Takefumi Kamakura

Expression and Translocation of Aquaporin‐2 in the Endolymphatic Sac in Patients with Meniere’s Disease

Meniere’s disease, characterised by episodic vertigo, fluctuating hearing loss and tinnitus, can occur under conditions of stress. Its pathology was first revealed to be inner ear hydrops through temporal bone studies in 1938. Although its pathogenesis has been proposed to be a disorder of water transport in the inner ear, subsequently, it remains unsolved, until now. A recent study revealed that both plasma stress hormone, vasopressin (pAVP) and its receptor, V2 (V2R) expression in the inner ear endolymphatic sac were significantly higher in Meniere’s patients. In the present study, to link V2R‐related molecules and inner ear hydrops, we examined V2R‐linked water channel molecule, aquaporin‐2 (AQP2) expression and translocation in human endolymphatic sac. AQP2 mRNA expression in the endolymphatic sac was significantly higher in Meniere’s patients by using real‐time polymerase chain reaction, as further confirmed by western blotting. AQP2‐like immunoreactivity (‐LIR) was translocated from luminal to basolateral side with endosomal trapping in the endolymphatic sac at the time of AVP exposure in human endolymphatic sac tissue culture. The similar AQP2‐LIR translocation was also demonstrated by forskolin and blocked by vasopressin/V2R specific antagonist, OPC31260 and protein kinase A (PKA) specific antagonists, H‐89 and KT‐5720. We concluded that in the pathogenesis of inner ear hydrops resulting in Meniere’s attacks, pAVP elevation as a result of stress and subsequent V2R‐cAMP‐PKA‐AQP2 activation and endosomal trapping of AQP2 in the endolymphatic sac, might be important as a basis of this disease. Further experimental and clinical studies are needed to better clarify the neuroscientific relationship between stress and Meniere’s disease.

Changes in endolymphatic hydrops after sac surgery examined by Gd-enhanced MRI.

Abstract Conclusion: Endolymphatic hydrops could be a reversible inner ear pathological condition. After sac surgery, hydrops was reduced and symptoms went into remission in some cases, although vertigo suppression was not always a result of the reduced hydrops. Objective: To examine the changes in endolymphatic hydrops detected by gadolinium (Gd) contrast-enhanced magnetic resonance imaging (MRI) before and 6 months after endolymphatic sac surgery in patients with unilateral Ménières disease. Methods: Fluid-attenuated inversion recovery MRI was obtained 4 h after intravenous administration or 24 h after intratympanic administration of Gd contrast medium. An enlarged negative stain corresponding to the cochlear duct and endolymphatic space of the vestibule was assessed as hydrops. Results: Of seven patients with hydrops confirmed by MRI before surgery, both cochlear and vestibular hydrops became negative in two, cochlear hydrops became negative in one, both hydrops were present, but reduced, in one, and there was no change in three patients. The number of vertigo spells was reduced in all cases at 6–12 months after surgery. As for the three cases of negative hydrops, vertigo was completely suppressed. In two cases in which hearing level improved, hydrops became negative after surgery.

Functional expression of TRPV1 and TRPA1 in rat vestibular ganglia

Both TRPV1 and TRPA1 are non-selective cation channels. They are co-expressed, and interact in sensory neurons such as dorsal root ganglia (DRG) and trigeminal ganglia (TG), and are involved in nociception, being activated by nociceptive stimuli. Immunohistological localization of TRPV1 in vestibular ganglion (VG) neurons has been reported. Although TRPA1 is co-expressed with TRPV1 in DRG and TG neurons, it is unclear whether TRPA1 channels are expressed in VG neurons. Moreover, it is unknown whether TRPV1 and TRPA1 channels are functional in VG neurons. We investigated the expression of TRPV1 and TRPA1 in rat VG neurons by RT-PCR, in situ hybridization, immunohistochemistry, and Ca(2+) imaging experiments. Both TRPV1 and TRPA1 RT-PCR products were amplified from the mRNA of rat VG neurons. In situ hybridization experiments showed TRPV1 and TRPA1 mRNA expression in the majority of VG neurons. Immunohistochemistry experiments confirmed TRPV1 protein expression. In Ca(2+) imaging experiments, capsaicin, a TRPV1 agonist, induced a significant increase in intracellular calcium ion concentration ([Ca(2+)]i) in rat primary cultured VG neurons, which was almost completely blocked by capsazepine, a TRPV1-specific antagonist. Cinnamaldehyde, a TRPA1 agonist, also caused an increase in [Ca(2+)]i, which was completely inhibited by HC030031, a TRPA1-specific antagonist. Moreover, in some VG neurons, a [Ca(2+)]i increase was evoked by both capsaicin and cinnamaldehyde in the same neuron. In summary, our histological and physiological studies reveal that TRPV1 and TRPA1 are expressed in VG neurons. It is suggested that TRPV1 and TRPA1 in VG neurons might participate in vestibular function and/or dysfunction such as vertigo.

Cisplatin-induced toxicity decreases the mouse vestibulo-ocular reflex

Cisplatin is a chemotherapeutic agent commonly used for the treatment of solid tumors, and its side-effects include vestibulotoxicity. Previous studies have reported cisplatin-induced vestibulotoxicity in various animal models, but no study has investigated in vivo mouse vestibular dysfunction after cisplatin. The aim of this study was to investigate cisplatin-induced vestibulotoxicity in C57BL/6J mice. Vestibular function was assessed by recording the vestibulo-ocular reflex (VOR). This was done during sinusoidal rotations in the horizontal plane at three frequencies (0.5, 1.0 and 2.5Hz). A high-resolution, high-frequency digital infra-red camera was used with eye-tracking algorithms. Cisplatin at 16mg/kg, but not 8mg/kg, decreased the VOR gain at 2.5Hz compared with the vehicle control. Following 16mg/kg cisplatin treatment, the animals showed no change in the optokinetic nystagmus response, suggesting that no major changes in visual or oculomotor functions had occurred. This mouse model may be useful for studying cisplatin-induced vestibulotoxicity and its treatment.

Chronic otitis media with cholesteatoma with canal fistula and bone conduction threshold after tympanoplasty with mastoidectomy.

Objective To understand the third mobile window effect of chronic otitis media with cholesteatoma with inner ear fistula on the bone conduction threshold, we examined changes in the bone conduction audiogram after tympanoplasty with mastoidectomy for chronic otitis media with cholesteatoma with canal fistula. Study Design Retrospective case review. Setting Tertiary referral center. Patients According to the intraoperative classification of Dornhoffer and Milewski, we focused especially on Type IIa (anatomic bony fistula with no perilymph leak). We checked the bone conduction threshold at least 3 times: just before, just after, and 6 months after surgery in 20 ears with Type IIa lateral semicircular canal fistula. Intervention Tympanoplasty with mastoidectomy. Main Outcome Measure Bone conduction thresholds before and after tympanoplasty with mastoidectomy. Results Compared with the preoperative bone conduction threshold, 6 cases were better, 12 cases were unchanged, and 2 cases were worse within the first postoperative week. Finally, 1 case was better, 15 cases were unchanged, and 4 cases were worse at the sixth postoperative month. Patients with a better bone conduction threshold in the low-tone frequencies immediately after surgery had a tendency to show no preoperative fistula symptoms. Postoperative spontaneous nystagmus had a tendency to be observed in patients with a worse bone conduction threshold in the high-tone frequencies. Conclusion The better bone conduction threshold at low-tone frequencies immediately after tympanoplasty with mastoidectomy and no preoperative fistula symptoms might imply the third mobile window theory. The worse bone conduction threshold in high-tone frequencies with spontaneous nystagmus after surgery might indicate inner ear damage.

Endolymphatic sac tumor with overexpression of V2 receptor mRNA and inner ear hydrops

Abstract Conclusion: We reported previously that hyperactivation of vasopressin type-2 receptor (V2R)-mediated signaling in the endolymphatic sac could affect endolymphatic fluid metabolism, resulting in the pathogenesis of endolymphatic hydrops. Taken together with the present endolymphatic sac tumor (ELST) study, it is suggested that disorder of V2R signaling in the endolymphatic sac for any reason could be involved in the pathogenesis of endolymphatic hydrops. Although it is due to tumor genesis in ELST, it is idiopathic in nature in Menieres disease. Objective: We encountered two cases of ELST showing Menieres disease-like symptoms. Both cases were suspected of having endolymphatic hydrops using neuro-otological examinations. To clarify the histopathological diagnosis of ELST and the molecular pathogenesis of endolymphatic hydrops, we performed histopathological and molecular biological examinations of the endolymphatic sac. Methods: ELSTs in two rare cases were removed completely through the transmastoidal approach. V2R mRNA expression was examined using real-time PCR. Results: The first case was diagnosed as inflammatory granulation adjacent to the endolymphatic sac, i.e. pseudo-ELST, and the second case was diagnosed as papillary adenoma of ELST. V2R mRNA expression was up-regulated in the endolymphatic sac of both cases as seen in Menieres disease compared with controls.

Assessment of endolymphatic hydrops and otolith function in patients with Ménière’s disease

Ménière’s disease is associated with hydrops of the inner ear endolymphatic space, and histopathologically, the cochlea and vestibule are usually involved. We used gadolinium-enhanced magnetic resonance imaging and measured cervical and ocular vestibular evoked myogenic potentials and the gain in the utricular induced linear vestibulo-ocular reflex to test the hypothesis that vestibular hydrops in Ménière’s disease patients is associated with otolith organ dysfunction. We evaluated 21 patients diagnosed with unilateral definitive Ménière’s disease using gadolinium magnetic resonance imaging to detect endolymphatic hydrops in the cochlea and vestibule. Cervical and ocular vestibular evoked myogenic potentials and the gain in utricular induced linear vestibulo-ocular reflex during eccentric rotation were measured to assess otolith organ function. For eccentric rotation, patients were rotated while displaced from the axis of rotation, while linear acceleration stimulated the utricle and induced the vestibulo-ocular reflex. Magnetic resonance imaging revealed vestibular hydrops in 14 of 20 patients (70%). Among the 14 patients, ten (71%) had abnormal cervical and three (21%) had abnormal ocular vestibular evoked myogenic potentials. Four patients (4/21, 19%) had abnormal linear vestibulo-ocular reflexes, three of whom also had abnormal ocular vestibular evoked myogenic potentials. Overall, 16 of 17 patients had normal linear vestibulo-ocular reflexes and normal ocular vestibular evoked myogenic potentials. Vestibular endolymphatic hydrops in Ménière’s disease patients caused otolith organ dysfunction, mainly in the saccule. The number of Ménière’s disease patients with abnormal ocular vestibular evoked myogenic potentials was low (19%), and they also had abnormal utricular induced linear vestibulo-ocular reflexes.

Transient low-tone air-bone gaps during convalescence immediately after canal plugging surgery for BPPV

OBJECTIVES The aim of the present study was to elucidate the time course and frequency patterns of transient low-tone air-bone gaps (ABGs) after canal plugging for intractable BPPV. METHODS We investigated eight patients with intractable BPPV who underwent canal plugging. Four were cases with posterior type (pBPPV) and the other four were those with horizontal type (hBPPV). Pure-tone audiometries (PTAs) were performed before and 7 days, 1 month and 6 months after surgery. ABGs (+) were defined as the three-tone-average ≥20dB formulated by (a+b+c)/3, where a, b, and c are ABGs at 0.25, 0.5, and 1kHz, respectively. RESULTS The ratio of the number of patients with ABGs (+) at the post-operative 7th day and 1st month was 100.0% (8/8). The ratio at the post-operative 6th month was 0.0% (0/8). There were no significant differences in the time course or frequency patterns of the ABGs between pBPPV and hBPPV. CONCLUSIONS We clearly demonstrated eight cases with intractable BPPV showing transient low-tone ABGs during convalescence immediately after canal plugging. During that period, patients also complained of motion-evoked dizziness. All these findings suggest that, during such a convalescence period, the plugged area might not be fixed yet and could still induce the dizziness and low-tone ABGs, as enlarged vestibular aqueduct syndrome and superior semicircular canal deficiency syndrome exhibit low-tone ABGs due to the third mobile inner ear window. More than one month after surgery, both the ABGs and dizziness could disappear according to fixation of the plugged area.

Effects of repeated optic flow stimulation on gait termination in humans

Abstract Conclusions: Because the basic strategies to stop walking are stored as motor programs, visual stimulation may have little influence on body deviation during gait termination and its time course. Walking velocity, however, demonstrated dynamic flexible changes, which may subserve the stable process of gait termination under variable circumstantial changes such as optic flow. Objective: The aim of this study was to examine the effect of repeated optic flow on body deviation and walking velocity during gait termination, which may be more complicated than continuous standing or walking. Methods: Twenty-three healthy subjects were instructed to start walking upon an acoustic cue and to stop walking when the scenery changed in a virtual reality environment. Subjects underwent eight control trials without optic flow and three sets of optic flow conditions including four trials each of optic horizontal and rotational movement randomly. Results: Repeated optic flow caused no significant change of body deviation or the time course of the gait termination process in comparison with that in the control. The walking velocity at the start of the termination process showed short-term flexibility that denoted a gradual increase over the trial for within-set and long-term flexibility that denoted a gradual decrease for between-set.

Changes in beta-2 adrenergic receptor and AMP-activated protein kinase alpha-2 subunit in the rat vestibular nerve after labyrinthectomy.

In the present study, to elucidate the role of vestibular ganglion (VG) after the unilateral labyrinthine damage, we examined quantitative changes in mRNA expression of beta-adrenergic receptors (bARs) and AMP-activated protein kinase alpha catalytic subunits (aAMPKs) in VG after unilateral labyrinthectomy (UL) in rats. Using the real-time PCR method, beta2 AR mRNA expression in bilateral VG and AMPK alpha2 mRNA expression in the ipsilateral VG were significantly up-regulated with the maximum increase at the postoperative 7 day and 1 day, respectively. The up-regulation of beta2 AR in bilateral VG was long-lasting until 28 days after UL and that of AMPK alpha2 in the ipsilateral VG was just transient within 7 days after UL. These mRNA changes were supported by immunohistochemical data. According to previous reports, both of bARs and aAMPKs could regulate mitochondrial uncoupling protein (UCP) mRNA expression in several kinds of tissues and therefore might have thermogenic neurotransmission and antioxidant neuroprotective roles in neuronal tissues. UL requires not only long-lasting response of VG for central vestibular neuro-plasticity around 2-4 weeks but rapid response of VG against apoptosis of peripheral vestibular epithelia-neuronal synapses. The present findings suggest that beta2 AR in bilateral VG and AMPK alpha2 in the ipsilateral VG might play important signaling roles after the unilateral labyrinthine damage.