Laboratory Investigation (2013) 93, 1001–1011 & 2013 USCAP, Inc All rights reserved 0023-6837/13 Morphological and functional changes in a new animal model of Me´nie`re’s disease Naoya Egami1, Akinobu Kakigi1, Takashi Sakamoto1, Taizo Takeda2, Masamitsu Hyodo2 and Tatsuya Yamasoba1 The purpose of this study was to clarify the underlying mechanism of vertiginous attacks in Me´nie`re’s disease (MD) while obtaining insight into water homeostasis in the inner ear using a new animal model. We conducted both histopatho- logical and functional assessment of the vestibular system in the guinea-pig. In the first experiment, all animals were maintained 1 or 4 weeks after electrocauterization of the endolymphatic sac of the left ear and were given either saline or desmopressin (vasopressin type 2 receptor agonist). The temporal bones from both ears were harvested and the extent of endolymphatic hydrops was quantitatively assessed. In the second experiment, either 1 or 4 weeks after surgery, animals were assessed for balance disorders and nystagmus after the administration of saline or desmopressin. In the first experiment, the proportion of endolymphatic space in the cochlea and the saccule was significantly greater in ears that survived for 4 weeks after surgery and were given desmopressin compared with other groups. In the second experiment, all animals that underwent surgery and were given desmopressin showed spontaneous nystagmus and balance disorder, whereas all animals that had surgery but without desmopressin administration were asymptomatic. Our animal model induced severe endolymphatic hydrops in the cochlea and the saccule, and showed episodes of balance disorder along with spontaneous nystagmus. These findings suggest that administration of desmopressin can exacerbate endolymphatic hydrops because of acute V2 (vasopressin type 2 receptor)-mediated effects, and, when combined with endolymphathic sac dysfunction, can cause temporary vestibular abnormalities that are similar to the vertiginous attacks in patients with MD. Laboratory Investigation (2013) 93, 1001–1011; doi:10.1038/labinvest.2013.91; published online 22 July 2013 KEYWORDS: animal model; aquaporin; desmopressin; endolymphatic hydrops; Me´nie`re’s disease; vasopressin; vestibular disorder Me´nie`re’s disease (MD) is a well-known inner ear disorder been observed.6 In other words, this model is insufficient for characterized by symptoms including recurring attacks of fully representing the common symptoms of MD. vertigo typically lasting for hours, fluctuating sensorineural Water homeostasis of the inner ear is essential for main- hearing loss, and tinnitus. Since the milestone findings on the taining the functions of hearing and balance. EH is con- temporal bones of MD patients,1,2 endolymphatic hydrops sidered to be the result of disruption of inner ear water (EH) has been considered as the histopathological origin of homeostasis, which involves excessive production of endo- MD, as characteristic morphological changes were reported lymph and/or reduced absorption of endolymph. Until to be produced by surgical obstruction of the endolymphatic recently, however, the detailed mechanisms underlying the sac (ES) in guinea-pig.3 This observation indicates that overaccumulation of endolymph were unclear. Since the malabsorption of endolymph in the ES is one of the possible discovery of aquaporin (AQP) water channels,7 it has been mechanisms underlying the development of EH. This proposed that precise regulation of water reabsorption experimentally induced EH has been frequently used as an largely depends on the regulation of AQP2 channels and animal model to investigate the pathogenesis of inner ear that water permeability can change rapidly in response to disorders associated with EH. In terms of vestibular function, vasopressin(VP).8 Recently, it has come to light that this while canal dysfunction and/or spontaneous nystagmus have mechanism has a crucial role not only in the kidney but also been occasionally observed in this animal model,4,5 episodes in the inner ear. In addition, the following evidence has of vertiginous attack or balance disorder have only rarely accumulated to suggest that VP is closely associated with the 1Department of Otolaryngology-Head and Neck Surgery, Faculty of Medicine, The University of Tokyo, Tokyo, Japan and 2Department of Otolaryngology-Head and Neck Surgery, Kochi Medical School, Kochi, Japan Correspondence: Dr A Kakigi, MD, Department of Otolaryngology, Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113 8655, Japan. E-mail: [email protected] Received 30 April 2013; revised 20 June 2013; accepted 22 June 2013 www.laboratoryinvestigation.org | Laboratory Investigation | Volume 93 September 2013 1001 Animal model of Me´nie`re’s disease N Egami et al formation of EH: (1) plasma levels of arginine VP are higher before euthanasia. In saline-treated animals, 25 ml/kg saline in patients with MD and may depend on the phase that the solution was administered subcutaneously 1 h before patient is in,9–11 (2) acute and chronic application of arginine euthanasia. All animals were killed under deep anesthesia VP produces EH in guinea-pigs and rats,12–14 (3) V2 receptor using ketamine and xylazine, and the temporal bones were mRNA is expressed in rat and human inner ear,15–18 and collected from both sides. As shown in Figure 1a, 48 temporal (4) expression of V2 receptor mRNA in the rat inner ear is bones harvested were classified into eight groups (n ¼ 6) downregulated by VP application.19 Such accumulated according to the presence or absence of surgery, the survival evidence has led to the assumption that production of duration, and type of treatment: (1) right temporal bones, endolymph is controlled by VP–AQP2 system. 1-week survival after left ear electrocauterization, administered In a previous study using an animal model of MD, saline (1 W Control group (RIGHT)); (2) left temporal Dunnebier et al20 reported chronic ES dysfunction induced bones of the above-mentioned animals (1 W Surgery group by destruction of the ES, and acute stress-induced endo- (LEFT)); (3) right temporal bones, 1-week survival after left lymph production by aldosterone administration revealed ear electrocauterization, administered desmopressin (1 W severe degrees of hydrops in the cochlea.20 In this Desmopressin group (RIGHT)); (4) left temporal bones of investigation, we successfully developed a new clinically the above-mentioned animals (1 W Combined group (LEFT)); relevant animal model for MD, in which desmopressin (5) right temporal bones, 4-week survival after left ear electro- (a VP type 2 receptor agonist) was administered after electro- cauterization, administered saline (4 W Control group cauterization of the ES. (RIGHT)); (6) left temporal bones of the above-mentioned We evaluated both morphological and behavioral altera- animals (4 W Surgery group (LEFT)); (7) right temporal tions of the vestibular system in this animal model to bones, 4-week survival after left ear electrocauterization, elucidate the role of VP in the pathogenesis of MD. administered desmopressin (4 W Desmopressin group (RIGHT)), and (8) left temporal bones of the above- MATERIALS AND METHODS mentioned animals (4 W Combined group (LEFT)). This study was composed of two experiments. Experiment 1 was designed to investigate the influence of desmopressin on Surgical procedure for Electrocauterization of the ES the development of EH morphologically. Experiment 2 was All experimental animals were anesthetized by an intra- designed to investigate the role of desmopressin on changes muscular injection of ketamine (35 mg/kg) and xylazine in vestibular function. The animals used in Experiment 1 (5 mg/kg). The animals received local anesthesia using xylo- were albino outbred English short-haired guinea-pig caine under sterile conditions. Subsequently, they were placed (Hartley). In Experiment 2, pigmented English short-haired in a prone position with a head holder and underwent guinea-pig were used. Pigmented animals were selected for dorsal midline scalp incision. The left occipital bone was Experiment 2 to facilitate eye movement recording using an removed for visual exposure of the ES via an epidural infrared-sensitive CCD camera. occipital approach. Thereafter, the extraosseous portion of These experiments were approved by Tokyo University the sac was cauterized electrically with a bipolar electro- Animal Care and Use Committee, and were conducted in coagulator (Surgitron Model FFPF; Ellman International, accordance with The Animal Welfare Act and the guiding Hewlett, NY, USA) so as not to injure the sigmoid sinus. principles for animal care produced by the Ministry of The operation was performed using a Carl Zeiss operation Education, Culture, Sports, and Technology, Japan. microscope. Experiment 1 Quantitative Assessment of the Endolymphatic Space Animals Under deep anesthesia by an intraperitoneal injection of A total of 24 guinea-pigs with a positive Preyer’s reflex and ketamine (35 mg/kg) and xylazine (5 mg/kg), all animals were weighing approximately 300 g were used in this experiment. perfused from the left ventricle with physiological saline All animals underwent electrocauterization of the ES of the left solution, and then fixed with 10% formalin. The temporal ear as reported previously.21,22 The right ear was not treated bones were removed on both sides and postfixed in 10% surgically. The surgical procedures are described below (in the formalin solution