A role for proprioceptors in sngception

Abstract

Proprioceptors are primary mechanosensory neurons to monitor the status of muscle contraction and/or body position (1). Although proprioceptors are known as non-nociceptive mechanoreceptors, they also express the pro-nociceptive acid-sensing ion channel 3 (ASIC3) (2-5). To probe the role for proprioceptors in sensing acidosis (or sngception) (6), we found that genetic deletion of Asic3 in proprioceptors but not in nociceptors abolished acid-induced chronic hyperalgesia in mice. Chemo-optogenetically activating proprioceptors resulted in hyperalgesic priming that favored chronic pain induced by acidosis. In humans, intramuscular acidification induced acid perception but not pain. Conversely, in a spinal cord-injured patient who lost pain sensation in the right leg, proprioception and sngception were remaining somatosensory functions, associated with the spinal dorsal column. Together, evidence from both mouse and human studies suggests a role for proprioceptors in sngception.

Fig. 1.. Effect of conditional knockout of Asic3 in proprioceptors and nociceptors on chronic sng pain.

(A to D) In a mouse model of chronic widespread pain, wild-type (Asic3^f/f) mice developed chronic widespread mechanical hyperalgesia after two injections of acidic saline (pH 4.0) to unilateral gastrocnemius muscle. Conditional knockout of Asic3 in nociceptors (Nav1.8-Cre::Asic3^f/f, as Nav1.8-Asic3^f/f) had no effect on the development of chronic hyperalgesia in hind paws and muscle [(A) and (B)], whereas conditional knockout of Asic3 in proprioceptors (Pv-Cre::Asic3^f/f, as Pv-Asic3^f/f) significantly impaired the development of chronic hyperalgesia [(C) and (D)] (control, n = 12; Nav1.8-Asic3^f/f, n = 7; Pv-Asic3^f/f, n = 8). Black arrows indicate the time of acid intramuscular injection. (E and F) In a second mouse model of chronic widespread pain induced by intermittent cold stress, Nav1.8-Cre::Asic3^f/f mice developed chronic widespread mechanical hyperalgesia in muscle comparable with wild-type controls (Nav1.8-Asic3^f/f, n = 8; Asic3^f/f, n = 10), whereas Pv-Asic3^f/f mice did not develop muscle hyperalgesia as compared with wild-type control (Pv-Cre::Asic3^f/f, n = 11; control, n = 8). Data were analyzed by two-way ANOVA (*P < 0.05, **P < 0.01, and ***P < 0.001 versus Asic3^f/f).

Fig. 2.. Effect of chemo-optogenetics activation of proprioceptors and nociceptors on the hyperalgesic priming to trigger acid-induced chronic hyperalgesia.

(A) Scheme illustrating the chemo-optogenetic design. (B and C) Experimental design of the dual intramuscular injections of CTZ-acid, CTZ-CTZ, acid-CTZ, and acid-acid. (D to G) The mechanical sensitivity of Nav1.8-Cre::LMO3 (Nav1.8-LMO3) and Pv-Cre::LMO3 (Pv-LMO3) mice was monitored before and after CTZ-acid injection to gastrocnemius muscle. One day after CTZ injection, an acid injection induced only transient hyperalgesia of hind paws and muscle in Nav1.8-LMO3 mice [(D) and (E), Nav1.8-LMO3, n = 9; control n = 6], but induced a long-lasting hyperalgesia in Pv-LMO3 mice [(F) and (G), Pv-LMO3, n = 10; control n = 10]. (H and I) CTZ had a dose-dependent effect on the hyperalgesic priming that allowed a follow-up acid injection to induce chronic hyperalgesia of hind paws and muscle in Pv-LMO3 mice (n = 6 to 10). Black arrows indicate intramuscular acid injection (pH 4.0). Orange arrows indicate intramuscular CTZ injection. All data presented as mean ± SEM. Data were analyzed by two-way ANOVA (*P < 0.05 and ***P < 0.001 versus control).

Fig. 3.. Involvement of PLD-coupled mGluR in the proprioceptor-mediated pain chronicity.

(A and B) The effect of PLD-coupled mGluR antagonism on CTZ-pH 4.0–induced chronic mechanical hyperalgesia was evaluated in Pv-LMO3 mice. In the CTZ-acid regime, mice were intramuscularly injected with CTZ with or without PCCG-13 (PC, 0.2, 1, or 2 nmol) followed by an acid (pH 4.0) injection, 1 day later. Orange arrows indicate the time point of CTZ injection, and black arrows indicate the time point of pH 4.0 injection. Mechanical responses were monitored via paw withdrawal responses (A) and muscle withdrawal threshold (B) at different time points (vehicle, n = 9; PCCG-13, 0.2 nmol, n = 6; 1 nmol, n = 6; and 2 nmol, n = 9). (C) PCCG-13 dose-dependently reduced CTZ-induced pERK expression in sensory neurons of Pv-LMO3 mice. Immunofluorescence images of pERK expression in L4 sensory neurons were analyzed 5 min after coinjection of CTZ (800 ng) with vehicle (n = 4) or different doses of PCCG-13 (0.2 nmol, n = 5; 1 nmol, n = 4; and 2 nmol, n = 4). Red arrows indicate pERK-positive neurons. Scale bars, 100 μm. (D) Summary data of pERK-positive neurons. Data are expressed as mean ± SEM and were analyzed by one-way ANOVA (*P < 0.05, **P < 0.01, and ***P < 0.001 versus vesicle).

Fig. 4.. Effect of Pv-Asic3 activation in acid-induced sng-pain chronicity.

(A and B) Coinjection of CTZ and [Sar⁹, Met(O₂)¹¹]-substance P (SM-SP; 0.8 nmol) diminished the CTZ-pH 4.0–induced chronic mechanical hyperalgesia of hind paws (A) and muscle (B) in Pv-Cre::LMO3 mice. Control group, n = 9; and SM-SP group, n = 7. (C) SM-SP–induced electrophysiological responses resulting from superfusion of 3 μM SM-SP for 4 s in Pv-positive and Nav1.8-positive DRG neurons. The number of neurons and percentage for each type of responses are shown. (D and E) In wild-type (Asic3^f/f) mice, dual acid injections of pH 6.5 and pH 4.0 1 day apart did not induce chronic hyperalgesia, whereas injection of pH 6.5 + RPRFamide (0.5 nmol) and pH 4.0 1 day later induced chronic hyperalgesia in hind paws (D) and muscle (E). The effect of pH 6.5 + RPRFamide was inhibited by coinjection of 2 nmol of PCCG-13. Asic3^f/f pH 6.5 group, n = 3; Asic3^f/f pH 6.5 + RF group, n = 6; Asic3^f/f pH 6.5 + RF + PC group, n = 6. (F and G) In Nav1.8-Cre::Asic3^f/f (as Nav1.8-Asic3^f/f) mice, the dual injections of pH 6.5 and pH 4.0 acidic saline 1 day apart induced chronic hyperalgesia in hind paws (F) and muscle (G), whereas coinjection of PCCG-13 (2 nmol) with the first acid injection significantly inhibited the development of chronic hyperalgesia. Orange arrows indicate the time point of CTZ + SM-SP injection. Red arrows indicate the time point of injection of pH 6.5 + RF (RPRFamide, 500 pmol) in wild-type or pH 6.5 in Nav1.8-Asic3^f/f mice. Black arrows indicate the time point of pH 4.0 intramuscular injection. Nav1.8-Asic3^f/f pH 6.5 + PC group, n = 7; all other groups n = 6. Data are expressed as mean ± SEM and were analyzed by two-way ANOVA (*P < 0.05, **P < 0.01, and ***P < 0.001 versus vehicle control). RF, RPRFamide; PC, PCCG-13.

Fig. 5.. Segregation of sng from pain in humans.

(A) The setup of intramuscular acid injection and sng-pain assessment. (B) Intramuscular acid injection induces sng but not pain in humans (n = 7 in each group). Data presented as mean ± SEM; *P < 0.05 and **P < 0.01 versus control. (C) A patient with ventral thoracic spinal cord herniation senses nothing but sng and proprioception in his right leg. The sensory examination revealed total sensory loss to light touch, pinprick, and temperature, as well as decreased sensation to vibration below the right T5 dermatome (orange area). In contrast, the motor function and proprioception were well maintained. Of note, the patient was not able to feel deep pain when his right first distal phalanx fractured, but he could feel sng in his right leg. (D) The T2-weighted MRI in sagittal view showed the ventral herniation of the spinal cord at T2–3 levels. The axial view at the level of the line marked with a “1” showed the spinal cord at T1–2 level was normally located within the dura matter. The axial view at the level of the line marked with a “2” showed that the ventral part of the spinal cord (red line) was out of the confinement of the dura matter (blue line).