Background & Aims
Colorectal cancer poses a leading health concern globally, ranking third in terms of incidence and second in mortality among all cancers. Oxaliplatin-induced cold hyperalgesia, a frequent and painful adverse effect experienced by up to 90% of patients treated by this prevalent chemotherapy agent, significantly restricts the permissible dosage and duration of therapy [1]. The underlying mechanisms of this side effect are yet to be fully understood. Aiming to expedite the research of oxaliplatin-induced cold hyperalgesia (OICH) in primary sensory neurons, we established a cellular model using primary culture and calcium imaging. First, we studied the characteristics of cold-sensitive neurons, followed by a comparison between oxaliplatin-treated and native neurons. We aim to contribute to a deeper understanding of the mechanisms behind chemotherapy-induced neuropathic pain and to manage treatment-related side effects, such as OICH, to improve patient outcomes and quality of life.
Methods
Dorsal root ganglia neurons from Sprague-Dawley rats were incubated with either oxaliplatin (100 μM), vehicle (DMSO, 100 μM) for 6-7h or 24-48h, or left untreated. All groups underwent a cooling protocol ex-vivo, containing a recurring cooling ramp made using a Peltier device (Warner Instruments) of 30°C to 13°C [2], allowing passive warming up through a heated platform. Thermal cooling was followed by chemical cooling using L-menthol (130 μM) and icilin (30 μM) as TRPM8/TRPA1 agonists. Neuronal viability and nociception were examined with KCl (140 mM) and Capsaicin (10 μM). Intracellular Ca2+ transients were measured using the ratiometric calcium indicator Fura-2 AM. The custom-made analysis pipeline utilized the machine learning algorithms Cellpose and Suite2p [4]. We trained a custom human-in-the-loop model of anatomical segmentation in Cellpose, which was then used in Suite2p for functional segmentation of experiments. The data was extracted and analyzed via Python and MATLAB scripts.
Results
We produced consistent, reproducible cold stimuli using our setup, used to examine a total of >2000 neurons in all groups. Preliminary results in a small subset of data show a reduction of 70% in cell viability after incubation with 100 μM oxaliplatin for 48 hours. Incubation with oxaliplatin did not increase the number of cold responders but did show prolonged responses to cooling. Co-expression of TRPV1/TRPM8 was observed in a marginal proportion of cells in all populations [3]. A comprehensive analysis of cold responses and their modification by oxaliplatin pre-incubation will be presented. Training of the custom model in Cellpose was made with 14 images and yielded an increase of 2.6x ROI than the standard
model. Suite2p automatically identified 68% of the total masks based on the trained Cellpose model.
Conclusions
The cellular model of primary sensory neurons has proven to be a viable method of studying the cold responses and oxaliplatin-induced cold hyperalgesia ex-vivo. Using machine learning algorithms enables a standardized, fast, and objective way of identifying ROI, resulting in a more reproducible analysis. Next, results from all experiments will be analyzed to quantify the impact of oxaliplatin on DRG neurons regarding threshold activation, response characteristics such as tachyphylaxis and sensitization, and TRP channels co-expression. This model and analysis could contribute to our understanding of the mechanisms involved in OICH, thereby making a step towards developing potential treatments for patients who suffer from it.
References
[1] Park, S. B., Goldstein, D., Krishnan, A. V., Lin, C. S., Friedlander, M. L., Cassidy, J., Koltzenburg, M., & Kiernan, M. C. (2013). Chemotherapy-induced peripheral neurotoxicity: a critical analysis. CA: a cancer journal for clinicians, 63(6), 419–437. https://doi.org/10.3322/caac.21204
[2] Descoeur, J., Pereira, V., Pizzoccaro, A., Francois, A., Ling, B., Maffre, V., Couette, B., Busserolles, J., Courteix, C., Noel, J., Lazdunski, M., Eschalier, A., Authier, N., & Bourinet, E. (2011). Oxaliplatin-induced cold hypersensitivity is due to remodelling of ion channel expression in nociceptors. EMBO molecular medicine, 3(5), 266–278. https://doi.org/10.1002/emmm.201100134
[3] Kobayashi, K., Fukuoka, T., Obata, K., Yamanaka, H., Dai, Y., Tokunaga, A., & Noguchi, K. (2005). Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with adelta/c-fibers and colocalization with trk receptors. The Journal of comparative neurology, 493(4), 596–606. https://doi.org/10.1002/cne.20794
[4] Pachitariu, M., & Stringer, C. (2022). Cellpose 2.0: how to train your own model. Nature methods, 19(12), 1634–1641. https://doi.org/10.1038/s41592-022-01663-4
Presenting Author
Carmel Kerem
Poster Authors
Carmel Kerem
MD
Department of Neurophysiology, Mannheim Center for Translational Neuroscience, Medical Faculty Mannheim, Heidelberg University
Lead Author
Alexander Dieter
PhD
Department of Neurophysiology, Medical Faculty Mannheim, Heidelberg University
Lead Author
Rolf-Detlef Treede
Department of Neurophysiology, Mannheim Center for Translational Neuroscience, Medical Faculty Mannheim, Heidelberg University
Lead Author
Wolfgang Greffrath
Department of Neurophysiology, Mannheim Center for Translational Neuroscience, Medical Faculty Mannheim, Heidelberg University
Lead Author
Topics
- Models: Acute Pain