Commercial cytokine assay on equine cerebrospinal fluid does not distinguish equine degenerative myeloencephalopathy from cervical vertebral stenotic myelopathy.

Overview

• This study assessed cerebrospinal fluid (CSF) cytokine levels in horses with two neurological diseases—equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) and cervical vertebral stenotic myelopathy (CVSM)—and compared them to healthy controls.
• The goal was to determine if a commercial cytokine assay could distinguish between these diseases based on CSF profiles and if cytokine levels correlated with clinical measures.

Study Objectives

• Measure and compare concentrations of multiple cytokines in equine CSF among three groups: horses with eNAD/EDM, horses with CVSM, and neurologically normal control horses.
• Evaluate whether cytokine profiles in CSF can serve as biomarkers to differentiate these two neurological diseases.
• Assess possible correlations between CSF cytokine levels and clinical disease parameters, such as neurofilament levels.

Methodology

• Samples: Banked CSF samples were used from 35 horses diagnosed postmortem with CVSM, 35 with eNAD/EDM, and 8 control horses without neurological disease.
• Diagnosis Confirmation: All included horses underwent a neurologic examination and postmortem confirmation of diagnosis.
• Cytokine Assay: An equine-specific cytokine/chemokine magnetic bead multiplex assay measuring 23 cytokines simultaneously (23-cytokine multiplex panel) was employed.
• Data Analysis:
  • Comparison of cytokine concentrations between the three groups.
  • Correlation analysis between cytokine concentrations and clinical disease measures, including CSF phosphorylated neurofilament heavy (pNF-H) protein, a biomarker of neuroaxonal damage.
• Investigation of methods to enhance cytokine detection, including increasing CSF sample volume and applying protein-concentrating techniques.

Key Findings

• The majority of cytokines in the multiplex assay were below the lower limit of detection in most samples across all groups, indicating generally low cytokine levels in CSF.
• Five cytokines—eotaxin, IL-10, interferon-γ-induced protein 10 (IP-10), granulocyte colony-stimulating factor (G-CSF), and tumor necrosis factor-α (TNF-α)—were detectable in at least five samples but showed no significant differences between eNAD/EDM, CVSM, and control groups.
• Efforts to improve detection by increasing CSF volume or using commercial protein-concentrating columns did not increase cytokine recovery or detection sensitivity.
• Within the eNAD/EDM group, IL-10 levels showed a correlation with CSF phosphorylated neurofilament heavy protein concentrations, suggesting some link with neurodegeneration or inflammation in this disease group.

Interpretation and Implications

• The commercial equine cytokine multiplex assay evaluated is not sufficiently sensitive or specific to differentiate between eNAD/EDM and CVSM based on CSF cytokine profiles.
• Low cytokine concentrations and limited assay sensitivity likely contribute to inability to detect disease-specific inflammatory signatures in CSF using this method.
• The correlation between IL-10 and neurofilament heavy protein in eNAD/EDM suggests that cytokines may still have potential as biomarkers if more sensitive assays are developed.
• These results highlight the need to optimize or develop more sensitive assays to better characterize neuroinflammation in equine neurological diseases, which may improve diagnostic capabilities and understanding of disease pathogenesis.

Conclusions

• Current commercial cytokine assays for equine CSF do not adequately distinguish between neurodegenerative and compressive spinal diseases like eNAD/EDM and CVSM.
• Methodological improvements are required for cytokine profiling to become a useful diagnostic or disease-monitoring tool in equine neurology.
• Further research is warranted to develop sensitive biomarker assays that can reflect the underlying neuroinflammatory processes in these diseases.