Fig 2: CALCA is transcriptionally regulated in the DRGs and spinal cord of aging mice. A, Depiction of a DRG with small (white), medium (teal), and large (yellow) diameter neurons outlined. B, Proportional distribution of DRG cells by diameter in mice 3, 12, and 24 months. Dotted lines demarcate approximate boundaries of small, medium, and large-diameter neurons. C, CALCA immunofluorescence in the L4/L5 DRGs of aging mice counterstained with DAPI. Scale bars, 100 µm. D, Median values of the mean fluorescent intensities (MFI) of cells within the L4 and L5 DRGs at indicated ages. Each point represents an individual animal. Black bars indicate group mean. E, Distribution of CALCA positive cells by cell size at indicated ages. F, qPCR analysis for Calca expression in L4-L6 DRGs normalized to Gapdh compared with age in a linear regression. G, qPCR analysis for Ramp1 expression in the lumbar spinal cord dorsal horn (SCDH) normalized to Gapdh compared with age. H, qPCR analysis for Calca expression in lumbar SCDH normalized to Gapdh compared with Linear regression line (solid black line) and 95% CI (dotted lines) are based on both sexes combined. Each point in a graph represents a single biological replicate

Fig 3: Model of response to aging in the mouse somatosensory nervous system. Damage to nerves and demyelination due to age-related degenerative diseases including intervertebral disc degeneration induces Atf3, possibly mediated by increased TRPA1 expression, which promotes expression of Npy and Bdnf. Nearby intact small diameter nerves respond to nerve damage by inducing Calca, resulting in increased CALCA expression which is transported to the SCDH which may induce further Calca expression in SCDH neurons, leading to central sensitization. Additionally, increased presence of TNFalpha in aging tissues or the circulatory system acts on its receptors, TNFR1/2, to induce NF?B pathway components, Rela and Nfkb1, which in turn increases expression of nociceptive sodium channels, NaV1.8 and NaV1.9. Higher sodium channel expression reduces thresholds and increases the stimulation of SCDH neurons

Fig 4: Results of positive expression of TRPV1, CGRP, CRLR, and RAMP1 in the trigeminal nerve of rats. (A) Positive expression of TRPV1, (B) Positive expression of CGRP, (C) Positive expression of CRLR, (D) Positive expression of RAMP1. All the values are expressed as mean ± S.D. Differentsymbols indicate significant differences (compared with the control group, ** p < 0.01; compared with the model group, + p < 0.05, ++ p < 0.01; compared with the same dose of XMT n-BuOH, # p < 0.05, ## p < 0.01; compared with the same dose of XMT EtOAc, ? p < 0.05, ?? p < 0.01).

Fig 5: Results of mRNA expression of TRPV1, CGRP, CRLR, and RAMP1 in the trigeminal nerve of rats. (A) mRNA expression of TRPV1, (B) mRNA expression of CGRP, (C) mRNA expression of CRLR, (D) mRNA expression of RAMP1. All the values are expressed as mean ± S.D. Differentsymbols indicate significant differences (comparedwith the control group, ** p < 0.01; compared with the model group, ++ p < 0.01; compared with the same dose of XMT n-BuOH, ## p < 0.01; compared with the same dose of XMT EtOAc, ▲▲ p < 0.01).