Temporal dynamics of mechanical hypersensitivity and dorsal root ganglion transcription in a bilateral CFA-Induced inflammation model in mice

This study aimed to explore the daily rhythmicity of mechanical sensitivity in a mouse model of induced subcutaneous inflammation, with the broader goal of understanding circadian oscillations that may influence pain conditions. Mice received unilateral injections of Complete Freund's Adjuvant (CFA) in one hind paw to induce localized inflammation; the contralateral paw received saline as a control. On days 3 post injection (acute stage) and 5 (persistent stage), mechanical sensitivity was assessed by paw withdrawal behaviour using calibrated monofilaments to apply defined force at three time points: ZT4 (early light phase), ZT12 (end of light phase), and ZT20 (dark phase). RNA was extracted from dorsal root ganglia (DRG) on days 4 and 6 for RNA-seq and droplet digital PCR (ddPCR). CFA-induced responsiveness to light mechanical force (0.16 g and 0.4 g) shifted from being time-of-day independent in the acute phase to time-of-day dependent in the persistent phase. The opposite pattern was observed with higher force (0.6 g), showing time-of-day dependency in the acute phase. Substance P transcription was upregulated at ZT4 relative to ZT12 on day 4 but not on day 6. Transcripts of two core clock components (Bmal1 and Per1) in the DRG were not significantly influenced by CFA-injection. Additional RNA-seq analysis identified differentially expressed genes at ZT4 as CFA-responsive independent of time-of-day. These findings reveal a circadian modulation of CFA-induced mechanical hypersensitivity to light mechanical force during the persistent stages of pain, which is associated with alterations in gene expression within the dorsal root ganglion (DRG).