Characterizing Tumor-Induced Ataxia in a Vestibular Schwannoma Mouse Model

Summary

We developed a panel of behavioral assays to study tumor-induced ataxia and to evaluate therapies that preserve neurological function in a mouse model of vestibular schwannoma.

Abstract

NF2-related schwannomatosis (NF2-SWN) is an autosomal dominant tumor predisposition syndrome caused by germline mutations in the NF2 tumor suppressor gene. The disease is characterized by the development of bilateral vestibular schwannomas (VSs), which progressively enlarge and result in hearing loss, imbalance, and ataxia-symptoms that profoundly affect patients' quality of life. No FDA-approved pharmacologic treatments are currently available. Among the neurological deficits, ataxia remains a particularly debilitating yet understudied manifestation, largely due to the lack of robust preclinical models for its evaluation. In this study, we developed a set of behavioral assays to systematically assess tumor-induced ataxia in an orthotopic mouse model of VS-associated hearing loss and ataxia. These tests allow quantitative measurement of balance and motor coordination deficits arising from tumor burden. This experimental platform enables mechanistic studies of tumor-induced cerebellar dysfunction and provides a valuable tool for preclinical evaluation of therapeutic strategies. By integrating behavioral assessments with tumor biology and hearing tests, this approach facilitates the characterization of both tumor-suppressive and neuroprotective treatment effects. Ultimately, these assays offer a versatile framework that can also be applied to other neurological disorders characterized by ataxia, expanding their utility beyond NF2-SWN.

Introduction

NF2-related schwannomatosis (NF2-SWN) is a rare, dominantly inherited tumor predisposition syndrome caused by mutations in the NF2 tumor suppressor gene, which encodes the cytoskeletal protein merlin¹. Loss of merlin function disrupts signaling pathways regulating cell proliferation, adhesion, and survival, predisposing affected individuals to the development of multiple benign tumors of the nervous system. The hallmark of NF2-SWN is the formation of bilateral vestibular schwannomas (VSs) - non-malignant Schwann cell-derived tumors that arise along the vestibulocochlear nerves. These tumors progressively enlarge, compressing adjacent neural structures and leading to sensorineural hearing loss, tinnitus, and facial nerve dysfunction².

Patients with bilateral VSs frequently suffer progressive damage to the vestibular nerve and inner ear vestibular apparatus, resulting in debilitating vestibular dysfunction manifested as chronic imbalance, ataxia, and gait instability, which severely impair mobility and quality of life³. The degree of vestibular dysfunction often correlates with tumor burden and nerve involvement. These symptoms often persist even after tumor control or hearing preservation efforts, suggesting complex neurobiological mechanisms beyond mechanical compression^4,5. Although clinical tools such as dizziness handicap inventories, gait analysis, vestibulo-ocular reflex (VOR) testing, and posturography are used to quantify vestibular impairment, the underlying mechanisms of tumor-induced ataxia remain poorly understood³. Despite advances in molecular understanding and the introduction of targeted therapies such as bevacizumab, there remains no FDA-approved treatment that reliably preserves both hearing and balance function in patients with NF2-SWN. Management of vestibular symptoms remains largely supportive, highlighting a major unmet clinical need.

A major barrier to improving clinical management of VS-associated ataxia is the lack of preclinical models that faithfully reproduce the vestibular and motor coordination deficits observed in patients. Most existing VS animal models focus on tumor growth or hearing loss, without recapitulating the characteristic balance and coordination impairments. Furthermore, standardized behavioral assays to objectively assess ataxia and vestibular dysfunction in preclinical settings are not well established. This gap hinders mechanistic investigation and limits the evaluation of candidate therapies aimed at preserving vestibular function.

Here, we report the development of a VS mouse model that exhibits measurable ataxia phenotypes, together with a panel of behavioral assays specifically designed to quantify tumor-induced ataxia. This model provides a much-needed platform for investigating the mechanisms underlying VS-associated vestibular dysfunction and for evaluating the efficacy of therapeutic interventions aimed at restoring balance, coordination, and overall neurological function in patients with NF2-SWN.

Protocol

Animal experiments were conducted in accordance with the protocol approved by the Institutional Animal Care and Use Committee of Massachusetts General Brigham (MGB).

1. Animal model

1. Inoculate Nf2^-/- cells (murine Nf2^-/- Schwann cells expressing Gaussia luciferase reporter gene, Gluc) into immunocompetent C57/FVB mice. Using a stereotactic injection device (Figure 1A), inject each mouse with 1 µL of 2,500 tumor cells into the CPA region of the right hemisphere^4,6.
2. Use both male and female mice (1:1 ratio) aged 8-12 weeks old to ensure sufficient statistical power and to examine any potential sex-related differences. House mice in groups of 4 per cage under a 12-h light/dark cycle, with food and water available ad libitum.
   NOTE: Given that schwannomas develop in patients' vestibular nerves, we employed the cerebellopontine angle (CPA) model. The CPA model recapitulates the intracranial microenvironment of VSs.
3. Sham group: To evaluate the effects of animal surgery and tumor cell implantation on ataxia, include a Sham surgery group, in which mice underwent sham surgery and injection of saline into the CPA region.
4. Include a non-tumor bearing group of mice as control.
5. To monitor tumor progression, use Nf2^-/- cells expressing Gaussia luciferase reporter gene (Gluc), and measure plasma Gluc every 3 days post-implantation.
6. Briefly, collect 13 µL of blood into the EDTA tubes to prevent clotting. Transfer the blood sample to a 96-well plate, and measure Gluc activity as previously described^6,7,8,9.

2. Ataxia tests

1. Perform the four behavioral assays in a fixed sequence to minimize variability across animals. Conduct the tests in the following order: (1) hind-limb clasping test, (2) ledge test, (3) gait evaluation, and (4) kyphosis assessment. Allow a minimum rest interval of 2-5 min between tests by returning the mouse to its home cage. Monitor the animal during rest periods and proceed to the next test only when normal exploratory behavior resumes. Discontinue testing if signs of excessive stress or fatigue are observed. Complete the full set of four ataxia assays for a cohort of 10 mice within approximately 45-60 min, including rest intervals between tests.
2. Hind-limb clasping test
   NOTE: The hind-limb clasping test evaluates neuromuscular coordination, motor tone, and postural control in mice. Mice with ataxia exhibit hind-limb retraction or clasping toward the abdomen, reflecting impaired motor coordination and muscle tone regulation⁵.
   1. Grasp the mouse gently by the base of the tail , ensuring a secure yet light hold to avoid discomfort. Support the animal briefly on the cage lid or hand before lifting to reduce startle response.
   2. Lift the mouse slowly so that it is suspended in the air with its body fully extended and free of any obstacles. Keep the mouse at least 10 cm above the cage or testing surface.
   3. Maintain the suspended position for approximately 10 s and observe the position of the hindlimbs and tail without rotating or swinging the animal.
   4. Assess hind-limb posture as described below.
      1. Identify normal posture when both hindlimbs extend outward and away from the abdomen.
      2. Identify abnormal posture when one or both limbs retract toward the abdomen or cross over each other, indicating hindlimb clasping associated with ataxia or neurological impairment.
   5. Record the behavior using a predefined scoring system or capture a short video for blinded post-hoc analysis.
      NOTE: Limit each test to ≤10 s to minimize stress.
3. Ledge test
   NOTE: The ledge test assesses balance, coordination, and fine motor control in mice. It measures the animal's ability to walk along a narrow ledge and safely descend to the home cage without losing balance. Mice with cerebellar dysfunction or vestibular ataxia typically exhibit slips, falls, or unstable posture during this task⁵.
   1. Place the cage on a stable, flat surface at bench height (~1 m).
   2. Connect one end of the testing cage (Figure 1C) to the housing case (Figure 1D), ensuring that there is no vertical gap or obstruction between cages to allow the mouse to descend smoothly and naturally into its home cage (Figure 1B) without jumping or falling.
   3. Position a video camera on a tripod facing the ledge at a 30°-45° angle to capture both lateral movement and descent.
   4. Adjust lighting to minimize glare and ensure clear visibility of the mouse's limbs and tail.
   5. Gently lift the mouse by the base of the tail and support its body with your other hand. Place the mouse in the center of the ledge so that all four paws are on the surface. Release the tail only when the mouse appears balanced and alert.
   6. Allow the mouse to walk freely along the ledge without external prompting. Record the mouse for at least 10 s or until it descends into the cage.
   7. Observe its ability to balance on the ledge without slipping and to walk along the ledge toward the cage or platform.
   8. Perform three independent trials per mouse, with a rest interval of ~2 min between trials. Clean the ledge surface between animals using 70% ethanol to remove scent cues.
   9. Conduct all tests in a quiet environment with consistent lighting and minimal background noise or vibration. Avoid repeating the test excessively on the same day, as fatigue and learning effects can influence performance.
4. Gait evaluation
   NOTE: The gait evaluation test assesses locomotor coordination, stride pattern, and postural stability in freely moving mice. Mice with ataxia often display shortened stride length, unsteady movements, and irregular paw placement, indicating deficits in coordination and balance control⁵.
   1. Line a straight, flat runway with white copy paper (8.5 × 11 inch). Place the home cage at the end of the paper to encourage the mouse to walk across the paper.
   2. Coat the forepaws and hind paws lightly with two contrasting colors (e.g., blue for forepaws, red for hind paws) using a non-toxic ink pad (Figure 1E).
   3. Place the mouse at the starting end of the runway, and allow it to walk freely across the paper without external prodding.
   4. Observe and measure the following parameters:
      Stride length: the distance between successive placements of the same paw.
      Base width: the lateral distance between left and right hind footprints.
   5. Perform two to three runs per mouse, allowing rest between tests.
      NOTE: Use non-toxic, fast-drying ink and ensure only paw pads are coated. Replace the paper after each trial to avoid smearing. Apply ink sparingly to avoid blurring footprints, as excessive ink reduces the accuracy of stride measurements.
5. Kyphosis evaluation
   NOTE: The kyphosis assessment evaluates spinal alignment and postural abnormalities during natural locomotion. Mice with neurological or muscular dysfunction often exhibit a persistent dorsal curvature or hunching of the spine⁵.
   1. Place the mouse on a surface and allow it to walk or explore freely for several seconds. Avoid startling the animal, as sudden movements may temporarily alter posture.
   2. Record a short video from the side view to document trunk curvature.
   3. Observe the mouse's posture during walking, focusing on spinal alignment and trunk curvature as described below.
      1. Identify normal posture when the mouse maintains a straight or slightly arched back with smooth body movement.
      2. Identify abnormal posture when the mouse displays kyphosis, characterized by an abnormally hunched or curved spine, a rigid trunk, and reduced flexibility during gait.

3. Ataxia test analysis

NOTE: Perform all behavioral scoring by an observer blinded to experimental group assignment to minimize observer bias.

1. Ledge test scoring
   1. Assign a score of 3 if the mouse walks along the ledge smoothly, maintains balance, and descends into the cage without slipping.
   2. Assign a score of 2 if the mouse shows mild impairment, such as hesitation or a minor paw slip, but recovers and descends safely.
   3. Assign a score of 1 if the mouse exhibits moderate impairment, including frequent slips, a crouched posture, excessive tail use for balance, or a delayed descent.
   4. Assign a score of 0 if the mouse shows severe impairment, is unable to balance, falls from the ledge, or refuses to move.
2. Hind-limb clasping test scoring
   1. Assign a score of 3 if the mouse consistently displays outward-splayed hindlimbs with no evidence of clasping.
   2. Assign a score of 2 if the mouse shows mild impairment, such as intermittent retraction of one hindlimb.
   3. Assign a score of 1 if the mouse exhibits moderate impairment, characterized by partial retraction of both hindlimbs maintained near the abdomen.
   4. Assign a score of 0 if the mouse shows severe impairment, with both hindlimbs tightly clasped against the abdomen for most of the observation period.
3. Gait scoring
   1. Assign a score of 3 if the mouse walks with a steady, coordinated gait and moves in straight lines.
   2. Assign a score of 2 if the mouse shows mild impairment, such as walking slowly with mild unsteadiness or lateral deviation, limping to one side, or traveling in broad, circling paths.
   3. Assign a score of 1 if the mouse exhibits moderate impairment, including clear gait disturbance, walking using only two or three paws, dragging part of the body, or moving in tight circles.
   4. Assign a score of 0 if the mouse shows severe impairment, is unable to ambulate effectively, remains stationary or shows uncontrolled rolling movements to the side.
4. Kyphosis scoring
   1. Assign a score of 3 if the mouse remains flat, straight back during both rest and walking.
   2. Assign a score of 2 if the mouse shows mild impairment, with a slight curvature of the back at rest that straightens during movement.
   3. Assign a score of 1 if the mouse exhibits moderate impairment, with a moderately hunched back at rest that remains curved during walking.
   4. Assign a score of 0 if the mouse displays severe impairment, with a severely hunched back throughout the observation period that restricts normal movement.

4. Inter-rater reliability assessment

1. Train observers using representative example videos prior to formal scoring to standardize the interpretation of scoring criteria.
2. To minimize observer bias, score all behavioral tests independently by two trained observers blinded to treatment group and experimental time point.
3. Quantify inter-rater reliability for each behavioral assay using Cohen's kappa coefficient. Interpret kappa values as follows: <0.40, poor agreement; 0.40-0.60, moderate agreement; 0.60-0.80, substantial agreement; and >0.80, near-perfect agreement.
4. Resolve any scoring discrepancies by joint review of the recorded videos and reach a consensus score⁵.

Results

CPA tumor growth causes ataxia in the VS mouse model

To evaluate tumor-induced ataxia, we established two experimental cohorts. First, to evaluate the effects of animal surgery and tumor cell implantation on ataxia, mice underwent sham surgery and injection of saline into the CPA region. Ataxia symptoms in the sham group mice were compared with those in unoperated control mice. In the second cohort, Nf2^-/- tumors were injected into the CPA region, and ataxi...

Discussion

The behavioral assays described here provide a robust and quantitative framework for assessing ataxia and motor coordination deficits in mouse models of VS and related vestibular disorders. Adapted from established protocols for neurodegenerative disease models, such as Huntington's disease and spinocerebellar ataxias¹⁰, these tests are well-suited for characterizing tumor-induced vestibular dysfunction in NF2-SWN. By combining multiple behavioral...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fetal bovine serum	ThermoFisher	A5670701
GloMax 96 Microplate Luminometer	Promega	GM3000
McKesson blue procedure underpad	McKesson	75402
Schwann cell growth supplement	ScienCell	1752