Cutaneous Hypersensitivity as an Indicator of Visceral Inflammation via C-Nociceptor Axon Bifurcation

Abstract

Pain on the body surface can accompany disorders in the deep tissue or internal organs. However, the anatomical and physiological mechanisms are obscure. Here, we provided direct evidence of axon bifurcation in primary C-nociceptive neurons that innervate both the skin and a visceral organ. Double-labeled dorsal root ganglion (DRG) neurons and Evans blue extravasation were observed in 3 types of chemically-induced visceral inflammation (colitis, urocystitis, and acute gastritis) rat models. In the colitis model, mechanical hypersensitivity and spontaneous activity were recorded in vivo from double-labeled C-nociceptive neurons in S1 or L6 DRGs. These neurons showed significantly enhanced responses to both somatic stimulation and colorectal distension. Our findings suggest that the branching of C-nociceptor axons contribute to cutaneous hypersensitivity in visceral inflammation. Cutaneous hypersensitivity on certain locations of the body surface might serve as an indicator of pathological conditions in the corresponding visceral organ.

Keywords: Axon bifurcation; C-nociceptor; Cutaneous hypersensitivity; Visceral inflammation.

Fig. 1

Retrogradely labeled primary sensory neurons in dorsal root ganglia (DRGs) innervating both skin and distal colon/urinary bladder. A Representative images of sensory neurons in bilateral L6 and S1 DRGs after injection of Dil in the distal colon and CTB-488 in the skin around the root of the tail [red, Dil-positive colon afferent fibers; green, CTB-positive skin afferent fibers; yellow, double-labeled cells (white arrows); scale bar, 25 μm; CTB-488, cholera toxin subunit B (recombinant) Alexa Fluor 488; DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetrame-thylindocar bocyanine perchlorate]. B Bar graph of the number of neurons labeled by DiI (distal colon) and CTB-488 (skin around the root of the tail) in bilateral T13, L1, L2, L6, and S1–S4 DRGs (T, thoracic; L, lumbar; S, sacral). C The Venn diagram showing the total number of DiI-labeled (red), CTB-488-labeled (green), and double-labeled (yellow) neurons in all DRGs (18 DRGs from 3 rats). D Numbers of neurons labeled by DiI (urinary bladder) and CTB-488 (skin around the root of the tail) in bilateral T10–13, L1, L5–6, and S1–2 DRGs. E Venn diagram showing the total number of DiI-labeled (red), CTB-488-labeled (green), and double-labeled (yellow) neurons in all DRGs (16 DRGs from 3 rats).

Fig. 2

Retrogradely labeled primary sensory neurons in DRGs innervating both skin and stomach. A Representative images of sensory neurons in bilateral T9 and T10 DRGs after injection of Dil into the stomach and CTB-488 into the thoracic back skin. Insets are higher magnification images. Scale bars, 100 μm (white); 25 μm (yellow). B Counts of DRG neurons labeled by DiI (stomach) and CTB-488 (back skin of thoracic segments) in bilateral T5–13 and L1 DRGs. C Venn diagram showing the total number of DiI-labeled (red), CTB-488-labeled (green), and double-labeled (yellow) neurons in all DRGs (30 DRGs from 3 rats).

Fig. 3

Histological changes, Evans blue extravasation and mechanical pain assessment in rat models of colitis, urocystitis, and gastritis. A–C Representative microscopic images showing H&E staining in control tissue of distal colon, urinary bladder, and stomach. D–F Representative microscopic images showing H&E staining of distal colonic, vesical, and gastric tissue sections from rat models. Scale bars in A–F, 100 μm. G and H Representative images showing Evans blue extravasation (blue dots in red circles) mainly at the root of the tail in rats with both colitis (n = 3) and urocystitis (n = 3). Scale bar in G, 2 cm. I Massive Evans blue extravasation (blue dots in red circles) in the thoracic back skin of rats with gastritis (n = 3). J–L Mechanical hyperalgesia in rats with colitis (n = 8), urocystitis (n = 6), or gastritis (n = 8). J Mechanical threshold before (baseline) and at 1–14 days after intrarectal TNBS instillation. K Mechanical threshold before (baseline) and at 1–14 days after transurethral delivery of H₂O₂ into the bladder. L Mechanical threshold before (baseline) and 30 min to 5 days after intragastric administration of ethanol plus HCL. *P < 0.05, **P < 0.01 vs. baseline, Student’s t-test.

Fig. 4

Mechanical threshold of double-labeled C-nociceptive neurons in S1 and L6 DRGs from control and TNBS-treated rats. A Bright-field image of the surface of an S1 DRG; red arrow indicates a small neuron. B Fluorescence image of the same cell under recording by an extracellular glass electrode (orange dotted line). Scale bar for B and A, 20 μm. C Typical response of a double-labeled small neuron to 50 mN mechanical stimulation. Action potentials (APs) in the original recording (Ie) are indicated by corresponding tick marks below. D and E This neuron also responds to nociceptive warm (51 °C) and cold (0 °C) stimulation. F. Measurement of conduction velocity (CV) of recorded neuron by stimulation of the receptive field (RF, red arrow). G Responses of double-labeled C-nociceptive neurons to Q-tip wiping and von Frey filaments of several forces (5, 10 mN, 30 mN, and 50 mN) in control and TNBS-treated rats. H Responses of double-labeled C-nociceptive neurons to expansion of the distal colon by a balloon at several pressures (20 mmHg, 40 mmHg, and 60 mmHg) in control and TNBS-treated rats. I and J. Action potential discharge rate s (AP/s) of double-labeled C-nociceptive neurons evoked by mechanical stimulation (I) and balloon expansion (J) in control (n = 6) and TNBS-treated (n = 7) rats *P < 0.05, **P < 0.01, TNBS vs. normal group (I and J), one-way ANOVA with Bonferroni post hoc test.

Fig. 5

Changes in spontaneous activity (SA) after different cutaneous stimuli in rat model of colitis. A An example of SA recorded in one S1 DRG neuron (conduction velocity, 0.72 m/s) changing with both non-nociceptive (Q-tip) and nociceptive (pinch) mechanical stimulation applied to both receptive field (RF) and non-RF areas (red, action potentials (APs) during 30-s stimulation). The neuron responds to both cutaneous stimulation and colorectal distention (CRD). B Quantification of SA frequency in L6 and S1 DRG neurons after application of a Q-tip (n = 8) and pinch (n = 10) to both RF and non-RF areas. Mean SA frequency within 3 min before application is defined as 100%. *P < 0.05, **P < 0.01, after application vs before application, one-way ANOVA. C Statistics of SA frequency after applying capsaicin (CAP, n = 4) and poking (poke, n = 5) to both RF and non-RF areas. *P < 0.05, **P < 0.01, after application vs. before application, one-way ANOVA.

Fig. 6

Schematic showing how axon branching of C-nociceptive neurons in dorsal root ganglia (DRGs) contributes to cutaneous hypersensitivity via neurogenic inflammation that occurs commonly in visceral inflammatory pain. The inflammation of visceral organs (colon and stomach) leads to discharges of C-nociceptive neurons in DRGs and further results in neurogenic inflammation and cutaneous hypersensitivity, which in turn is an indicator of visceral pain.