The Role Of Spinal Glial Gap Junction In The Maintenance Of Neuropathic Pain In Rats After An L5Spinal Nerve Iniury

ObjectiveAfter nerve injury, neuronal excitability is increased in the dorsal horn, making it a key site for nociceptive transmission and modulation. The neighboring astrocytes may also play an important role in exaggerated spinal nociceptive transmission. A principal mechanism of communication between astrocytes is through gap junctions. While the role of spinal glial gap junctions, especially those formed by connexin43expressed preferentially in astrocytes, in the maintenance of neuropathic pain remains unclear. In this study, we aim to examine whether pharmacologic blockade of glial gap junctions and downregulation of Cx43expression in the spinal cord could inhibit neuropathic pain manifestations and dorsal horn wide-dynamic range (WDR) neuronal activity in rats after an L5spinal nerve ligation (SNL).Materials and Methods1. CBX was administrated intrathecally in SNL model to examine whether it could alleviate mechanical hypersensitivity and heat hypersensitivity. Because CBX-induced pain inhibition may also result from drug actions other than inhibition of glial gap junctions, intrathecal injection of GCA (an analogue of CBX that does not affect glial gap junctions) and MFQ (a selective neuronal gap junction decoupler) was also administrated to examine if they could alleviate mechanical hypersensitivity in SNL rats. Repetitive CBX injection was also administrated to test if rats would develop tachyphylaxis to pain inhitition.2. Single dorsal horn neuron activity was recorded on SNL rats. The effect of CBX on the neuron activities of WDR neurons to mechanical stimuli was examined. Windup of C-component induced by electrical stimulation was recorded, and effect of CBX on the action potentials in C-component and windup was also determined.3. The expressions of GFAP, Cx43and OX42in lumbar spinal cord of sham and SNL group rats were examined by immunofluorescence.4. Cx43siRNA and control siRNA were constructed. Rats are grouped as:sham, Cx43siRNA and control siRNA group. The effect of siRNA on mechanical hyperalgesia was observed. The expressions of Cx43and GFAP in lumbar spinal cord of rats were determined by western blot.Results1. Intrathecal injection of CBX (0.1μg、0.5μg、5μg、25μg、50μg) could dose-dependently inhibit mechanical hyperalgesia induced by SNL. At the same doses (0.81nmol、8.1nmol、81nmol), CBX significantly inhibited mechanical hyperalgesia compared with the vehicle-treated group, whereas GCA or MFQ did not (although GCA significantly inhibited mechanical hyperalgesia at the highest dose of81nmol, rats in that group exhibited severe signs of discomfort). Intrathecal injection of CBX (5μg) also significantly inhibit heat hypersensitivity. Rats received repetitive intrathecal injections of CBX (25μg/day) exhibited a carryover effect, in which the pre-drug PWTs on the second and third CBX treatment day were significantly higher than that on the first day.2. Spinal topical administration of CBX in SNL rats attenuated WDR neuronal response to mechanical stimuli and to windup-inducing electrical stimuli.3. Immunofluorecence showed that after SNL, markers of reactive astrocytes (GFAP) and microglia (0X42) were clearly seen in the lumbar spinal dorsal horn of rats, whereas the Cx43immunoreactivity was significantly decreased. There was colocalization between GFAP and Cx43, but not0X42and Cx43.4. Western blot results showed that SNL induced decreased expression of Cx43, which was further decreased by intrathecal Cx43siRNA treatment. Mechanical hypersensitivity in SNL rats was also inhibited by Cx43siRNA treatment, whereas increased expression of GFAP induced by SNL remained unchanged after Cx43siRNA injection.Conclusions1. Intrathecal treatment of CBX could dose-dependently inhibit manifestations of neuropathic pain, of which the underlying mechanism is through inhibiting glial gap junctions.2. Spinal topical application of CBX in SNL rats attenuated the evoked response of WDR neurons to peripheral mechanical stimuli, and attenuated windup in WDR neurons, which suggested a potential neuronal mechanism for intrathecal CBX to inhibit mechanical hypersensitivity.3. After SNL, expression of both GFAP and OX42in lumbar spinal cord was increased, which suggested that both astrocytes and microglial cells were activated after SNL. Although SNL decreased the expression of Cx43in the spinal cord, further downre gulation of spinal Cx43expression by intrathecal Cx43siRNA alleviated mechanical hypersensitivity in SNL rats. Thus, an enhanced function and not increased expression of spinal glial gap junctions, may contribute to neuropathic pain in rats after SNL.4. In conclusion, enhanced functions of glial gap junctions, especially those formed by Cx43, may contribute to the maintenace of neuropathic pain after SNL. These results above provides new clues for the mechanisms study of neuropathic pain, and an important target for clinical treatment of neuropathic pain.