Background & Aims

Neuroinflammation and oxidative dysfunction, and their reciprocal interplay, are critically involved in the pathophysiology of chronic neuropathic pain (NeuP). Numerous studies have investigated the crosstalk between inflammatory biomolecules such as cytokines, chemokines, and prostaglandins, as well as neuronal cells. However, the impact of immunomodulatory lipoproteins and lipids in NeuP pathophysiology is far less explored. The aim of this study was to explore the inflammatory profile in patients suffering from chronic peripheral neuropathic pain (NeuP) at different layers of integrated biological systems, concurrently, using a combination of proteomic techniques together with multivariate data analysis.

Methods

Density-gradient ultracentrifugation was used to isolate LDL and HDL from EDTA plasma as described previously with minor modifications (1)(2). Briefly, 200?g LDL and 240?g HDL were separated by isoelectric focusing on 11 cm immobilized pH gradient (IPG) strips (pH 3-10) at 48000 vHr (max 8000 V) overnight. For the analysis of oxidized HDL and LDL, an additional derivatization step was added prior to equilibration of IPG strips. After separation in the second dimension, the gel was cut and electroblotted for analysis of oxidized lipoproteins. Proteins in a master gel of pooled samples were fixed and visualized using fluorescent Lumitein. The concentration of cytokines in plasma was analyzed using U-PLEX assay based on an electrochemiluminescent detection method (Meso Scale Diagnostics, Rockville, MD, USA)(3). Oxylipins were extracted and determined using an UPLC-MS protocol previously published (4).

Results

The proteome profile analysis of low-density lipoproteins (LDL) and high-density lipoproteins (HDL) revealed lower levels of protective apolipoproteins combined with higher levels of acute-phase serum amyloids in patients compared to healthy controls. The constitutively expressed SAA4 was detected in 6 isoforms in patients, but only 2 isoforms were detected in healthy controls. The bacteriolytic enzyme lysozyme C was detected in patient LDL only. Analysis of carbonylation in HDL, revealed six oxidized protein spots, three unique to NeuP patients. No oxidized proteins were found in LDL. An inverse relationship of significantly lower levels of oxylipins, specifically octadecanoid lipid mediators, and higher levels of cytokines was found in patients compared to healthy controls.

Conclusions

Combined, these results present a previously unexplored network of integrated alterations of lipoproteins and oxylipins in patients suffering from NeuP, indicative of aberrant immuno-protective functioning in several biological systems including lipid metabolic processes and inflammation.

References

1. Karlsson H, Lindahl M. Lipoproteomics I: Mapping of proteins in low-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry. [cited 2023 Mar 8]; Available from: https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/pmic.200300938
2. Karlsson H, Lindahl M. Lipoproteomics II: Mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry. Proteomics [Internet]. 2005 [cited 2023 Mar 8];5:1431–45. Available from: https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/pmic.200401010
3. Jönsson M, Gerdle B, Ghafouri B, Bäckryd E. The inflammatory profile of cerebrospinal fluid, plasma, and saliva from patients with severe neuropathic pain and healthy controls-a pilot study. BMC Neurosci. 2021;
4. Kolmert J, Fauland A, Fuchs D, Säfholm J, Gómez C, Adner M, et al. Lipid Mediator Quantification in Isolated Human and Guinea Pig Airways: An Expanded Approach for Respiratory Research. Anal Chem [Internet]. 2018 Sep 4 [cited 2024 Jan 22];90(17):10239–48. Available from: https://pubs.acs.org/doi/abs/10.1021/acs.analchem.8b01651
5. Cruccu G, Truini A. A review of Neuropathic Pain: From Guidelines to Clinical Practice. Pain Ther. 2017;

Presenting Author

Mika Jönsson

Poster Authors

Mika Jönsson

BSc(Hons)

Linköping University

Lead Author

Emmanuel Backryd

Pain and Rehabilitation Centre

Lead Author

Stefan Ljunggren

Lead Author

Antonio Checa

Lead Author

Craig E. Wheelock

Lead Author

Björn Gerdle

Lead Author

Bijar Ghafouri

Linkoping University

Lead Author

Topics

  • Specific Pain Conditions/Pain in Specific Populations: Neuropathic Pain - Peripheral