Experimental Design.

We investigated whether activation of the inner ear AVP-V2R-AQP2 signaling pathway is potentially involved in the development of motion sickness and whether blocking V2R is more effective in reducing motion sickness. We adopted two motion sickness models using dogs and rats, respectively, to test the anti–motion sickness effects of AVP V2R antagonist mozavaptan (OPC-31260).

Animals and Chemicals.

Sprague-Dawley rats (b.wt. 200–220 g) of both genders and pregnant dams were obtained from the Experimental Animal Center of Nantong University, Nantong, China. Six male and 14 female beagle dogs (age 8.0–8.5 months, b.wt. 7.5–8.8 kg) were purchased from Shanghai Jia-Gan Biologic Science and Technology Co., Ltd., which was qualified and certified by the Shanghai Laboratory Animal Management Committee [SCXK (Hu) 2015-0005]. An additional 12 beagle dogs (six males and six females, age 8.5–9 months, b.wt. 7.8–10.2 kg) were purchased for vestibular training from Yangzhou Sifang Experimental Animal Technology Co., Ltd., which was qualified and certified by the Jiangsu Laboratory Animal Management Committee [SCXK (Su) 2008-0006]. All procedures used in this study were in accordance with our institutional guidelines, which comply with the Animal Research: Reporting of In Vivo Experiments guidelines and were approved by the Animal Care and Use Committee of Nantong University, Nantong, China.

Adult rats were fed under 12-hour light/dark cycles (light, 08:00–20:00 hour; darkness, 20:00–08:00 hour) at room temperature 22–24°C with free access to standard rat chow and tap water. Beagle dogs were housed in kennels at the Experimental Animal Center of Nantong University. Dogs were fed under natural light/dark cycles at a temperature of 16–26°C and 40%–70% relative humidity with free access to tap water. Standard canine food was provided in the morning and afternoon. The dogs were under the care of a licensed veterinarian.

Common inorganic salts were purchased in China. Culture medium was purchased from Invitrogen (Carlsbad). The following primary antibodies were purchased: anti-V2R and monoclonal anti-GAPDH from Merck Millipore (Temecula); monoclonal anti-β-actin from Sigma-Aldrich (Saint Louis); anti-p-AQP2 Ser 256 from Abcam Trading (Shanghai) Company Ltd. (China); anti-AQP2 and anti-cytokeratin 7 from Santa Cruz Biotechnology, Inc. (Dallas); and anti-PKA C-α, anti-p-PKA C-Thr 197, anti-CREB (48H2), and anti-p-CREB Ser 133 from Cell Signaling Technology (Danvers). V2R agonist desmopressin acetate (DDAVP), AVP, V2R antagonist mozavaptan (hydrochloride), poly-d-lysine, PKA inhibitor H89 dihydrochloride, cytosine β-d-arabinofuranoside, SDS, and other chemicals except those indicated elsewhere were purchased from Sigma-Aldrich.

Motion Sickness–Provoking Rotatory Stimuli and Vestibular Training.

Because they lack an emetic reflex, rats do not develop the symptoms of nausea and vomiting. However, rats will produce conditioned taste aversions (CTAs) after exposure to a vestibular stimulus, which has been used as a behavioral index (“being sick”) substitute for motion sickness in rats (Ossenkopp, 1983; Fox and McKenna, 1988; Sutton et al., 1988; Gallo et al., 1999; Li et al., 2005).

The rotatory stimulator for use in small animals shown in our previous study (Li et al., 2005) was manufactured according to the report by Crampton and Lucot (1985). For the induction of CTA, rats were rotated in the stimulator in alternating acceleration and deceleration modes. The acceleration rate was 16°/s² for 7.5 seconds with a maximal velocity of 120°/s, and the deceleration rate was 48°/s² for 2.5 seconds. The clockwise and counterclockwise rotations were alternately repeated for a total time of 120 minutes. Before rotatory stimulation, in addition to tap water, the rats were also supplied with 0.15% saccharin sodium solution (SSS) as a novel fluid for drinking for 48 hours. When the rotatory stimulation was completed, a supply of 0.15% SSS was maintained for 3 days. Intake volume of 0.15% SSS per 24 hours was measured. The rats with intake volume decreases less than 10% were considered to be not susceptible to motion sickness. In contrast, the rats with intake volume decreases more than 15% were considered to be susceptible.

For the induction of motion sickness in beagle dogs, the animals were rotated in the same way as with the rats. The clockwise and counterclockwise rotations were alternately repeated until the development of the first vomiting action. Before vomiting, saliva dropping was seen commonly. Salivation and vomiting served as the indices of motion sickness in the dogs. The same rotatory test was repeated 1 week later. The latencies to salivation and vomit in the two tests were averaged as control values. The dogs with a vomit latency of 5–15 minutes were selected for further drug tests and vestibular training.

To perform the vestibular training, rats susceptible to motion sickness identified after the above motion sickness–provoking rotatory stimulus (provoking test) were rotated once a day in the same way with the duration gradually increased from 10 to 60 minutes over a month (10-minute increase per 5 days). For vestibular training with the dogs, eight susceptible dogs were rotated similarly to the rats once every 2 days for 3 months, and the rotation duration was increased from 4 to 12 minutes with a 2-minute increase every 2 weeks.

At 1 week after the end of the vestibular training, the animals were tested with the provoking test, and loss of CTA in the rat or a significant extension of the vomit latency in the dog was considered to be an acquisition of an adaption against this provoking test.

Drug Tests in the Dogs and Rats for Motion Sickness Induced by Rotatory Stimuli.

For drug tests on motion sickness in the dogs, 12 susceptible dogs were selected from 20 dogs, and a 6 × 6 Latin square design was used with one test every week. For example, 12 dogs were randomly allocated to six groups, including the control group; groups receiving 0.018 and 0.036 mg/kg scopolamine (a positive control drug for anti-motion sickness); and groups receiving 0.9, 1.8, and 3.6 mg/kg mozavaptan (an antagonist of V2R), with a total observation time of 6 weeks. Rotatory stimulus was performed as described above for 30 minutes or until vomiting was induced. The drugs were orally administered 1 hour before the rotatory stimulus, and control dogs were given an equal volume of solvent (0.2 ml/kg normal saline).

To investigate whether the AVP V2R agonist promotes the induction of motion sickness due to rotatory stimulus in the dogs, 15 beagle dogs were selected from 20 dogs and allocated to two groups with vomiting latencies less or more than 10 minutes. In the following 3 weeks, each dog randomly received one treatment of control or 0.25 or 0.5 μg/kg DDAVP each week. Rotatory stimulus was described as above for 30 minutes or until vomiting was induced. The V2R agonist DDAVP was injected intramuscularly 1 hour before the rotatory stimulus, and control dogs were given an equal volume of solvent (0.1 ml/kg normal saline).

For the drug tests on motion sickness in rats, susceptible rats were randomly allocated to three groups, including the control group and groups receiving 2.5 and 5.0 mg/kg mozavaptan. Mozavaptan was delivered via intraperitoneal injection 45 minutes before 2 hours of rotatory stimulus, and the control rats received an equal injection volume of solvent (2 ml/kg normal saline).

CTA Induction in the Rats via Intraperitoneal Injection of AVP and V2R Agonist and Inhibition by V2R Antagonist.

To induce CTA in rats, AVP was intraperitoneally injected at doses of 100, 200, and 400 μg/kg. In addition to drinking water, 0.15% SSS was also supplied at the same time for the rats to drink for 2 days before AVP injection and 2 days after AVP injection. The mean intake volume of 0.15% SSS before and after AVP injection was measured. To further investigate whether the induction of CTA by AVP in rats is mediated by V2R, a V2R agonist, DDAVP (0.45 and 0.9 μg/kg, i.p.), was used to see whether it could also induce CTA. Similarly, the reduction in 0.15% SSS intake volume was measured after DDAVP injection.

Additionally, an antagonist of V2R, mozavaptan, was used to determine whether it could block the CTA-inductive effects of DDAVP. Mozavaptan was intraperitoneally injected at doses of 3.2 and 6.4 mg/kg. Control rats were injected intraperitoneally with an equal volume of solvent (2 ml/kg normal saline).

Middle Ear Injection through the Tympanic Membrane.

Middle ear injection of drugs through the tympanic membrane (intratympanic injection) was performed under ether anesthesia according to previous reports (Hunt et al., 1987; Takumida and Anniko, 2006; Liu et al., 2016; Zou and Pyykko, 2016). Rats were placed on a heating pad in a prone position, the tympanic membrane was penetrated under an operating microscope at the upper edge of the posterior upper quadrant with a 30-gauge needle, and a total of 50 μl of drug solution for each side was respectively injected into both middle ear cavities. The animals received subsequent treatments 2 hours after the injections.

Epithelial Cell Culture of the Endolymphatic Sacs.

Epithelial cell culture of the endolymphatic sacs was conducted based on other reports (Dahlmann and von During, 1995; Agrup et al., 2001; Kumagami et al., 2009). Rat pups at 10 days after birth were used for primary culture of the endolymphatic sac epithelial cells. After ether anesthesia, the temporal bones from both sides were removed and placed in a cold (4°C) endolymph-like solution containing 150 mM KCl, 5 mM NaCl, 2 mM KH₂PO₄, 1 mM MgCl₂, 3 mM glucose, 25 mM HEPES, and 1 mg/ml of albumin. The endolymphatic sacs were carefully isolated with the aid of a dissecting microscope. The endolymphatic sacs were divided into small pieces, transferred to culture dishes, and attached as explants to poly-lysine–coated cover slips with the gentle pressure of a scalpel. The explants were cultured for 5 days at 37°C in a humidified 5% CO₂ incubator. The culture medium used was Dulbecco’s modified Eagle’s medium with the addition of 15% fetal calf serum, penicillin (85 IU/ml), streptomycin (85 μg/ml), and epithelial growth factor (100 μg/ml). Half of the culture medium was changed every 3 days. On day 6 in vitro, the explants were digested with 0.125% trypsin, dispersed gently with a heat-polished pipette, and then cultured with the above culture medium containing Bromodeoxyuridine (0.1 mM). On day 11 in vitro, the cultures were digested once more with 0.125% trypsin containing EDTA∙4Na (190 mg/l; Gibco, Shanghai, China) and centrifuged at 1500 rpm for 4 minutes. The supernatants were discarded, and the cells were resuspended with the above culture medium and seeded on poly-lysine–coated cover slips or culture dishes. On days 15–16 in vitro, the cultured epithelial cells from the endolymphatic sac were used for the following experiments. The cultured cells were identified via the immunofluorescent observation of the following characteristic markers: 1) cytokeratin-7 (a maker of epithelial cells), 2) AQP2, and 3) V2R (Supplemental Fig. 1). A purity of >90% for the cultured epithelial cells from the endolymphatic sacs was obtained.

Real-Time Quantitative Polymerase Chain Reaction.

Rats were decapitated under 10% chloral hydrate anesthesia (400 mg/kg, i.p.), the temporal bones from both sides were removed and placed in cold (4°C) PBS (0.1 M, pH 7.4), and the inner ears and endolymphatic sacs were isolated under a dissecting microscope. The inner ears and endolymphatic sacs or the cultured epithelial cells from the endolymphatic sacs were homogenized in Trizol reagent (Invitrogen), and total RNA was purified. Potential DNA contamination was removed using DNase and, 1 μg RNA was used for first-strand cDNA synthesis by retrotranscription using oligo(dT) primers and the PrimeScript RT Reagent Kit (Tiangen Biotech Co., Ltd, Beijing, China) according to the manufacturer’s instructions. Real-time quantitative polymerase chain reaction (qRT-PCR) was performed in a Rotor-Gene PCR machine (RG-3000A; Corbett Research Proprietary Limited, New South Wales, Australia), and the detailed cycling conditions were as follows: 1) an initial denaturation step at 95°C for 5 seconds and 2) 45 cycles of a 95°C denaturation for 15 seconds, 55°C annealing for 30 seconds, and 72°C extension for 20 seconds. The amount of cDNA per sample was determined using a SYBR Premix Ex Taq kit (Roche, Basel, Switzerland). PCR reaction progression was assessed by changes in the SYBR green dye fluorescence (Roche) attached to the double-stranded DNA. All values were normalized to the housekeeping gene β-actin or GAPDH. The following primer pairs were used for qRT-PCR: 1) AQP2, forward, 5′-TGA​GTT​CTT​GGC​CAC​GCT​CCT​TT-3′, and reverse, 5′-ATG​GAG​AGG​GCA​GGG​CTA​CC-3′ and 2) V2R, forward, 5′-GCC​ACT​GAC​CGC​TTC​CAT-3′, and reverse, 5′-CAC​CCC​ACT​GCC​ATT​TCC-3′.

Western Blot Analysis.

The inner ears and endolymphatic sacs isolated from the rat temporal bones or the cultured epithelial cells from the endolymphatic sacs were lysed in tissue or cell lysis buffer containing phenylmethanesulfonyl fluoride (Beyotime, Nantong, China), homogenized, and then centrifuged at 14,000 rpm for 20 minutes at 4°C. The protein contents of the supernatants were determined spectrophotometrically using the bicinchoninic acid method. Equal amounts of protein (40 μg per lane) from each sample were loaded on a 10% SDS-PAGE, electrophoresed, and transferred onto polyvinyledene difluoride membranes (Merck Millipore). These membranes were blocked in 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 2 hours at room temperature and then incubated overnight at 4°C with the primary antibodies against AQP2 (1:100), p-AQP2 (1:300), CREB (1:1000), p-CREB (1:1000), PKA (1:1000), p-PKA (1:1000), V2R (1:100), β-actin (1:5000), or GAPDH (1:5000). Finally, the membranes were incubated with a second antibody (1:6000; Bioworld, Nanjing, China) conjugated with horseradish peroxidase for 2 hours at room temperature, and the immunoreactive bands were visualized by enhanced chemiluminescent agents (Pierce Biotechnology, Rockford) and captured using a Tanon 5200 multi system (Tanon, Shanghai, China). Expression level of each protein was quantified using Image-J analysis software and normalized to the β-actin, CREB, or GAPDH value in the same lane. The same samples were repeatedly analyzed at least two times.

Immunofluorescent Observations.

Rats were anesthetized with 10% chloral hydrate (400 mg/kg, i.p.) and perfused transcardially with 200 ml of normal saline containing sodium heparin (2000 IU) and subsequently 500 ml of 4% paraformaldehyde in PBS (0.1 M, pH 7.4). The temporal bones were then isolated, postfixed in 4% paraformaldehyde for 12 hours, and immersed in decalcification solution containing 0.2 M EDTA-Na₂ at 37°C for 28 days. After dehydration with ethanol, the tissues were embedded in paraffin. Paraffin-embedded tissues were then sectioned at 5-μm thickness and mounted on slides. Prior to immunostaining, selected paraffin sections were dewaxed using xylenes. The sections were quenched in a 3% hydrogen peroxide methanol solution, pretreated to promote antigen retrieval through steaming with citrate buffer solution (pH 6.0, 95°C), and incubated with 5% normal goat serum for 1 hour and then with primary antibodies at different dilutions overnight at 4°C. The cultured epithelial cells from the endolymphatic sacs were fixed with 4% paraformaldehyde at room temperature for 15 minutes and then incubated with precooled methanol at −20°C for 8 minutes. After incubation with 10% bull serum albumin for 1 hour, the cells were incubated with primary antibodies at different dilutions overnight at 4°C. Simultaneously, a negative control was performed without each primary antibody. For immunofluorescent observation of the target molecule distribution in the inner ears or their expression in cultured endolymphatic sac epithelial cells, tissue sections or cultured cells were incubated with donkey anti-rabbit Alexa-488, donkey anti-mouse Alexa-594, or donkey anti-rabbit Alexa-647 secondary antibodies (1:1000; Jackson, West Grove) for 1–2 hours at room temperature. The immunostained images were collected using a laser confocal microscope (TCS SP8; Leica Microsystems, Wetzlar, Germany) at room temperature and processed with the software LASX 2.0.

ELISA.

Blood was obtained within 30 minutes after the end of rotatory stimulus from the cephalic vein of the anterior legs of dogs and at different times after rotation from the left ventricle of rats under 10% chloral hydrate anesthesia (400 mg/kg, i.p.). Blood samples (2 ml of each) from rats and dogs were all placed in test tubes containing 200 μg of dried sodium heparin, shaken gently, and centrifuged at 4000 rpm at 4°C for 5 minutes. The inner ears and endolymphatic sacs were isolated from the rat temporal bones under a dissecting microscope and homogenized in tissue lysis buffer (4°C). The cultured endolymphatic sac epithelial cells were isolated and homogenized in 0.1 M of HCl/cell lysis buffer (1:5, 4°C). The homogenized tissues or cells were centrifuged at 4°C and 14,000 rpm for 15 minutes. The supernatants were then collected. All of these samples were frozen and stored at −80°C for subsequent analyses. The AVP, cAMP, and AQP2 contents were assayed in duplicate using an AVP assay kit (R&D Systems, Minneapolis), a cAMP assay kit (R&D Systems), and an AQP2 assay kit (CUSABIO, Baltimore), respectively, according to the manufacturers’ guidelines.

Statistical Analysis.

All data are presented as the mean ± S.E.M. After the homogeneity of variance test, Student’s t test was used for the comparison of two groups, two-way ANOVA was used for the comparison of data from Latin design experiments, and one-way ANOVA was used for the comparison of data from the other expreiments with three or more groups, with the least significant difference test for post hoc comparisons between two groups. Differences were considered statistically significant at a level of P < 0.05.