Chronic Low-Frequency Electromagnetic Field Exposure as a Modifier of Ligament Injury Susceptibility and Early Post-Injury Inflammation

1. Summary / Rationale

Low-frequency electromagnetic fields (ELF-EMF) generated by electrical infrastructure are very prevalent in modern environments. Correlations between ELF-EMF exposure and musculoskeletal injury have been reported, yet controlled experimental evidence linking ELF-EMF exposure to connective tissue integrity and injury susceptibility is limited. This study will test whether pre-injury exposure to ELF-EMF alters baseline joint immune and extracellular matrix (ECM) programs, thereby modifying early biological responses following anterior cruciate ligament rupture (ACLR). Using a controlled, non-invasive tibial compression injury model that captures key features of human non-contact injury, whole-joint RNA sequencing and multiplex cytokine profiling will be used to assess inflammation and matrix remodeling pathways relevant to tissue integrity and injury susceptibility.

2. Specific Hypothesis

Primary hypothesis:
Chronic pre-injury exposure to low-frequency electromagnetic fields (60 Hz, 50 mG RMS) alters baseline joint immune and extracellular matrix gene programs, resulting in an altered early inflammatory and matrix remodeling response following ACL rupture.

If stable generation of 50 mG is limited by coil constraints, a reduced exposure of 20 mG RMS will be used, with field strength verified in-cage.

Support for the hypothesis would include:

Differential expression of ECM organization and matrix remodeling pathways in whole joints from EMF-exposed versus sham-exposed mice prior to injury.
Altered early post-injury inflammatory and chemokine programs following ACLR in EMF-exposed mice.
Corresponding changes in cytokine profiles measured by multiplex immunoassay.

Refutation of the hypothesis would include:

No significant differences in whole-joint transcriptomic or cytokine profiles between EMF-exposed and sham-exposed mice at baseline or following injury.

3. Experimental Design

Overview

A 2×2 factorial design will be used to assess the effects of ELF-EMF exposure and ligament injury:

Group	EMF Exposure	ACLR
A	Sham	No
B	EMF	No
C	Sham	Yes
D	EMF	Yes

All injured animals will be evaluated at a single early post-injury timepoint (Day 3).

Animal Model

Species: Mouse
Strain: C57BL/6J
Sex: One sex for this pilot study (balanced sex analysis planned for future work)
Housing: Standard ventilated rack housing with routine animal facility care

Animals will be randomly assigned to exposure and injury groups, with multiple cages represented per group to minimize cage-level confounding. Sample processing and data analysis will be performed blinded to group.

ELF-EMF Exposure Protocol

Exposure type: Sinusoidal magnetic field
Frequency: 60 Hz
Field strength: 50 mG RMS (20 mG RMS if required by coil limitations), verified within cages using a gaussmeter
Exposure schedule: 4 hours/day for 4 weeks prior to injury
Exposure delivery: External, removable Helmholtz-style coils positioned adjacent to a designated rack bay
Sham condition: Identical coil configuration with field cancellation (anti-Helmholtz configuration)

Coils will not contact cages or interfere with airflow, feeding, or husbandry. Field uniformity and temperature will be monitored to confirm non-thermal exposure.

ACLR Injury Model

Following the exposure period, mice in ACLR groups will undergo non-invasive anterior cruciate ligament rupture using a standardized tibial compression protocol. Injury parameters (e.g., force–displacement data) will be recorded when available as exploratory measures of injury severity.

4. Outcome Measures

Primary Outcome: Whole-Joint RNA Sequencing

Whole knee joints will be harvested at Day 3 post-ACLR (or matched timepoint for non-injured groups), cleaned of skin and muscle, snap frozen, homogenized, and processed for bulk RNA sequencing.

Preregistered pathway-level analyses will focus on:

Extracellular matrix organization and remodeling
Collagen fibrillogenesis, ECM structural constituents, and matrix turnover
Matrix degradation and repair (MMP, ADAMTS, TIMP families)
Inflammatory and immune programs (chemokine signaling, macrophage activation, innate immune response)
Stress and mechanotransduction-related pathways

Differential expression will be assessed using a preregistered pipeline (e.g., DESeq2), with false discovery rate correction applied.

Secondary Outcome: Multiplex Cytokine Profiling (Luminex)

Multiplex immunoassays will be used to quantify inflammatory cytokines and chemokines in serum. Targets will include canonical early inflammatory mediators (e.g., IL-1β, IL-6, TNF-α, CXCL1, CCL2), providing protein-level validation of transcriptomic findings.

Exploratory Outcomes

Injury severity metrics derived from ACLR force–displacement data (if available)
Integration of transcriptomic and cytokine data to identify coordinated inflammatory–matrix remodeling signatures
Exploratory ex vivo micro-CT at Day 3 post-ACLR on harvested limbs/joints (same animals used for RNA-seq and serum collection) to assess acute periarticular bone morphology and early structural injury features. Cartilage degeneration outcomes, which typically require later timepoints, are reserved for follow-up studies.

Sample Size

RNA-seq: n = 6 mice per group (total n = 24)
Luminex: All available animals will be analyzed when sample volume permits

This sample size is appropriate for discovery-focused, preregistered transcriptomic analyses within feasibility constraints.

Statistical Analysis

Primary contrasts:

EMF vs sham (no injury)
EMF vs sham (post-ACLR)

Statistical methods:

Differential gene expression analysis with FDR correction
Pathway enrichment analyses using preregistered gene sets
Cytokine comparisons using parametric or non-parametric tests as appropriate

Significance threshold: adjusted p < 0.05.
All analyses will follow the preregistered plan without post hoc modification of endpoints.

Timeline (≤ 6 months)

Month 1: Coil construction, calibration, sham validation, animal habituation
Months 2–3: Daily ELF-EMF exposure period
Month 4: ACLR procedures and tissue collection
Month 5: RNA-seq library preparation, sequencing, Luminex assays
Month 6: Data analysis and preregistered report preparation

5. Data Sharing Plan

All raw sequencing data, processed expression matrices, cytokine data, metadata, and analysis scripts will be deposited in an open-access repository (e.g., GEO, OSF, or Zenodo) in accordance with ResearchHub data-sharing requirements.

6. Impact

This study will provide controlled experimental evidence addressing whether infrastructure-relevant ELF-EMF exposure can plausibly alter joint tissue biology and susceptibility to ligament injury. By combining whole-joint transcriptomics with cytokine profiling, this work replaces speculation with mechanistic data relevant to connective tissue integrity and injury risk.

Chronic Low-Frequency Electromagnetic Field Exposure as a Modifier of Ligament Injury Susceptibility and Early Post-Injury Inflammation

Funding Progress

Nonprofit Recipient

Funders

Topics

DOI

Funding Progress

Nonprofit Recipient

Funders

Topics

DOI

Chronic Low-Frequency Electromagnetic Field Exposure as a Modifier of Ligament Injury Susceptibility and Early Post-Injury Inflammation

Funding Progress

Nonprofit Recipient

Funders

Topics

DOI