19: Spatial Transcriptomic Insights: Astrocyte Reactivity in a Rat Model of Trigeminal Neuropathic Pain

Topic: Basic Science
Subtopic: Pain category (ICD-11), Chronic neuropathic pain, , , , , , , , , , Omics (Proteomics/Metabolomics/Transcriptomics/Genomics)

Background and Aims: Chronic trigeminal neuropathic pain is particularly distressing and disabling, with current treatments largely ineffective. Part of the difficulty in treating trigeminal neuropathic pain is a consequence of the lasting functional changes within brain regions involved in pain processing. At the cellular level, the transcriptional changes in trigeminal recipient brainstem nuclei are largely unknown. Here we use spatial transcriptomic approaches to investigate the cellular and molecular changes in the medullary dorsal horn of the spinal trigeminal nucleus (SpVN) that may underpin the development and maintenance of trigeminal neuropathic pain.

Methods: Trigeminal neuropathic pain was modelled in male and female rats using the infraorbital nerve ligation model [ION-CCI] (M=6; F=6) [1,2] and compared to naïve, uninjured (M=6; F=6), or sham-injured (M=6; F=6) rats. Twenty-eight days after ION-CCI, rats were deeply anaesthetised (pentobarbitone 130mg/kg, IP), perfused transcardially with cold 4% paraformaldehyde, and the brains cryoprotected until sectioning. The SpVN was cut coronally at 10µm, and a custom 480-gene panel was applied using the 10X Genomics Xenium assay. Xenium data were analysed on R using unsupervised clustering with Seurat’s LoadXenium function before normalisation with SCTransform.

Results: The gene expression profiles of astrocyte-specific clusters in the medullary dorsal horn of the SpVN ipsilateral to ION-CCI were compared with sham-injured and naïve conditions, using the FindMarker function in Seurat, with statistical significance determined via nonparametric Wilcoxon Rank Sum Test. Our Xenium spatial transcriptomics data revealed that ION-CCI in female rats dramatically increased complement C3 by 7.5-fold when compared to naïve rats and a 2-fold increase when compared to sham-injured rats.

Conclusions: Our findings provide the first high resolution transcriptional and spatial analysis of the astrocyte-specific response to ION-CCI in the medullary dorsal horn of the SpVN. We reveal at this region, the astrocytic complement system may play a critical role in the development and maintenance of trigeminal neuropathic pain. Identification of a known neuroinflammatory pathway may identify one therapeutic target that can be targeted to better manage chronic trigeminal neuropathic pain.

References: 1. Vos, B. P., & Strassman, A. M. (1995). Fos expression in the medullary dorsal horn of the rat after chronic constriction injury to the infraorbital nerve. The Journal of comparative neurology, 357(3), 362–375. https://doi.org/10.1002/cne.903570304
2. Kang, J. W. M., Davanzo, O. I., Emvalomenos, G. M., Mychasiuk, R., Henderson, L. A., & Keay, K. A. (2024). Infraorbital nerve injury triggers sex-specific neuroimmune responses in the peripheral trigeminal pathway and common pain behaviours. Brain, behavior, and immunity, 118, 480–498. https://doi.org/10.1016/j.bbi.2024.03.022

Presenting Author: James WM Kang
Submitting Author: James Kang

Senior Author: Kevin A Keay

IASP 2026 Global Year theme of Neuropathic Pain: Yes

• James WM Kang: PhD - Neuroscience, School of Medical Sciences, Faculty of Medicine and Health, University of Sydney; Brain and Mind Centre, University of Sydney.
• Lipin Loo: PhD - School of Life and Environmental Sciences, Faculty of Science, University of Sydney; Charles Perkins Centre, University of Sydney.
• Hyunsol Lim: BSc - Neuroscience, School of Medical Sciences, Faculty of Medicine and Health, University of Sydney; Brain and Mind Centre, University of Sydney.
• Greg G Neely: PhD - School of Life and Environmental Sciences, Faculty of Science, University of Sydney; Charles Perkins Centre, University of Sydney.
• Luke A. Henderson: PhD - Neuroscience, School of Medical Sciences, Faculty of Medicine and Health, University of Sydney; Brain and Mind Centre, University of Sydney.
• Kevin A Keay: PhD - Neuroscience, School of Medical Sciences, Faculty of Medicine and Health, University of Sydney; Brain and Mind Centre, University of Sydney.