Animal Studies

Inflammation during the neonatal period can exacerbate pain severity following reinjury in adulthood. This is driven by alterations in the postnatal development of spinal and supraspinal nociceptive circuitry. However, the contribution of alterations in peripheral nociceptor function remains underexplored.

Capsaicin is an agonist of transient receptor potential cation channel subfamily V member 1 (TRPV1). Strong TRPV1 stimulation with capsaicin causes mitochondrial damage in primary sensory neurons. However, the effect of repetitive and moderate exposure to capsaicin on the integrity of neuronal mitochondria remains largely unknown. Our electron microscopic analysis revealed that repetitive stimulation of the facial skin of mice with 10 mM capsaicin induced short-term damage to the mitochondria in small-sized trigeminal ganglion neurons. Further, capsaicin-treated mice exhibited decreased sensitivity to noxious heat stimulation, indicating TRPV1 dysfunction, in parallel with the mitochondrial damage in the trigeminal ganglion neurons. To analyze the capsaicin-induced mitochondrial damage and its relevant cellular events in detail, we performed cell-based assays using TRPV1-expressing PC12 cells. Dose-dependent capsaicin-mediated mitochondrial toxicity was observed. High doses of capsaicin caused rapid destruction of mitochondrial internal structure, while low doses induced mitochondrial swelling. Further, capsaicin induced a dose-dependent loss of mitochondria and autophagy-mediated degradation of mitochondria (mitophagy). Concomitantly, transcriptional upregulation of mitochondrial proteins, cytochrome oxidase subunit IV, Mic60/Mitofilin, and voltage-dependent anion channel 1 was observed, which implied induction of mitochondrial biogenesis to compensate for the loss of mitochondria. Collectively, although trigeminal ganglion neurons transiently exhibit mitochondrial damage and TRPV1 dysfunction following moderate capsaicin exposure, they appear to be resilient to such a challenge. Our data show a dose-response relationship in capsaicin-mediated mitochondrial toxicity. We postulate that induction of mitophagy and mitochondrial biogenesis in response to capsaicin stimulation play important roles in repairing the damaged mitochondrial system.

Cav3 channels play an important role in modulating chronic pain. However, less is known about the functional changes of Cav3 channels in superficial spinal dorsal horn (SDH) in neuropathic pain states. Here, we examined the effect of partial sciatic nerve ligation (PSNL) on either expression or electrophysiological properties of Cav3 channels in superficial SDH. Our in vivo studies showed that the blockers of Cav3 channels robustly alleviated PSNL-induced mechanical allodynia and thermal hyperalgesia, which lasted at least 14 days following PSNL. Meanwhile, PSNL triggered an increase in both mRNA and protein levels of Cav3.2, but not Cav3.1 or Cav3.3 in rats. However, in Cav3.2 knockout (KO) mice, PSNL predominantly attenuated mechanical allodynia but not thermal hyperalgesia. In addition, the results of whole-cell patch-clamp recordings showed that both the overall proportion of Cav3 current-expressing neurons and the Cav3 current density in individual neurons were elevated in spinal lamina II neurons from PSNL rats, which could not be recapitulated in Cav3.2 KO mice. Altogether, our findings reveal that the elevated functional Cav3.2 channels in superficial SDH may contribute to the mechanical allodynia in PSNL-induced neuropathic pain model.

Recently, studies have focused on the antihyperalgesic activity of the A3 adenosine receptor (A3AR) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Here, we investigated the expression and functional effects of A3AR on the excitability of small- to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats. Real-time quantitative polymerase chain reaction experiments and immunofluorescence analysis revealed A3AR expression in DRG neurons. Patch-clamp experiments demonstrated that 2 distinct A3AR agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca-activated K (KCa) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca influx via N-type voltage-dependent Ca channels, as Cl-IB-MECA-induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca currents, and its effect was inhibited by 56% in the presence of A3AR antagonist MRS1523, demonstrating that the majority of adenosine's effect is mediated by this receptor subtype. Current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A3AR activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca measurements confirmed the inhibitory role of A3AR on DRG neuronal firing. We conclude that pain-relieving effects observed on A3AR activation could be mediated through N-type Ca channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A3AR-based therapy as a viable approach to alleviate pain in different pathologies.

Following destabilization of the medial meniscus (DMM), mice develop experimental osteoarthritis (OA) and associated pain behaviors that are dependent on the stage of disease. We aimed to describe changes in gene expression in knee-innervating dorsal root ganglia (DRG) after surgery, in order to identify molecular pathways associated with three pre-defined pain phenotypes: "post-surgical pain", "early-stage OA pain", and "persistent OA pain".