Background & Aims
In small fiber neuropathy (SFN), patients suffer from a spectrum of sensoalgesic clinical symptoms due to the impairment of the A-delta and C-nerve fibers (1, 2). Diabetes and prediabetes are common causes of SFN (3). In this context, hyperglycemia plays an essential role in the pathogenesis of (pre-)diabetic SFN. The exact pathogenesis is not yet understood, but it has been shown that hyperglycemia leads to increased oxidative stress, which damages neurons (4). So far, research has mainly focused on in vivo animal models or in vitro models based on immortalized cell lines or rodent primary tissue cultures (5). We aimed to develop a human in vitro hyperglycemia model based on sensory neurons derived from induced pluripotent stem cells to investigate the pathophysiology of (pre-)diabetic SFN.
Methods
Human sensory neurons were differentiated from induced pluripotent stem cells, which had previously been generated from fibroblasts of a healthy subject (6). Neuronal maturation medium contains a glucose concentration of 17 mM, which was considered “normoglycemic”. For hyperglycemia, we incubated cells with defined glucose concentrations (30, 40, and 60 mM) for 24 h. As an osmotic control, we used mannitol (40 mM). To validate the model, we assayed intracellular reactive oxygen species (ROS) levels as a marker for oxidative stress via live cell imaging. Cytoplasmic protein expression of selected stress markers, namely p38 mitogen-activated protein kinase (p38 MAPK), phosphorylated p38 MAPK, nitric oxide synthase 2 (NOS2) and superoxide dismutase 2 (SOD2), was investigated via immunocytochemistry. To analyze apoptosis, caspase 3/7-activation was detected using live cell imaging.
Results
We observed higher intracellular ROS levels in sensory neurons incubated with 40 and 60 mM glucose compared to 17 mM (p < 0.001), while no such increase was observed at 30 mM. To ensure that the increase in ROS levels at 40 and 60 mM was glucose-specific and not due to mere osmotic stress, we incubated sensory neurons with mannitol and observed no increase in ROS levels compared to 17 mM glucose. Using immunocytochemistry, we detected higher cytoplasmic protein expression of p38 MAPK, phosphorylated p38 MAPK, NOS2, and SOD2 at 40 mM compared to 17 mM glucose (p < 0.0001). In live cell imaging, activated caspase-3/7 did not differ between 17 and 40 mM, indicating that there was no increased apoptosis at 40 mM glucose.
Conclusions
We pioneer a human in vitro hyperglycemia model to study the pathophysiology of (pre-)diabetic SFN. We show that hyperglycemia induces an increase of oxidative stress in cultured neurons accompanied by elevated expression of further stress related proteins, but does not induce increased apoptosis. Our model provides a platform for further analysis like a transcriptome analysis of cultured neurons, which will reveal valuable insights into the pathophysiology of (pre-)diabetic SFN. This will help to improve the prevention of (pre-)diabetic SFN so that SFN occurs less frequently in (pre-)diabetes or later in the course of the disease and will help to identify targeted treatments.
References
1.Devigili G, Tugnoli V, Penza P, et al. The diagnostic criteria for small fibre neuropathy: from symptoms to neuropathology. Brain 2008;131:1912-1925.
2.Gross F, Uceyler N. Mechanisms of small nerve fiber pathology. Neurosci Lett 2020;737:135316.
3.de Greef BTA, Hoeijmakers JGJ, Gorissen-Brouwers CML, Geerts M, Faber CG, Merkies ISJ. Associated conditions in small fiber neuropathy – a large cohort study and review of the literature. Eur J Neurol 2018;25:348-355.
4.Bonhof GJ, Herder C, Strom A, Papanas N, Roden M, Ziegler D. Emerging Biomarkers, Tools, and Treatments for Diabetic Polyneuropathy. Endocr Rev 2019;40:153-192.
5.Gardiner NJ, Freeman OJ. Can Diabetic Neuropathy Be Modeled In Vitro? Int Rev Neurobiol 2016;127:53-87.
6.Klein T, Grüner J, Breyer M, et al. Small fibre neuropathy in Fabry disease: a human-derived neuronal in vitro disease model. bioRxiv 2023:2023.2008.2009.552621.
7.Bakkers M, Faber CG, Hoeijmakers JG, Lauria G, Merkies IS. Small fibers, large impact: quality of life in small-fiber neuropathy. Muscle Nerve 2014;49:329-336.
8.Elafros MA, Andersen H, Bennett DL, et al. Towards prevention of diabetic peripheral neuropathy: clinical presentation, pathogenesis, and new treatments. Lancet Neurol 2022;21:922-936.
Presenting Author
Cara Sophie Fellmann
Poster Authors
Cara Sophie Fellmann
Department of Neurology, University Hospital of Würzburg
Lead Author
Christoph Erbacher
University Hospital Würzburg
Lead Author
Nicole Schottmann
University Hospital of Würzburg
Lead Author
Katharina Klug
MSc
Department of Neurology, University Hospital of Würzburg
Lead Author
Luisa Kreß
Dr. med.
University Hospital Würzburg
Lead Author
Nurcan Üçeyler
MD
Department of Neurology, University of Würzburg, Germany
Lead Author
Topics
- Mechanisms: Biological-Systems (Physiology/Anatomy)