ischemia is also potentially an etiology, and there are several studies suggesting that MD is closely associated with dysfunctional cochlear blood flow. Microvascular ischemia may result in basement membrane damage and perivascular fibrosis seen in vestibular end organs from intractable MD9 and may also cause the otolithic membrane damage.18 -Histine increases cochlear stria vascularis flow in a dose-dependent manner,39 which may be one of its mechanisms of action in MD. The ability to image EH in MD and in other otopathologies will allow for a better understanding of the relationship between hydrops of the inner ear and the pathophysiology of MD. Conclusions The histopathological findings of the vestibular neuroepithelium in MD are significant for severe
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damage with hair cell loss, basement membrane thickening, and perivascular endothelial cell damage, possibly consistent with microvascular damage. The overlying otolithic membrane demonstrates atrophy and degeneration in MD, and in particular in MD with vestibular drop attacks. Cytochemical findings include alteration of AQP4 and AQP6 expression, mitochondrial translocase expression, and cochlin. Altered AQP expression may cause EH, but it remains unknown how the attacks of vertigo and hearing fluctuation occur in MD. Currently, we have developed imaging techniques to visualize EH, which may enable us to study the relationship between EH and MD. Acknowledgments
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15. 16. 17.
18.
The authors thank Dr. Robert W. Baloh, whom we admire for his insight and inspiration in our studies of MD. We have greatly enjoyed working together with him and are looking forward to our further collaborations.
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Conflicts of interest The authors declare no conflicts of interest.
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A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S Issue: Dizziness and Balance Disorders
Meniere’s disease: histopathology, cytochemistry, and imaging Gail Ishiyama,1 Ivan A. Lopez,2 Ali R. Sepahdari,3 and Akira Ishiyama2 1
Department of Neurology, Reed Neurological Research Center, David Geffen School of Medicine at UCLA, Los Angeles, California. 2 Department of Head and Neck Surgery, Department of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, California. 3 Department of Radiology, David Geffen School of Medicine at UCLA, Los Angeles, California Address for correspondence: Gail Ishiyama, M.D., Department of Neurology Reed Neurological Research Center, UCLA David Geffen School of Medicine, 710 Westwood Blvd., Box 951769, Los Angeles, CA 90095. [email protected]
Meniere’s disease is a poorly understood, disabling syndrome causing spells of vertigo, hearing fluctuation, tinnitus, and aural fullness. In this paper, we present a review of the histopathology, cytochemistry, and imaging of M´eni`ere’s disease. Histopathology is significant for neuroepithelial damage with hair cell loss, basement membrane thickening, and perivascular microvascular damage. Cytochemical alterations are significant for altered AQP4 and AQP6 expression in the supporting cell, and altered cochlin and mitochondrial protein expression. Current developments include imaging techniques to determine the degree and presence of endolymphatic hydrops, and future studies will endeavor to correlate the observance of hydrops with clinical findings. Keywords: endolymphatic hydrops; inner ear; Meniere’s disease; vestibular; MRI imaging; aquaporins
Introduction Meniere’s disease (MD) is a disabling syndrome characterized by fluctuating hearing loss, episodic vertigo, ear fullness, and tinnitus. In 1861, Prosper M´eni`ere was accredited with describing this clinical syndrome as an inner ear labyrinthine dysfunction, rather than a central neurological disorder.1 It was not until almost 80 years later that the most prominent and consistent pathological correlate of MD was revealed in postmortem human temporal bone studies: endolymphatic hydrops (EH), the dilation of the membranous labyrinth of the inner ear. In two simultaneous publications on temporal bone histopathology by Yamakawa2 and Hallpike and Cairns,3 these independent findings of EH were noted in subjects that had MD. Schuknecht conducted detailed postmortem temporal bone histopathology on subjects with a history of MD and reported various abnormal pathologies, including distension of Reissner’s membrane, a ballooning out of the saccular wall, ruptures of the membranous labyrinth, and
sometimes fistulae. However, he reported the relative sparing of the neuroepithelia, the region of the vestibular end organ containing the receptors for balance, the vestibular hair cells.4 It had been proposed that EH leads to the symptoms of MD—vertigo spells and hearing loss—because of potassium intoxication of the hair cells during membrane ruptures, allowing the potassium-rich endolymph to paralyze the sensory hair cells that are located within the perilymph. Temporal bone studies demonstrated that nearly all cases of MD are associated with EH;4 however, not all EH cases are associated with the symptoms of MD.5 In a study using an archival human temporal bone bank, all 28 cases with classical symptoms of MD demonstrated hydrops. However, there were nine cases with idiopathic hydrops and 10 cases with secondary hydrops that did not exhibit MD during their lifetimes. Of note, in all these cases, there was ipsilateral sensorineural hearing loss, sometimes fluctuating, indicating that they may have had what was once called “cochlear Meniere’s disease.” In that study, it was proposed that EH is an
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epiphenomenon of MD, rather than the etiology of symptoms.5 The currently accepted American Academy of Otolaryngology–Head and Neck Surgery (AAO– HNS; 1995) criteria for definite MD include two or more spontaneous episodes of vertigo lasting 20 min or longer, audiometrically documented hearing loss on at least one occasion, tinnitus or aural fullness in the treated ear, and other causes excluded. Probable MD is similar, except there is only one episode of vertigo, whereas possible MD includes episodic vertigo of MD type without hearing loss or hearing loss without definitive vertigo episodes (such as disequilibrium spells).6 One of the difficulties in the previous archival temporal bone studies is that, in many cases, the prior history may not have always been detailed as in prospective or concurrent histopathological studies. Second, postmortem histopathological analyses, by necessity, occur many years and sometimes several decades after the symptoms of MD. Many theories have been proposed regarding the pathophysiology of MD, including anatomic abnormalities affecting endolymph resorption, vascular abnormalities, postviral autoimmune mechanisms, and factors relating to water homeostasis.7,8 Although EH is nearly universally present in postmortem human temporal bones of subjects with a history of MD, the relationship between EH and MD symptoms remains unclear. The study of the vestibular end organs from subjects with a history of intractable MD may reveal clues to an underlying pathophysiology that causes both the symptoms of MD and EH. Previous studies had reported that surgically obtained end organs from MD patients at the light microscopic level exhibited a relative preservation of the vestibular neuroepithelium.4,9,10 Rivzi9 reported that there was severe dilatation of the ampullary walls and proposed that this interfered with cupular movement, accounting for caloric paresis in the early stages. This mechanism was proposed because of the relatively spared neuroepithelia in the histopathological studies. Similarly, Ylikoski et al.10 reported the relative preservation of the vestibular neuroepithelium under light microscopy in intractable MD. Previous studies had systematically and predominantly studied the utricular maculae. Therefore, we carried out a study
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of the horizontal cristae ampullares and otolithic organs from subjects with intractable end-stage MD. Histopathology and electron microscopy findings were reported from 17 patients (10 male, 7 female) with an average age of 62 years (range: 29–83 years) and an average duration of MD of 7 years, and with intractable MD with profound ipsilateral hearing loss. All patients, except one, had varying degrees of ipsilateral caloric paresis, ranging from 25% to 90% ipsilateral caloric paresis. McCall et al.11 demonstrated varied degrees of degeneration of the neuroepithelium, the region of the vestibular end organ that contains the vestibular hair cells. Often, there was partial or complete replacement of the normal epithelial cytoarchitecture by a monolayer of cells: 92% of the horizontal cristae (n = 13) and 75% of the saccular maculae (n = 4), contrasting with only 24% of the utricular maculae (n = 17). In the majority of the cristae, there was severe hair cell loss, basement membrane thickening, and fibrosis (Fig. 1A). There were also nonspecific changes, including the loss of hair cell stereocilia and cellular vacuolization, which were equally present in the utricular maculae and the cristae ampullaris. Electron microscopy demonstrated perivascular basement membrane thickening, an increased number of fibrocytes, and endothelial cell cytoplasm vacuolization in both cristae and utricular maculae (Fig. 1B). The finding of severe neurodegeneration and loss of hair cells is at odds with previous studies that reported relatively normal cytoarchitecture under light microscopy.9,10,12,13 The neuroepithelial degeneration was associated with the presence of thickening of the underlying basement membrane. Furthermore, neuroepithelial degeneration of the horizontal crista was associated with a caloric paresis. Of note, most prior studies had systematically analyzed only the utricular maculae, and thus, the relative sparing of the utricular maculae from neuroepithelial degeneration may explain why previous studies reported relatively normal neuroepithelium in MD. Alternatively, the thickness of the temporal bone specimens and processing of celloidin-embedded specimens may affect histopathological analysis. The relative resistance of the utricular maculae to degenerative damage has been noted additionally in both age-related neurodegeneration and
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Figure 1. Histopathology of the vestibular end organs in Meniere’s disease (courtesy of McCall et al.11 ). (A) The vestibular end organ (superior crista) from a patient with Meniere’s disease demonstrates degeneration and disorganization. The sensory epithelia demonstrated disorganization of the cytoarchitecture. The epithelium demonstrated significant vestibular hair cell (HC) loss and degenerative changes. Supporting cell (SC) cytoplasm was expanded into areas of HC loss, and the stroma nerve fibers (NF) appeared normal. Thickening of the BM underneath the subepithelium (SE) was prominent (asterisk). Bar in A = 50 m. (B) Transmission electron micrograph of the basement membrane (BM) and stroma beneath the sensory epithelia from patients with Meniere’s disease (BM from a Meniere’s utricular macula). The thickening of the BM appeared to be a disarray of collagen-like fibrils (arrows). The stromal fibroblast (FB) cytoplasm demonstrated mild vacuolization. The stromal perivascular BMs surrounding the blood vessels (BV) was also thickened ({}), and endothelial cell cytoplasm was vacuolated (arrowheads). Bar in B = 5 m.
intratympanic gentamicin ototoxicity. In our case, a report on the histopathology of the vestibular end organs after intratympanic gentamicin failure in intractable MD, a relative sparing of the utricular macula was noted. The utricle exhibited 7.3% of type I hair cells and 4.9% of type II hair cells remaining, when compared with age-matched normative controls. In contrast, there was a near complete loss
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of hair cells in the cristae ampullaris, which correlated with a 100% caloric paresis.14 Human temporal bone studies have indicated that, in the early stages of MD, the hydrops is predominantly in the cochlear duct and the saccule, and later can involve the entire endolymphatic system.4 Correspondingly, there was nearly always severe degeneration of the saccule, often rendering the small organ atrophied in appearance at surgery. Although the utricular macula is relatively spared, intraoperatively the otolithic membrane overlying the utricle appeared to be friable and degenerating. A related phenomenon, the vestibular drop attack, also known as a Tumarkin fall, occurs in a small subset of patients with MD. In 1936, Tumarkin described the sudden fall, without loss of consciousness, in patients and proposed that the otolithic organs were to blame, calling the falls “an otolithic catastrophe.”15 Patients describe being pushed down to the ground, often with tilting of the surrounding environment, and the multiple falls tend to occur in the same direction.15–17 These falls are referred to as an otolithic crisis because they are attributed to the sudden mechanical stimulation of the saccule or utricle, triggering the vestibulospinal reflex. Another characteristic that is evidence for otolithic organ involvement is that a subset of patients experience an ocular tilt reaction at the same time as the fall.17 In a study of otolithic membrane histopathology in MD, there was evidence that the instability of the utricular otolithic membrane may be the cause of Tumarkin falls, causing sudden otolithic stimulation, and that MD is associated with otolithic membrane damage.18 Figure 2 is reproduced from Calzada et al.,18 demonstrating otolithic atrophy and degeneration in MD and vestibular drop attacks. This comparative study examined the degenerative changes and thickness of the otolithic membrane in intractable MD; delayed EH, drop attacks, or acoustic neuroma (AN) were studied.18 Using archival temporal bones, the thickness of the membrane from subjects with MD was compared with age-matched normative controls. In MD, there was significant atrophy of the otolithic membrane (average thickness = 38 m) compared with normative controls (average thickness = 11.45 m), and the otolithic membrane in MD appeared to be degenerated and disorganized. Surgical observations had noted that 100% of those with drop attacks had
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Figure 2. Thickness of the utricular otolithic membrane in archival temporal bone specimens of Meniere’s disease compared with age-matched normative controls (courtesy of Calzada et al.18 ). The utricular otolithic membrane thickness was evaluated in five temporal bone specimens from patients with documented normal audiovestibular function and in five specimens from patients with documented MD. The mean thickness of the otolithic membrane in the five archival temporal bone MD specimens was 11.45 m, versus 38 m in healthy specimens (P = 0.001).
disrupted otoconial membranes. Six of seven patients had disruptions, and one otolithic membrane could not be identified owing to severe atrophy and damage. Approximately 50% of the patients with delayed EH and 56% of those with MD had disrupted otoconial membranes, whereas no otolithic membranes from patients with AN were damaged. These data support the hypothesis that damage to the otolithic organs occurs as part of the underlying pathophysiology of MD and may also play a role in otolithic or vestibular drop attacks18 (Fig. 2). Given the nearly universal finding of EH in MD, the overproduction of endolymph secondary to a disruption of fluid homeostasis has been proposed, because of an alteration in the expression of water and ion channels.19,20 Aquaporins (AQPs) play a fundamental role in mediating bidirectional water transport across membranes, regulating the volume and internal osmotic pressure of cells, and
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are found in all living organisms, including bacteria, plants, and animals.21 The distribution of AQPs 1, 4, and 6 in the human inner ear revealed a tightly conserved expression of AQPs across mammalian animal models.22 AQP1 is an osmotic water channel in the kidney, brain, and vascular system and has been proposed to function as a cGMP-gated anion channel.23 AQP1 is localized to nonsensory epithelial cells in the spiral ligament and the subbasilar tympanic cells in humans. In the macula utricle and crista ampullaris, AQP1 is localized to the fibrocytes beneath the sensory epithelium. AQP4 is critical for the movement of water from the blood or cerebrospinal fluid into and out of the brain, and is abundantly expressed in astrocyte foot processes, with the highest water permeability.24 The AQP4 knockout mouse exhibits significant hearing loss with hair cell dysfunction.25 In the normal human inner ear and in rodents, AQP4 is expressed
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in cochlear supporting cells and the basal region of vestibular supporting cells.22 Finally, AQP6 is a water channel of unknown physiological significance and is expressed in renal cells. We discovered the expression of AQP6 in apical supporting cells of the vestibular neuroepithelium in humans and rodents.22 MD has been proposed to be secondary to an alteration of AQP expression in the inner ear, causing an accumulation of endolymphatic fluid, or EH. Surgically acquired utricular maculae from subjects with intractable MD and ipsilateral profound hearing loss were evaluated for alterations in AQP1, AQP4, and AQP6 expression. As a normative control, age-matched postmortem normal human temporal bone specimens were studied, and as a surgical normative control, surgical specimens from AN were examined. The expression of AQP1 in subepithelial fibrocytes of the vestibular end organs was unchanged in MD. However, within supporting cells of the vestibular end organ in MD, AQP4 expression was significantly decreased and AQP6 expression was significantly increased compared with both AN and postmortem normative. The AQP6 expression exhibited a loss of polarity, being spread throughout the cell, rather than being polarized to the apical supporting cell. The effect of a loss of polarization likely has physiological consequences26 (Fig. 3). Although the alteration in AQP expression is likely most relevant to the final outcome of increased endolymphatic fluid and EH, we have noted other related alterations in cytochemical expression in the inner ear from subjects with MD. Using archival human temporal bones, diminished expression of the translocase of the outer mitochondrial membrane (Tom20) in human cochlea from MD patients has been reported,27 as well as upregulated expression of mRNA and cochlin protein28 and altered regional expression of glutamate aspartate transporter (GLAST) within human cochlea from MD patients.29 The stria vascularis and spiral ligament demonstrate significant atrophy compared with age-matched controls.30 Part of the difficulty in establishing causative factors for MD and in determining its relationship to EH is that most of the studies of MD inner ear have been conducted using postmortem human temporal bone. Many of the cytochemical findings may be part of the universal pathway that occurs in
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neurodegeneration of the vestibular epithelium. For example, the diminished expression of Tom2027 and GLAST28 appear to occur with aging as well. However, some cytochemical changes appear to be specific to MD and do not occur with aging or with other otological diseases. An example of an apparent MD-specific cytochemical alteration is that of AQP4 and AQP6 expression. Understanding the role of altered AQP expression in other systems is noteworthy. After cerebral injury, the astrocytic expression of AQP4, normally highly enriched at perivascular endfeet (the outermost layer of the blood–brain barrier), becomes dysregulated and spreads throughout the astrocytic plasmalemma, and is associated with cerebral edema.24 Thus, the loss of polarized AQP4 expression appears to be a principal component of pathological cerebral edema. The ability to detect inner ear structural changes, such as the degree of EH, would be an invaluable tool to begin to understand the relationship between EH and MD. Only since 2007 has the technology been developed for the visualization of EH using magnetic resonance imaging (MRI) techniques, and these studies demonstrate EH in clinically defined MD, originally using intratympanic (IT) gadolinium by researchers in Japan.31 Other researchers have used IT gadolinium and MRI to demonstrate a lack of correlation between EH and caloric paresis.32,33 In some of the prior studies, a significant minority of subjects with MD did not demonstrate EH of the vestibular space.34 In the study performed by Barath et al.,35 90% of the subjects with MD had ipsilateral EH. Gurkov et al.36 demonstrated that the degree of hearing loss correlated with cochlear hydrops, using an observerbased grading of EH on a Likert scale (0–3). Recently, objective quantitative criteria for EH using delayed intravenous gadolinium 3D fluid attenuation inversion recovery (3D-FLAIR) MRI have been developed, and the degree of EH was significantly correlated with the degree of hearing loss. In Sepahdari et al., vestibular endolymphatic space (VES)/vestibule ratio is used as a quantitative indicator of the degree of EH. There was a strong positive correlation with PTA in subjects with MD. Threedimensional analysis was able to evaluate for EH in the anterior/inferior vestibule. It was found that the degree of EH strongly correlated with auditory hearing loss based on the pure tone average in the setting of MD (Spearman = 0.89, P = 0.0003).37
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Figure 3. Aquaporin (AQP) 1, 4, and 6 expression in the vestibular end organs of Meniere’s disease (MD) compared with acoustic neuroma (AN) and normative (N1) (courtesy of Ishiyama et al.26 ) AQP1 expression was not different in MD compared with N1. AQP1 (red) is localized to the fibrocytes beneath the overlying sensory epithelium (se, arrow) and to the fibrocytes in the underlying stroma (st, double arrows) in the utricular maculae (lu, luminal region) in MD (A), AN (a ), and Nl (a ). The AQP4 expression in the utricular macula from subjects with MD (B) was significantly diminished in the sensory epithelium (arrows) when compared with that in AN (b , arrows) and in Nl (b , arrows). Quantitative immunoreactivity analysis (b ) shows a statistically significant decrease in AQP4 immunoreactivity in MD when compared with AN and N1. The AQP6 (red) expression was altered in MD (C) and diffusely distributed throughout the supporting cells (arrows), compared with a subapical polarized expression in the supporting cell in AN (c ) and Nl (c ). Quantitative immunoreactivity analysis (c ) shows a statistically significant increase in AQP6 immunoreactivity in Meniere’s disease specimens when compared with acoustic neuroma and autopsy specimens. Phalloidin Oregon green staining (green) was used to identify actin at the apical portion of the sensory epithelium. No colocalization was observed between AQP6 (red) and phalloidin (green), corroborating a subapical distribution in the supporting cell of utricular maculae derived from acoustic neuroma (B) and normative (C) samples. Bars 50 m (a–a , b–b , c–c ). 6
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Figure 4. 3D maximum-intensity projection (MIP) image in unilateral Meniere’s disease (methodology as described in Ref. 37). Shown are results from a 60-year-old man with right-sided definite Meniere’s disease. A 3D MIP image from a heavily T2-weighted 3D-FLAIR sequence (TR = 9000 ms, TE = 534 ms, TI = 2350 ms) was obtained at a 4-h delay after double-dose intravenous contrast administration. The cochlear basal turn (BT), lateral semicircular canal (LSC), posterior semicircular canal (PSC), and medulla are labeled. The superior semicircular canals are en face in this projection and not well seen. The vestibular endolymphatic structures (VES) are outlined in blue, and the vestibules are outlined in yellow, with respective cross-sectional areas noted on the image. The right inner ear shows a dilated utricle and saccule, effacing the vestibular perilymph, with a VES/vestibule ratio of 67%. The normal left side shows a small utricle, with a VES/vestibule ratio of 25%. Increased signal is also seen in the perilymph of the cochlear basal turn on the right side. (Image courtesy of Ali Sepahdari, M.D., David Geffen School of Medicine at UCLA, Department of Radiology.)
Figure 4 illustrates imaging methodology to quantify the degree of EH in patients. The image uses 3D maximum-intensity projection images (MIPs) from a heavily T2-weighted 3D-FLAIR sequence obtained at a 4-h delay after double-dose intravenous gadolinium administration. Illustrated is imaging of the internal auditory canals of a 60-year-old man with unilateral right-sided definite MD. The technique allows for visualization of the VES, outlined in blue, and of the vestibule, outlined in yellow. By evaluating the fraction of the vestibule occupied by the VES, a quantitative indication of EH was obtained. A high VES/vestibule ratio is indicative of EH. In this case, the normal left side shows a VES/vestibule ratio of 25%, whereas the right side, with MD, demonstrates a dilated utricle and saccule, effacing the vestibular perilymph, with a VES/vestibule ratio of 67%. Also of note is the increased contrast signal intensity seen in the perilymph of the cochlear basal turn on the MD side. The development of a quantitative method using MRI during life will allow for the study of other related phenomena, including vestibular migraine, migraine in MD, delayed EH, and secondary
hydrops. There is the possibility that other otopathological entities exhibit hydrops during the active phase of the illness and then the hydrops dissipate with time. It is known that a multitude of insults can trigger the development of EH: postinfectious,38 autoimmune,38 and acoustic trauma. Microvascular ischemia is also potentially an etiology, and there are several studies suggesting that MD is closely associated with dysfunctional cochlear blood flow. Microvascular ischemia may result in basement membrane damage and perivascular fibrosis seen in vestibular end organs from intractable MD9 and may also cause the otolithic membrane damage.18 -Histine increases cochlear stria vascularis flow in a dose-dependent manner,39 which may be one of its mechanisms of action in MD. The ability to image EH in MD and in other otopathologies will allow for a better understanding of the relationship between hydrops of the inner ear and the pathophysiology of MD. Conclusions The histopathological findings of the vestibular neuroepithelium in MD are significant for severe
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damage with hair cell loss, basement membrane thickening, and perivascular endothelial cell damage, possibly consistent with microvascular damage. The overlying otolithic membrane demonstrates atrophy and degeneration in MD, and in particular in MD with vestibular drop attacks. Cytochemical findings include alteration of AQP4 and AQP6 expression, mitochondrial translocase expression, and cochlin. Altered AQP expression may cause EH, but it remains unknown how the attacks of vertigo and hearing fluctuation occur in MD. Currently, we have developed imaging techniques to visualize EH, which may enable us to study the relationship between EH and MD. Acknowledgments
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15. 16. 17.
18.
The authors thank Dr. Robert W. Baloh, whom we admire for his insight and inspiration in our studies of MD. We have greatly enjoyed working together with him and are looking forward to our further collaborations.
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Conflicts of interest The authors declare no conflicts of interest.
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