Sensitization of P2Χ3 Receptors by Cystathionine β-Synthetase Mediates Persistent Pain Hypersensitivity in a Rat Model of Lumbar Disc Herniation

Abstract

Lumbar disc herniation (LDH) is a major cause of discogenic low back pain and sciatica, but the underlying mechanisms remain largely unknown. Hydrogen sulfide (H2S) is becoming recognized for its involvement in a wide variety of processes including inflammation and nociception. The present study was designed to investigate the roles of the H2S signaling pathway in the regulation of expression and function of purinergic receptors (P2XRs) in dorsal root ganglion (DRG) neurons from rats with LDH. LDH was induced by implantation of autologous nucleus pulposus (NP), harvested from rat tail, in lumbar 5 and 6 spinal nerve roots. Implantation of autologous NP induced persistent pain hypersensitivity, which was partially reversed by an intrathecal injection of A317491, a potent inhibitor of P2X3Rs and P2X2/3Rs. The NP induced persistent pain hypersensitivity was associated with the increased expression of P2X3Rs, but not P2X1Rs and P2X2Rs, receptors in L5-6 DRGs. NP implantation also produced a 2-fold increase in ATP-induced intracellular calcium signals in DRG neurons when compared to those of controls (P < 0.05). Interestingly, NP implantation significantly enhanced expression of the endogenous hydrogen sulfide producing enzyme, cystathionine-β-synthetase (CBS). Systematic administration of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, suppressed the upregulation of P2X3R expression and the potentiation of ATP-induced intracellular calcium signals in DRG neurons (P < 0.05). Intrathecal injection of AOAA markedly attenuated NP induced- persistent pain hypersensitivity. Our results suggest that sensitization of P2X3Rs, which is likely mediated by CBS-H2S signaling in primary sensory neurons, contributes to discogenic pain. Targeting CBS/H2S-P2X3R signaling may represent a potential treatment for neuropathic pain caused by LDH.

Inhibition of cystathionine β-synthetase suppresses sodium channel activities of dorsal root ganglion neurons of rats with lumbar disc herniation

“…injection of sodium pentobarbital at dose of 50 mg/kg body weight, as described previously4. Briefly, NP was harvested from the disc level between the second and the third coccygeal intervertebral disc of rat tail.…”

“…DRG neurons innervating the hindpaw were labeled by injection of 1, 1′-dioleyl-3, 3, 3′, 3-tetramethylindocarbocyanine methanesulfonate (DiI, Invitrogen) into the rat left hindpaw, as described previously412. In short, rats were anesthetized with a cocktail of sodium pentobarbital (50 mg/kg body weight, ip).…”

“…Rat hindpaw withdrawal threshold (PWT) in response to stimulation of von Frey filaments was determined as described previously4. To measure thermal hyperalgesia, rats were housed in a Plexiglas box on top of a glass platform, and the withdrawal latency (PWL) from a thermal stimulus was obtained by using the radiant heat test with a Model 336 Paw/Tail Stimulator Analgesia Meter (IITC/life Science Instruments, CA, USA), which was set at 2% idle light intensity and 50% working light intensity.…”

“…However, the exact causes of low back pain and sciatica have not been fully elucidated and effective therapeutics for the primary symptoms has been unavailable. Recent studies in rodents found that autologous nucleus pulposus (NP) transplantation induced rats to develop pain hypersensitivity34. Therefore, autologous NP transplantation in rats has been used as an animal model of LDH to study the mechanisms of chronic pain.…”

“…Sensitization of primary sensory neurons is maintained by a number of ion channels such as transient receptor potential channels5, purinergic P2X3 receptors4, and voltage-gated sodium, potassium and calcium channels678. VGSCs are integral membrane glycol-proteins that are essential for AP generation and conduction of in excitable cells, thus playing a crucial role in regulating neuronal excitability.…”