Intranasal SARS-CoV-2 infection changes the transcriptome of the mouse trigeminal ganglion and brainstem: potential mechanisms underlying headache and trigeminal pain presentation in COVID-19.

Multiple symptoms have been observed in COVID-19 patients, including migraine and facial pain which may result from the sensitization of the trigeminal ganglion (TG) and brainstem. Recent studies suggest that SARS-CoV-2 may invade trigeminal nerve endings in the nasal cavity. However, despite these insights, the precise underlying mechanisms remain poorly understood. Here, we investigated the cellular and molecular changes in the TG and brainstem with a special attention for the spinal trigeminal nucleus in the K18-hACE2 mouse model infected with SARS-CoV-2. We first confirmed the expression of the cellular proteins playing a role in SARS-CoV-2 cell entry (ACE2, TMPRSS2, and NRP1) in both structures. We reported the expression of the viral nucleocapsid (N) and spike (S) proteins in TG and brainstem at 6 days post infection by multimodal approaches (RNAseq, RNAscope, and immunofluorescence). In the TG, S and N proteins were detected in nerve fibers as well as in TRPV1 and CGRP nociceptive neurons. Transcriptomic analyses of the TG from infected K18-hACE2 revealed significant changes in gene expression, including Ccl2, Atf3, Cxcl10, Saa3, and Plin4 genes. Additionally, increased immunoreactivity for ATF3 and Iba1 was detected in the TG of infected mice. In the brainstem, SARS-CoV-2 protein was exclusively found in neurons, with no detection in astrocytes or microglial cells, the latter exhibiting an activated form in SARS-CoV-2 mice. Bulk RNA-Seq analysis revealed a robust inflammatory response characterized by cytokine and chemokine storm, inflammasome activation (Gsdmd, Casp11, Casp1, and Nlrp3), markers of neuronal activation (Jun, Fos, Fosb), neuronal injury (Atf3), and pain-associated genes (Tacr1, Gfra1, Ntrk1, Slc17a6, Ptgs2). As found in the TG, we observed that infected neurons were found within a dense network of CGRP nociceptive nerve fibers in the trigeminal brainstem. Finally, we provided gene interaction networks and identified specific SARS-CoV-2 biomarkers Saa3, Cxcl10, Ccl2, Atf3, and Plin4 in TG and brainstem, which could serve as potential indicators of disease severity. In conclusion, this study reports a robust set of transcriptomic and cellular changes triggered by SARS-CoV-2 in the TG and brainstem, offering a potential mechanistic explanation for sensory abnormalities, such as migraine and pain, observed in COVID-19 patients. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12974-026-03722-5.