An Integrated System for Comprehensive Mouse Peripheral Vestibular Function Evaluation Based on Vestibulo-ocular Reflex

Abstract

Purpose

In the fields of both vestibular and auditory research, reliable vestibular function tests are essential. However, unlike the auditory function tests, which use standard Auditory Brainstem Response (ABR) equipment, there is no equivalent widely adopted apparatus for vestibular tests. Vestibulo-ocular reflexes (VORs) are the compensatory ocular reflexes that ensure stable vision during head motion. VORs are widely used in clinics to diagnose vestibular deficits. In the research, VORs have been used by various groups to evaluate the mouse vestibular function. However, the effectiveness of VOR tests has not been systematically evaluated with appropriate mouse models, and the lack of commercial equipment hampers its accessibility, confining vestibular testing to a select few labs.

Methods

In this study, we developed an integrated and surgery-free instrument system with both angular VOR (aVOR) and off-vertical axis rotation (OVAR) modes for evaluating mouse vestibular function. In addition, the eye rotation calibrations used in this study standardize the data between instruments. To demonstrate its validity and efficacy of the testing equipment, we evaluated four mouse models, including both genders, with peripheral vestibular deficits: 1) mice injected with the vestibulotoxic drug 3,3’-iminodiproprionitrile (IDPN, 2 mg/g and 4 mg/g, 3 male/3 female per group); 2) Critical MET-related mutant mice (Cdh23^v2J/v2J, 4 male/4 female and TMC1^−/−, 6 male/5 female); 3) Vestibular-specific mutant mice (Zpld1^−/−, 6 male/6 female, for semicircular canal dysfunction and Otop1^tlt/tlt, 3 male/2 female, for otoconia deficient); 4) Unilateral vestibular lesion (UVL) mouse model (3 male/3 female per group) where gentamicin was injected into horizontal semicircular canal.

Results

The results showed: 1) Quantification of vestibular deficits can be achieved as a daily routine; 2) Both the horizontal semicircular canal and otolith organs can be assessed, respectively; and 3) The lesion side of UVL can be identified.

Conclusion

These test results reveal the potential of our system as standard equipment for evaluating common vestibular deficits in mice.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10162-025-01007-x.

Introduction

The vestibular system detects head and body position changes, controls gaze, and facilitates higher-level cognitive perception functions [1]. It consists of three semicircular canals and two otolith organs (utricle and saccule), which mediate sensitivity to rotational and linear (including gravity) head movements, respectively [2]. In recent years, studies on the pathological mechanisms underlying vestibular dysfunction and their treatments have gained momentum [3, 4]. Furthermore, due to their similar hair cell function [5] and developmental processes [6, 7], vestibular organs and the cochlea provide a reciprocal frame of reference in studies such as hair cell regeneration [8, 9] and hair cell planar cell polarity establishment [10, 11]. However, it is worth noting that standard, efficient, and comprehensive vestibular function tests for mice remain elusive, which prevents the results of different studies from being easily compared and is therefore an obstacle in vestibular-related research.

Mice are the most commonly used animal models in research. Typical approaches for assessing vestibular function in mice include post-mortem morphological evaluation, behavioral tests, vestibular evoked potentials (VEP), and vestibulo-ocular reflexes (VORs). Morphological evaluation [12, 13] is relatively time-consuming, often requires euthanizing mice, and is therefore not suitable for studies involving significant numbers of animals, such as drug screening [14] and longitudinal studies [15]. Due to the involvement of multiple neural pathways [16], commonly used behavioral signs, such as circling and head-tossing, cannot be regarded as vestibular-specific. And, the behaviors, such as circling and head tilt, often rely on observer judgments and thus lead to subjective results and possible errors [17–19]. While the vestibular evoked myogenic potentials (VEMPs) are valuable clinical tools for assessing vestibular function, their utility in mice is limited due to technical difficulties [20, 21]. The short latency linear vestibular sensory evoked potentials (VsEPs) have been a commonly used method to analyze otolith function in mice [22–24]. However, Jones. et al. [25] reported that VsEP signals could be compromised if not controlled for auditory brainstem response (ABR) to the acoustic stimuli emitted from the mechanical shaker during acceleration pulse production. Also, VsEP does not evaluate semicircular canal activity. The VORs are commonly used in clinical testing [26] and have also been part of standard vestibular function tests in primate studies [27–29]. VORs can be classified into angular VOR (aVOR) induced by rotational accelerations and primarily driven by signals from horizontal semicircular canal cristae(HSCC) [30, 31], and translational VORs (tVORs), such as linear VOR (LVOR) and off-vertical axis rotation (OVAR), can provide otolith stimulus [32, 33]. OVAR typically entails constant rotational velocity of the mouse head about an axis that is tilted to the Earth's vertical axis. After achieving steady state rotation, the semicircular canal response decays, and the resulting ocular response is from otolithic origin [27, 33]. LVOR response is too small to provide reliable estimates of otolith function [34], but OVAR signals are large enough, and OVAR stimulus could be easily combined with aVOR stimulus into one machine. Therefore, the combination of aVOR and OVAR can form the basis of a complete functional test for the peripheral vestibular organ. The commonly used techniques for recording VOR include video oculography (VOG), search coil, and electro-oculography (EOG), with VOG being the most widely used recording method due to its high resolution and being non-invasive [30].

TDT (Tucker-Davis Technologies) provides commonly used equipment for hearing evaluation in mice, which allows for standardized tests. There is no similar standard equipment for vestibular function tests on mice. Each laboratory requires vestibular testing of mice using custom-made equipment, and therefore difficult to compare results across laboratories. Developed from a previous aVOR system [35], we implemented a comprehensive mouse vestibular peripheral functional evaluation system, integrating both aVOR and OVAR tests in a compact and commercially available instrument. The eye rotation calibrations used in this study unify the data between each instrument. UVL can also be identified with a newly developed velocity steps stimulus. To validate the system, we systematically tested the vestibular function of representative mouse models used in vestibular research and demonstrated their accuracy and repeatability in evaluating common vestibular dysfunctions. The vestibular deficit mouse models we used were: 1) Dose-dependent vestibulotoxic drug model produced by intraperitoneal injection of 3,3’-iminodiproprionitrile (IDPN) at two different doses (0, 2, and 4 mg/g) [36]. 2) Unilateral vestibular lesion (UVL) model produced by injecting ototoxic gentamicin through the semicircular canals. 3) Transgenic mice affecting critical MET-related activity (Cdh23^v2J/v2J, TMC1^−/−). 4) Vestibulo-specific transgenic mice (Zpld1^−/− for semicircular canal dysfunction, Otop1^tlt/tlt for otolith deficit). Predicted results were achieved, which demonstrated the potential as a standard system for mouse vestibular function evaluation.

Materials and Methods

The Vestibular Function Test System

A schematic overview of the binocular VOG-based VOR test system (Shenzhen Giant Tek Co., Ltd) shown in Fig. 1, consists of a microcontroller unit (MCU) and a servo motor system to generate rotational motion of various profiles and used as the vestibular stimulus. The MCU interfaces with an LCD touchscreen for inputting test parameters for rotary motion profiles, which are listed in the table in Fig. 1A. The MCU also controls the video camera for simultaneous recording mouse's eye movement. The recorded video can be downloaded via a WIFI to a PC or edge computing device for processing. A similar system has been previously described [35]. The eye movement video recording and the data processing are automated. No human intervention is required. The video recording is synchronized with the rotational stimulus. The stimulus parameters (for aVOR: frequencies and speeds) set up the test profiles. The eye movement during each test profile is recorded in a separate video file.

Fig. 1.

The mouse vestibular function evaluation system used in the studies. (A) The rotation modes of aVOR and OVAR and the hardware architecture diagram of the VOR detection system, as used in these studies. (B) Overview of the components of the system. (C) The noninvasive animal-immobility setup. In the setup, mice are head-fixed at ~ 30° nose-down to maintain the horizontal semicircular canals parallel to the yaw plane. (D) Scheme of rotation paradigm for the angular horizontal vestibulo-ocular reflex (aVOR) stimulus, a sinusoidal counter rotation, and velocity step stimulus in the horizontal plane. (E) Schematic view of apparatus for Off Vertical Axis Rotation (OVAR) stimulus, clockwise (CW) and counter-clockwise rotations (CCW) in yaw plane with the platform tilted off the vertical axis of rotation by θ degrees, at a rotation of speed of 30, 50, 80°/s. θ indicates the tilt angle of 17° and 30° to the rotation axis during OVAR. (F) The linear calibration of the eye movement recording system. (G) The horizontal angle calibration of the eye movement recording system. (H) The vertical angle calibration of the eye movement recording system

Figure 1B shows that the platform supports the core of the test system: the animal and eye tracking system. This includes the servo motor that rotates the platform, which delivers the vestibular stimulus. Eye movements were recorded by two infrared-red motion cameras (EZVIZ S6, China) with 1920 × 1080 resolution at 120 frames per second. Illumination of the video recording was provided by two infrared LEDs, one for each eye. Another pair of LEDs flashed in synchrony with the rotational platform and marked each rotational cycle to calculate the eye movement phase delay compared to the sinusoidal rotational stimulus. The mouse was restrained in a custom-built holder adapted to its size and weight (Fig. 1C) and then attached to the rotary platform. Developed from a noninvasive animal-immobility setup [35], the mouse holder allows fixing the animal position without anesthesia and surgery for headpost implantation, simplifying the preparation. The magnetic holder allows for animal restraint within 30 s per animal (see the YouTube videos: https://www.youtube.com/watch?v=YSHMsHDkunQ&list=LL). Pilocarpine eye drops (2%) were applied to the eyes to maintain pupil constriction in darkness.

The Test Modes for Evaluating Vestibular Functions

Two commonly used vestibular testing modes, angular vestibulo-ocular reflex (aVOR) (Fig. 1D) and off-vertical axis rotation (OVAR) (Fig. 1E), were integrated into this system.

The aVOR is generally used to assess HSCC function [30]. Two types of stimuli, horizontal sinusoidal oscillation and velocity step, were implemented to measure the HSCC function and identify the side of the unilateral vestibular lesion (UVL). During the HSCC test, the animal was stimulated by the sinusoidal oscillation of the platform at frequencies of 0.2, 0.5, 0.8, 1.6, 3.2, and 5.0 Hz, with peak velocities of 40°/s. During the UVL test, the velocity step or trapezoid (144°/s) stimulus was used to identify the lesion side. The VOR turntable platform was accelerated for one second, then at a constant velocity for 32 s, and finally, the velocity decreased to zero within one second. Horizontal nystagmus was observed during platform acceleration and deceleration, from which the vestibular time constant was calculated as described in the previous study [37].

The OVAR test has been used to evaluate otolith organ function in mice [31, 38]. To accommodate the new OVAR test, the equipment system is hinged to a 50*50*2 cm aluminum base, which holds a motorized push rod to raise the back of the system and create a pitch of set angles. During the OVAR test, the equipment was tilted at 17° or 30°, as shown in Fig. 1E. The platform rotated at constant velocities of 30, 50, and 80°/s in clockwise (CW) or counterclockwise (CCW) directions. The horizontal and vertical (as described in this paper) eye movements during the test were analyzed to evaluate the otolith function. The vertical velocity has no bias component, and the eye movement is relatively smooth. The horizontal eye movement includes a modulation component and a bias component with sustained nystagmus. In this paper, we focus on vertical eye movement because, compared with horizontal eye movement, vertical eye movement data analysis is easier and more accurate. Vertical eye movements reflect the changes of the head relative to the direction of gravity, and can also accurately evaluate the function of otolith organs in mice.

Eye Rotation Calibration

A triangular head model was used to calibrate the zoom factor of the camera system, as shown in Fig. 1F. The zoom length of the two cameras in the system was adjusted to ensure the field of view of both cameras is adjusted to 6 mm. The rotational angle of the eye movement was calibrated with an eyeball model attached to a protractor, as shown in Fig. 1G for horizontal and Fig. 1H for vertical eye movement. The diameter of the eyeball modal is 3 mm, about the same size as the eyeball of a 20 g mouse [39]. We measured the eye diameters of mice weighing 20 g, 25 g, and 30 g to be approximately 3 mm. The eyeball diameter can also be changed according to the size of the mouse. During the calibration, the artificial eyeball is rotated at precise angles with the protractor, and the image of the artificial eyeball is recorded at different angles. The angular calibration scale can be calculated based on the translational distance of the pupil in the picture and the angular readout from the protractor. With the horizontal and vertical calibration gadget, we verified that the angular calibration scale is the same in both axes. The standard equipment and test procedure, as well as these calibration tools, ensured the repeatability between different systems, making it possible to compare results from other labs.

Eye Movement Analysis

Each experiment would contain many test sessions, which feature different modes (aVOR or OVAR), rational frequencies, and velocity stimuli. During each session, the eye movement was recorded as an individual video file. The computing unit reads this video file via WIFI and starts processing right after the first session finishes, in parallel with the video recording of the second test session. After the second session is finished, the second video file will be read and processed. And the third session starts. As shown in Fig. 2A, usually the processing time is less than the test time for each session. This parallel processing ensures that the experimental results can be attained a few minutes after the last (N) test session.

Fig. 2.

The workflow and outputs of the eye movement analysis. (A) The workflow of eye movement analysis. Each experiment would contain many test sessions. During each session, the eye movement was recorded as an individual video file. The computing unit reads this video file via WIFI. The parallel processing ensures that the experimental results can be attained a few minutes after the last (N) test session. The eye movement analysis processing was constituted of three parallel threads: 1) A CPU thread reads the video file for the last test session and each frame was extracted, shown as the black block; 2) A GPU thread processed each frames of the video to recognize the pupil using a pre-trained YOLOv5 DNN (Deep Neural Networks), shown as the white block; 3) Another CPU thread segmented the pupil from the recognized region (the red square) and an ellipse fitted the resulting binary image to calculate the pupil center position, shown as the grey block. (B) During low-frequency aVOR testing, both the fast and slow phases of eye movements are present, and we filter out the fast-phase component and preserve the slow-phase one. (C) The example outputs of aVOR responses (0.2 Hz) and the trigger signal generated at a specific position in each cycle of the rotating table. (D) The example outputs of aVOR responses (5.0 Hz) and the trigger signal. Gain (E) and phase (F) for each testing frequency were computed from the aVOR waveforms. (G) The fast-slow alternating nystagmus and eye velocity were recorded at the beginning of the velocity step stimulus (The black arrow indicates a slow phase). (H) The horizontal slow phase velocity, highlighted in Fig. 2H, was fitted, and the vestibular time constant was calculated. (I) The example outputs of measurable eye movements evoked by OVAR (80°/s) include horizontal eye position and vertical eye position

The eye movement analysis is implemented in an embedded computer: Jetson Orin Nano, as shown in Fig. 2A. Jetson Orin Nano is a multi-core processor with both CPU cores for classical imaging processing and GPU cores for implementing deep neural networks (DNN) for image classification. The eye movement analysis processing was constituted of three parallel threads: 1) A CPU thread reads the video file for the last test session and extracts each frame for processing, shown as the black block in Fig. 2A; 2) A GPU thread processes each frame of the video to recognize the pupil using a pre-trained YOLOv5 DNN, shown as the white block in Fig. 2A; 3) Another CPU thread segmented the pupil from the recognizes region (the red square) and an ellipse fits the resulting binary image to calculate the pupil center position, shown as the grey block in Fig. 2A.

During low-frequency sinusoidal oscillation in aVOR testing, both the fast and slow phases of eye movements are present, as depicted in Fig. 2B. However, the gain and phase metrics we need can be derived from the slow-phase eye movements alone. Hence, we first filter out the fast-phase component and preserve the slow-phase one using a proper method [40].

To calculate the phase, we selected 10–20 good (more regular) eye-movement cycles from the first 30-cycle period following the onset of rotational stimulation. As shown in Figs. 2C and 2D, a trigger signal (red pulse) is extracted from the eye movement video. The trigger was generated at the left-most position of the rotating table in each cycle, by flushing an infrared LED for 10 ms. In this way, the synchronous trigger is printed onto each eye movement video with an intensity pulse at each rotational cycle. Using the trigger to slice the eye movement data at a consistent phase, then performing Fast Fourier Transform (FFT) analysis, we obtained the phase difference between the eye movements and the rotational table movement (head movement of the mouse). Under low-frequency conditions, a phase lead is observed, while a phase lag is exhibited at high frequencies (Fig. 2F).

In the aVOR mode presented in this study, gain is calculated as the ratio of the maximum slow-phase eye movement velocity to the maximum table velocity, which is 40°/s under aVOR testing, as shown in Fig. 2E.

The angular velocity step at 144°/s stimulus was employed to evaluate the response of the vestibular system to HSCC input. A fast-slow alternating nystagmus will appear at the beginning and end of the stimulus. As shown in Fig. 2G, the eye velocity includes fast-phase and slow-phase velocity. The fast-phase component is removed to calculate the vestibular time constant, and an exponential fit is applied to the slow-phase eye velocity, as shown in Fig. 2H. A curve ($f\left(x\right)=a\ast e_{}^{b\ast x}$) was best fit to the slow phase velocity. The vestibular time constant is calculated as $\tau =-1/b$. By calculating the time constants of the vestibular system in both CCW and CW directions, it can reveal any asymmetry of peripheral input, and this can be used to assess the presence of UVL.

OVAR stimulation induces both vertical and horizontal eye movements in mice. Vertical eye movements lack of fast phase, while horizontal eye movements include it. By filtering out the fast phase, as done in aVOR data processing, we obtain relatively smooth horizontal slow-phase eye movements, as shown in Fig. 2I.

Mouse Models of Peripheral Vestibular Deficits

The Animal Care and Ethics Committee of the Southern University of Science and Technology approved all surgical and experimental procedures. We have attempted to abide by the ARRIVE guidelines throughout the manuscript [41]. We used common mouse models of peripheral vestibular deficits with genetic or vestibulotoxic or surgical intervention to validate the system's efficacy in vestibular function evaluation. Table 1 provides details of these various models. 6–8-week-old C57BL/6J mice, including both genders, were used to investigate vestibulotoxicity drugs. The mutant mice and their wild-type littermates, including both genders, were also used at 6–8 weeks old.

Table 1.

The mouse models used in the study

		Name	Drug delivery strategies	Concentration	Time points (day)
Vestibulotoxicity drugs	Vestibular dysfunction	3,3′-iminodiproprionitrile (IDPN, 3 male/3 female per group)	Intraperitoneal injection	2 mg/g, 4 mg/g	0, 2–7, 9, 11, 14, 17, 21, 28, 42, 56 (post-injection day)
	Unilateral vestibular lesion (UVL)	Gentamicin (3 male/3 female per group)	Injection through semicircular canals (canalostomy)	8 mg/ml 15ul	7 (post-injection day)
Mutant mouse models- Deaf mice	Hearing loss with vestibular dysfunction	Cdh23v2J/v2J (4 male/4 female)			56 (postnatal day)
	Hearing loss without vestibular dysfunction	Tmc1−/− (6 male/5 female)			56 (postnatal day)
Mutant mouse models- vestibular dysfunction mice	Absent otoconia	Otop1tlt/tlt (3 male/2 female)			56 (postnatal day)
	Semicircular canal dysfunction	Zpld1−/− (6 male/6 female)			56 (postnatal day)

Two mutant mouse models of mechano-electrical transduction deficits, Cdh23^v2J/v2J and TMC1^−/− were tested with the system. The Cdh23 protein is localized in the tip links of hair cell stereocilia and is proposed to regulate the activity of mechanically gated ion channels in mice [42]. The Tmc1 protein is a pore-forming component of mechanosensory transduction channels in hair cells [43]. Cdh23^v2J/v2J mice (The Jackson Laboratory, 002552) are characterized by circling, head-tossing, deafness, and hyperactivity. TMC1^−/− mice were generated using the Biocytogen Extreme Genome Editing System (EGE®) (Biocytogen, Beijing, China) in a C57BL/6 background. TMC1^−/− mice are deaf but with no reported vestibular defects due to the compensation of its closely related paralog Tmc2 [44].

Two other mutant mouse models with dysfunction in different vestibular organs, Otop1^tlt/tlt and Zpld1^−/− were tested to demonstrate the system's specificity. It has been reported that the zona pellucida-like domain protein (Zpld) is one of the major structural proteins of the overlying cupula [45]. Moreover, Vijayakumar S. et al. [46] have reported that spontaneous mutations of the Zpld1 gene in mice cause semicircular canal dysfunction but do not impair gravity receptor or hearing functions. Otop1 has been regarded as a critical protein in regulating multiple aspects of otoconia and otolith development [47]. The Otop1^tlt/tlt mice (obtained from Jackson Labs, 001104) carry single-point mutations that specifically impair the morphogenesis of otoconia without affecting other inner ear organs [48]. Zpld1^−/− mouse model was generated using the Biocytogen Extreme Genome Editing System (EGE®) (Biocytogen, Beijing, China) in a C57BL/6 background.

The vestibulotoxic drug 3,3’-iminodiproprionitrile (IDPN, Tokyo Chemical Industry, I0010, d = 1.02 g/mL) was used to establish a vestibular dysfunction mouse model with quantified damage levels. IDPN, as a nitrile, is a neurotoxic and vestibulotoxic compound, and was used to induce vestibular damage [49–51]. Each animal received a single intraperitoneal injection of IDPN at either 2 or 4 mg IDPN per gram of body weight or saline (NS) as a control. Vestibular function was evaluated before and after the drug exposure. Over the next 56 days, the three groups of animals, each receiving a different dose of IDPN or saline, were tested at multiple time points. This design allowed us to demonstrate the progressive damage and recovery/compensation of the vestibular function, providing valuable insights into the effects of IDPN on vestibular function.

A unilateral vestibular lesion (UVL) mouse model is established by delivering gentamicin (Macklin, G810322) via a canalostomy, which is a surgical approach for local drug delivery into semicircular canals [52, 53]. Briefly, mice were anesthetized by intraperitoneal injection of Dexmedetomidine Hydrochloride Injection (Dexdomitor®, 0.08 mg/kg) and Zoletil 50® (32 mg/kg). Surgery occurred either on the left or right ear. The post-auricular region was prepared by shaving with a clipper and depilatory cream, and a post-auricular incision was made along the retroarticular groove. The muscle covering the temporal bone was reflected using micro-forceps, revealing the posterior semicircular canal (PSCC) and HSCC. A small hole was created in the middle portion of the HSCC using a 26 G needle, and perilymphatic fluid leakage confirmed the perforation of the bony labyrinth. Subsequently, 15 μl of gentamicin (8 mg/ml) was injected into the HSCC via polyimide tubing (inner diameter 114.3 μm, outer diameter 139.7 μm) using a micro-infusion pump (3 μl/min). After the injection, the hole was plugged with a small piece of muscle and covered with an adhesive agent. Finally, the skin incision was sutured.

Auditory Brainstem Responses (ABRs)

The ABR measurements were conducted in a soundproof chamber. The tone bursts ranging from 4 to 32 kHz were delivered through a speaker (Tucker-Davis Technologies) placed in front of the animal 5 cm away from the vertex. The rising and falling times of the stimulus sound were set at 1 ms, and the total duration of stimulation was 3 ms. ABRs were recorded via three needle electrodes: the active electrode was placed at the vertex of the skull, the reference electrode was placed at the mastoid area of the right ear, and the ground electrode was placed at the left hind leg. The response signals were digitized and acquired using BioSigRZ software (Tucker-Davis Technologies). The ABR threshold was the lowest sound stimulus level to evoke a precise and repeatable waveform.

Statistical Analysis

The “n” in the figures refers to the number of mice per group. All data are presented as the mean ± SEM. Data analysis was performed using GraphPad Prism 6 software. Student's t-test was used for comparing two groups, while one- or two-way analysis of variance (ANOVA) tests were used for comparisons involving more than two groups. The detected difference was defined to be significant when p < 0.05.

Results

Quantification of IDPN-Induced Vestibular Dysfunction and its Progress

The IDPN-injected (NS, 2mg/g and 4 mg/g) mice were tested at different VOR stimulus levels for 56 days (Fig. 3A). There were three groups and six mice per group. We tested 18 mice per day with two VOR machines, one for aVOR and one for OVAR. Typical aVOR waveforms from the three groups of mice are shown in Fig. 3B. Gain and phase for each testing frequency were computed from the recorded eye movement data. Typical waveforms of vertical eye movement in the OVAR test are shown in Fig. 3C. Vertical eye movement amplitude in degrees was calculated from the waveforms.

Fig. 3.

The vestibular dysfunction after IDPN injection. (A) Time course of vestibular system evaluation after IDPN injection. The red arrow means mice were treated with IDPN at day0 (D0). The black arrows mean the time-points of aVOR & OVAR tests. The red arrowheads mean the time-points (Day3 and Day14) that we will highlight in this figure. (B) Representative aVOR waveforms of the IDPN injected mice (NS, 2 mg/g, and 4 mg/g) at Day14 (D14) were induced by sinusoidal rotation of 0.8 Hz, peak velocity 40°/s. (C) Vertical eye position waveforms of the IDPN injected mice (NS, 2 mg/g, and 4 mg/g) at Day14 (D14) were induced by OVAR stimulus (constant velocity of 80°/s in CW direction at the 17° tilted table). (D) The time-course characteristics of aVOR gain in the IDPN injected mice (NS, 2 mg/g, and 4 mg/g, n = 6 per group) at 3.2 Hz, 40°/s. The dashed box highlights the aVOR gain at Day3. (E) The time-course characteristics of vertical eye positions in the IDPN injected mice (NS, 2 mg/g, and 4 mg/g) at a constant velocity of 50°/s in CW direction at the 17° tilted table. Gains (F) and phases (G) of aVOR, at different frequencies, in the IDPN injected mice (NS, 2 mg/g, and 4 mg/g) at day3 (D3). Gains (I) and phases (J) of aVOR in the IDPN injected mice at D14. Amplitudes of vertical eye positions in the IDPN injected mice (NS, 2 mg/g, and 4 mg/g) at 30, 50, and 80°/s CW OVAR stimulus at D3 (H) and D14 (K). All data presented in mean ± SEM, two-way ANOVA, * p < 0.05

The progress of aVOR gain for the three groups under a rotational stimulus of 3.2 Hz and 40 degree/s is shown in Fig. 3D. The gains at other frequencies, 0.2 Hz, 0.5 Hz, 0.8 Hz, 1.6 Hz, and 5.0 Hz, were shown in Supplemental Figure S1A-E. All showed a similar progression pattern. After IDPN administration, the aVOR gain of the 2 mg/g group shows a sharp decline on day5 and a slow recovery from day11. The aVOR gain of the 4 mg/g group showed an early sharp decrease on day3 and stayed at virtually zero after that. A similar progression pattern was shown in the vertical movement amplitude in the OVAR test of the CW-50°/s stimulus (Fig. 3E). The amplitudes using CW-30°/s, CW-80°/s, and counterclockwise (CCW) stimulus are shown in Supplemental Figure S1F-J. All showed a similar progression pattern. Test results on two individual days (D3 and D14) were shown in Fig. 3F, G and I, J for the aVOR tests, and Fig. 3H and K for the OVAR tests. The aVOR gain and OVAR test results demonstrated a dose-dependent decrease, except for the aVOR responses on day3. aVOR gain of the 2mg/g group increased on day3 compared to the NS group under all stimulus modes. In prior work, we also found aVOR gain of the 2 mg/g group increased early in the post-injection period [35]. The phases of the 2 mg/g group are generally not significantly different from those of the NS group on day3 (Fig. 3G). The aVOR gain and OVAR of the 2 mg/g group are recovering on day14 (Fig. 3I, K). There is no significant difference between the phases of the NS and 2 mg/g group on day14 at 0.2 Hz, 0.5 Hz, 0.8 Hz, and 3.2 Hz (Fig. 3J).

These dose-dependent results and the progression pattern demonstrated the quantification capability of the test system for the vestibular function.

Different Vestibular Phenotypes in MET-Related Mutant Mice (Cdh23^v2J/v2J and TMC1^−/−)

Both adult Cdh23^v2J/v2J and TMC1^−/− mice are profoundly deaf (Fig. 4A, B). The results of Cdh23^v2J/v2J mice are shown in Fig. 4C-G. Cdh23^v2J/v2J mice have no obvious and recordable eye movements under all aVOR and OVAR stimuli (Fig. 4C, F) but have spontaneous nystagmus (data not shown). Data shown in Fig. 4D summarize the gains of the aVOR responses for the Cdh23^v2J/v2J mice and their wild-type littermates. While the WT group exhibited high-gain compensatory responses at all the frequencies, Cdh23^v2J/v2J mice exhibited virtually zero gain responses (below the noise floor) at the same frequencies. Therefore, the phase of Cdh23^v2J/v2J mice cannot be determined (Fig. 4E). As for the OVAR response at 17° by Cdh23^v2J/v2J mice, they exhibited no eye movement responses (below the noise floor) to head rotation (Fig. 4G). The OVAR responses at 30° tilted table were proportionally similar to those of 17° (Figure S2A).

Fig. 4.

aVOR and OVAR responses in MET-related mutant mice (Cdh23^v2J/v2J and TMC1^−/−). The mean ABR thresholds of Cdh23^v2J/v2J mice (A) and TMC1^−/− mice (B) indicate that both mice have severe hearing loss at all stimuli. The black arrows mean that mice showed no response at 90 dB SPL, the upper limit of the ABR recording. (C) aVOR waveforms in response to 0.8 Hz 40°/s stimulation of Cdh23^v2J/v2J and control WT mice. Gain (D) of aVOR is plotted as a function of head rotation frequency for the WT group (purple) and Cdh23^v2J/v2J mice (green). (E) Cdh23^v2J/v2J mice exhibited virtually zero gain responses (under the Noise floor), so the phase cannot be determined. (F) OVAR waveforms (vertical eye positions) in response to constant velocity of 80°/s in CW direction at the 17° tilted table stimulation for Cdh23^v2J/v2J mice. (G) Amplitudes of vertical eye positions in Cdh23^v2J/v2J mice at 30, 50, 80°/s OVAR stimulus. (H) aVOR waveforms in response to 0.8 Hz 40°/s stimulation of the WT (purple) and TMC1^−/− (green) mice. Gain (I) and phase (J) of aVOR for WT group and TMC1^−/− mice. (K) OVAR waveforms (horizontal and vertical eye positions) in response to constant velocity of 80°/s in CW direction at the 17° tilted table stimulation for TMC1^−/− mice. (L) Amplitudes of vertical eye positions in TMC1^−/− and control WT mice at 30, 50, 80°/s OVAR stimulus. All data presented in mean ± SEM, unpaired t-test, * p < 0.05

The results of TMC1^−/− mice are shown in Fig. 4H-L. TMC1^−/− mice exhibited compensatory aVOR and OVAR that were similar to the responses of the WT mouse (Fig. 4H, K). Gains of TMC1^−/− mice were not significantly different from those of the WT group at all frequencies tested (Fig. 4I). The phases were nearly identical to those of the WT group (Fig. 4J). Similar OVAR responses were seen in WT and TMC1^−/− mice across all stimulus modes (Fig. 4L). Consistent with the results of 17°, there is no significant difference between TMC1^−/− and WT mice under all stimulus modes at 30° OVAR (Figure S2B).

These data demonstrated the capability of the test system in screening for the genetic mutants affecting vestibular function.

Functional Evaluation of the Semicircular Canals and Otolith Organs Respectively

The results of Zpld1^−/− mice are shown in Fig. 5A-E. Zpld1^−/− mice have decreased aVOR response (Fig. 5A) but normal OVAR response (Fig. 5D). On average, across frequencies of 0.2–1.6 Hz, aVOR gain was significantly lower in the Zpld1^−/− mice than in WT mice (Fig. 5B). At 0.2 Hz, gains for Zpld1^−/− mice were near zero, and as a result, phase estimates were unreliable and thus not included. There was a significant increase in aVOR phases between Zpld1^−/− and WT mice under 0.5–5.0 Hz stimulus modes (Fig. 5C). In response to head rotation, robust OVAR responses at all stimulus modes were seen in Zpld1^−/− mice. There was no significant difference in vertical eye movements between Zpld1^−/− and WT mice at both 17° and 30° tilted tables (Fig. 5E, S2C).

Fig. 5.

aVOR and OVAR responses in semicircular canal dysfunction mice (Zpld1^−/−) and otoconia-deficient mice (Otop1^tlt/tlt). (A) aVOR to sinusoidal head rotations with representative eye responses to 0.8 Hz head rotation for Zpld1^−/− mice. aVOR gains (B) and phases (C) for the WT group (purple) and Zpld1^−/− mice (green). (D) Representative eye position waveforms to constant velocity of 80°/s in CW direction at the 17° tilted table stimulation for Zpld1^−/− and control WT mice. (E) Amplitudes of vertical eye positions of OVAR are shown for the WT group (purple) and Zpld1^−/− mice (green). (F) aVOR to sinusoidal head rotations with representative eye responses to 0.8 Hz head rotation for Otop1^tlt/tlt and control WT mice. aVOR gains (G) and phases (H) for the WT group (purple) and Otop1^tlt/tlt mice (green). (I) Representative eye position measurements to the constant velocity of 80°/s in CW direction at the 17° tilted table stimulation for Otop1^tlt/tlt and control WT mice. (J) Amplitudes of vertical eye positions in the WT group (purple) and Otop1^tlt/tlt mice (green) at 30, 50, and 80°/s OVAR stimulus. All data presented in mean ± SEM, unpaired t-test, * p < 0.05

The results of Otop1^tlt/tlt mice are shown in Fig. 5F-J. Otop1^tlt/tlt mice exhibited normal aVOR responses (Fig. 5F) but impaired OVAR responses (Fig. 5I). Their aVOR gains (Fig. 5G) and phases (Fig. 5H) were not statistically different from those of the WT mice at all frequencies. The Otop1^tlt/tlt mice exhibited virtually no eye movement responses to 17° and 30° tilted OVAR stimuli (Fig. 5J, S2D). These results suggested that the test system could distinguish between the functions of the otolith system and the semicircular canals, respectively.

Identification of the Lesion Side on UVL Mice

The VOR results of the unilateral vestibular lesion (UVL) mouse model are shown in Fig. 6A-H. Typical examples of aVOR recordings from each group are shown in Fig. 6A. Compared with the control group, aVOR gains in the UVL groups were significantly reduced at all tested frequencies (Fig. 6B), as would be expected. There was no significant difference between the left and right eyes in the UVL groups. The phases of the UVL groups were generally not significantly different from those of the control group (Fig. 6C). Figure 6D shows examples of OVAR responses from each group 7 days after the lesion. The OVAR responses in UVL groups were significantly impaired at all stimulus modes (Fig. 6E, S2E-S2G) compared with the control group, and there was also no significant difference between the left and right eyes in the UVL groups.

Fig. 6.

Impaired vestibular function in unilateral vestibular lesion (UVL) mice. (A) Typical aVOR waveforms of the control group (green), left UVL group (purple), and right UVL group (blue) were induced by sinusoidal rotation of 0.8 Hz, peak velocity 40°/s. Gain (B) and phase (C) of the left eye and right eye aVOR for the control group (green), left UVL group (purple), and right UVL group (blue). (D) Representative raw traces of vertical and horizontal eye movements of the control group (green), left UVL group (purple), and right UVL group (blue) at 80°/s OVAR stimulus. (E) Amplitudes of the left eye and right eye vertical eye positions in the control group (green), left UVL group (purple), and right UVL group (blue) at 17° tilted platform OVAR stimulus in CW direction, * p < 0.05 compared with Control-Right eye; # p < 0.05 compared with Control-Left eye. (F) Velocity step or trapezoid (144°/s) stimulus elicited changes in horizontal eye position. (G) The time constant for the control group, left UVL group, and right UVL group in CCW and CW direction (paired t-test). (H) The normalized time constant for the control group (green), left UVL group (purple), and right UVL group (blue). The time constant for contralesional stimulation (CW for left UVL, CCW for right UVL) was standardized to 1. All data presented in mean ± SEM, two-way ANOVA, * p < 0.05

During velocity step stimuli, while the time constant obtained at the beginning of CW and CCW rotation was symmetric in the control group, in UVL mice it was asymmetrical and lateralized to the unaffected side rotation (CW in left UVL and CCW in right UVL) (Fig. 6F, G). To simplify the comparison of the time constant between control and UVL mice, the time constant at the unaffected side rotation (CW for left UVL, CCW for right UVL) was taken as 1 (Fig. 6H). These results suggest that the time constant can determine if the lesion is contra- or ipsilateral.

Discussion

In our study, we have successfully developed a vestibular function evaluation system based on VOR, and efforts have been made to establish standard vestibular tests across laboratories. The advantages of our system are as follows:

(1) Our system can evaluate different vestibular deficits, making it applicable in various vestibulo-related studies. The system integrates both aVOR and OVAR tests in a compact and commercially available instrument. The integrated aVOR and OVAR modes allowed us to assess the otolith organs and horizontal semicircular canals separately. UVL can be identified by quantifying the time constant during velocity step stimuli. In comparison, VsEP, a widely used vestibular function test, only tests the function of otolith organs [54, 55]. The efficacy of our system was thoroughly evaluated with various vestibular (canal, otolith, UVL) deficit models. We also developed calibration tools to ensure the consistency among different test units.

(2) The operation of the system is simple and fast, making it convenient for daily vestibular tests and suitable for high-throughput tests if needed. Our animal holders ensure a quick and convenient VOR test without anesthesia and surgery. In comparison, most VOR systems [56–59] require headpost implantation surgery, and the tests cannot begin until at least 48 h after the surgery. The data processing in our system is automated, and results can be attained within a few minutes after experiments. As an example, our system allowed us to conduct hundreds of experiments within two months to monitor the progress (deterioration and recovery) of the mouse vestibular functions after ototoxic drug IDPN treatment.

Low-Dose IDPN Treatment Resulted in Increased aVOR Gain at the Early Stage and Significant Functional Recovery at the Later Stage

Recent studies have also explored the effects of different IDPN concentrations on VOR in mice [36, 60]. Zeng et al. evaluated the VOR test 7 days, 1 month, and 3 months after IDPN injection. And, in Schenberg et al.'s work, the VORs were quantified every two weeks using canal-specific and otolith-specific tests. In their works, the observation interval is long, thus, the changes in vestibular function in the early administration stage were not reported and potentially missed. In traditional VOR tests, headpost implantation surgery is necessary, and the tests cannot begin until at least 48 h after the surgery. Our animal holders help us test immediately without anesthesia and surgery. Due to its convenience, our system allowed us to monitor vestibular function changes continuously following various treatments. For example, during the IDPN toxicity experiment, we observed increased aVOR gain after administration of 2 mg/g from Day 2 to Day 4 compared with the baseline before treatment. Our previous study also observed this phenomenon [35]. While the precise reason for this phenomenon remains unknown, it has been reported that hair cells may not be the primary target of IDPN [61]. Type I hair cells are susceptible to IDPN toxicity, and their calyx junction dismantlement and synaptic uncoupling are early events in the mouse vestibular sensory epithelium post-exposure [62–64]. In addition, studies have shown that exposure to IDPN may also lead to alterations in the responses of vestibular afferent neurons to acetylcholine (ACh) [65]. ACh is a neurotransmitter of the vestibular efferent nervous system, in which cholinergic receptors activate Type II hair cells and afferent fibers. We suggest that in the early phase of post-IDPN administration, the vestibular efferent system is altered, possibly leading to disinhibition of Type II hair cells. Consequently, there is an increased response to head rotations, leading to a transient increase in aVOR at the 2 mg/g dosages. Further studies are needed to determine the precise mechanism.

We observed significant recovery of vestibular function starting from Day 9 after 2mg/g IDPN injection. In our study, mice were administered a single intraperitoneal injection. Zeng et al. administered the IDPN to mice in the same protocol and mentioned that eye movement almost recovered to normal at 3 months after a 2 mg/g IDPN body weight injection. And, in the 4 and 6 mg/g IDPN groups, only partial recovery was seen [51]. They propose that the recovery of vestibular function depends on the remaining hair cells and supporting cells. In Schenberg et al.’s study, IDPN was sub-chronically administered to mice through drinking water. And, a significant recovery of VOR responses was observed during the washout period, accompanied by the number of type I HC labelled in the IDPN-treated mice recovering to the Sham group level, which suggest recovery of the vestibular function quantified by VOR measures is correlated with the number of type I HC in both canals and otoliths [60]. The recovery in the number of type I HC observed is more likely the result of molecular repair, not cell regeneration. We speculate that the peripheral input from the vestibular nuclei increases after vestibular organ injury[66], which may also adjust the VOR gain recovery. Sub-chronic IDPN treatment leads to reversible dismantlement of calyceal junction, followed by reversible synaptic uncoupling at the early stage [63]. Whether these reversible structure changes correlate with the recovery of low-dose IDPN treatment at the later stage should be future studies on acute IDPN treatment.

VOR is a More Reliable Measure of Vestibular Function than Behavioral Tests

Behavioral tests are also a common measure of vestibular function for mice. During the Zpld1^−/− experiment, only a few mice (two out of twelve) exhibited observable behavioral patterns such as circling and head bobbing. Vijayakumar and his colleagues also reported this highly variant behavioral pattern in the Zpld1 spontaneous mutant mice, but unfortunately, they were unable to perform VOR tests on the Zpld1 knockout mice [46]. All Zpld1^−/− mice consistently show reduced aVOR gain (Fig. 5B) and increased phase lead (Fig. 5C) in our work. This result suggests that VOR measurements are a more reliable measure of vestibular function.

Here we report the first VOR results of the Zpld1^−/−mice. Compared with Zpld1 spontaneous mutant mice [46], Zpld1 knockout mice have more severe semicircular canal dysfunction during aVOR testing. We observed Zpld1^−/−mice have normal gain at the two high frequencies (3.2 and 5.0 Hz), which may suggest that under high-frequency stimulation, hair cells are directly impacted by the inner lymph fluid, which deflects to produce nerve impulses, and drives eye movement through the three-neuron pathway. Even without the cupula, the above-mentioned process can also produce near-normal eye movement reflex in Zpld1^−/− mice.

VOR Identifies the Lesion Side of UVL with an Asymmetric Time Constant

Although it has been reported that VOR is not able to detect UVL, our data suggest that the time constant during the velocity step or trapezoid (144°⁄s) stimulus provides a simple metric for detecting UVL. In unilateral damage to the vestibular organ, there is a reduction or even silencing of resting firing frequency on the lesioned side, creating an imbalance between the normal resting firing rates on the contralesional and ipsilesional sides [67]. This imbalance mimics a constant rotation toward the contralesional side, resulting in asymmetric responses.

Difference of the aVOR Gain and Phase

Compared to the gain, the phase of the aVOR response is less sensitive to the vestibular dysfunction in most peripheral vestibular deficit models in this study. Only Zpld1^−/− mice showed a clear phase increase compared to the wild-type ones. In the Zpld1 spontaneous mutant mice, the VOR phase showed an increased tendency compared to the wild-type mice [45], and there is little explanation for this phenotype. Previous studies have shown that unilateral labyrinthectomy also results in a phase increase [66]. Our unilateral vestibular lesion model didn’t show phase change, probably because the damage degree in our study is lower than the unilateral labyrinthectomy, which completely destroyed the sensory epithelia of the semicircular canals and otolith organs by aspiration and excision. As discussed before, after a unilateral vestibular lesion, the hair cells'recovery and the efferent vestibular system's compensation may help with VOR recovery.

Summary

This paper introduced a mouse vestibular function test system, and its efficacy was systematically tested with standard peripheral vestibular deficit mouse models. The design of the animal holder, together with the automatic test procedure, allows researchers to evaluate vestibular function on a daily basis. This system may provide standardized testing of mouse vestibular function and may also help with the study of the development and regeneration of the inner ear. Developed from an aVOR system [35], we extended it to include OVAR and velocity step or trapezoid (144°/s) stimulus to test otolith organs and UVL. Other stimulus paradigms may be expanded in the future. Based on the system, mouse models of vestibular central deficit may also be evaluated by applying a new stimulus profile and extracting more eye movement features.

Supplementary Information

Below is the link to the electronic supplementary material.

Authors Contributions

Conceptualization: F.C., T.Z.; Methodology: T.Z., S.X., W.L.; Project administration: F.C.; Software: J.Z.; Data curation: T.Z., B.S.; Writing—original draft: T.Z., F.C.; Writing—review and editing: T.Z., F.C., S.X.

Funding

This study was supported by grants from the National Key R&D Program of China (2024YFC2417900), the National Key Research and Development Program of China (2023YFF1203505), Shenzhen Science and Technology Program (KJZD20230923114909019), and Shenzhen Municipal Science and Technology Innovation Council (202110293000007). POE (https://poe.com/) was used between October and November 2023 to refine the academic English in the introduction, methods, and discussion sections of this article. The authors take full responsibility for the accuracy and integrity of the content. AI was not used to generate data or figures.

Data Availability

The data will be available from the corresponding author upon reasonable request.

Code Availability

The MATLAB code will be available from the corresponding author upon request.

Declarations

Ethics Approval

The Animal Care and Ethics Committee of the Southern University of Science and Technology (SUSTech-JY202303083) approved all animal surgical and experimental procedures.

Conflict of interest

The VOR test system used in our study was developed by Giant Tek Ltd. W.L. is an employee of Giant Tek, and F.C. is its technical advisor. The remaining authors declare no competing interests.