Involvement Of Glia In Tetanic Sciatic Stimulation-induced Persistent Pain: Study On Peripheral And Spinal Mechanisms

Tetanic stimulation of the sciatic nerve (TSS) elicits long-term potentiation (LTP) of C-and A-fiber-evoked field potentials in the spinal cord and long-lasting pain hypersensitivity in rats. The study was focused on:(Ⅰ), perpheral mechanisms underlying tetanic sciatic stimulation-induced persistent pain in rats; (Ⅱ), the effect of interrupting spinal glial function on electroacupuncture analgesia.Tetanic stimulation of the sciatic nerve elicits long-term potentiation (LTP) of both C-and A-fiber-evoked field potentials in the spinal cord and long-lasting mechanical allodynia in rats. Consequently, spinal LTP is considered as a substrate of central sensitization in the sensory pathway of the spinal cord. The central mechanism underlying these processes has been largely explored. However, their peripheral mechanism is still poorly understood. The present study investigated tetanic stimulation-induced nerve injury and the following changes in sensory neurons and satellite glial cells (SGCs) in the related dosal root ganglion (DRG), which may contribute to spinal LTP and pain hypersensitivity induced by tetanic stimulation of sciatic nerve (TSS). Using experimental techniques including behavioral tests, immunofluorescence, confocal microscopy, histological staining and electron microscopy, the present study revealed that TSS induced bilateral mechanical allodynia and ipsilateral thermal hypergesia for at least 5 weeks in rats. Following TSS, the expression of ATF3, a neuronal injury marker, was sparing at 12 hours and maximal from 1 day to 14 days after TSS in the ipsilateral DRG neurons, and faded away 5 weeks thereafter. In the spinal cord, ATF3 was expressed in the motoneurons in the ventral horn but not in the dorsal horn in rats with tetanic sciatic stimulation. Fast Blue staining and electron microscope showed that myelinated and unmyelinated fibers had degenerative changes in the sciatic nerve in TSS rats. Meanwhile, proliferation of Schwann cells and irregular myelin sheaths at the stimulated site and distal parts were observed in the sciatic nerve of rats 7 days after TSS. Double immunofluorescence showed that ATF3 was mainly co-stained with NF200, a marker for large neurons with myelinated fibers and CGRP, a marker for peptide-containing neurons with unmyelinated fibers, on day 1,4,7 and 14 detected, whereas ATF3 was co-stained with IB4, a marker for non-peptide-containing small neurons, within 4 days after TSS. Immunofluorescence showed that the percentage of NF200-positive neurons in the ipsilateral DRG at lumber 4 (L4) did not change after TSS. The percentage of IB4-positive neurons in the L4 DRG decreased significantly on day 4,7 and 14 after TSS. The percentage of CGRP-positive neurons in the L4 DRG decreased greatly on day 7 and 14 after TSS. GFAP-positive satellite glial cells which are almost invisible in naive DRG largely appeared in the ipsilateral DRG at L4 in rats with tetanic sciatic stimulation. The double labeling of ATF3 with GFAP showed that some GFAP-positive cells surrounded injured neurons. The expression of P2X7 receptor, an ATP-sensitive ligand-gated cation channel, increased significantly in the L4 DRG after TSS. Confocal microscopy revealed that expression of P2X7 receptor in GFAP-positive satellite cells also increased significantly in the ipsilateral DRG. Further, activation of microglia and astrocyte was observed in the spinal cord after TSS. These results suggest that tetanic stimulation-induced nerve injury and subsequent glial activation may be the substrates for persistent pain induced by TSS. Previous studies indicated that spinal glial cells might be involved in the modulating effects of electroacupuncture (EA). Disruption of glial function could enhance EA analgesia in arthritic rats. Propentofylline is a methylxanthine derivative previously found to attenuate glial activation and has been shown to be antiallodynic in a rodent model of neuropathic pain. Using experimental techniques including behavioral tests and immunofluorescence, the present study examined whether propentofylline potentiates the EA-induced antiallodynia in neuropathic pain rats induced by tetanic stimulation of sciatic nerve (TSS). TSS induced decrease in ipsilateral paw withdrawal threshold (PWT) in rats on day 2,4,7 after stimulation. On day 7 after tetanic stimulation, EA treatment for 30 minutes showed analgesic effect for an hour on mechanical allodynia. Intrathecal administration of propentofylline (0.1,1 and 10μg) dose-dependently relieved mechanical allodynia induced by TSS. However, the co-application of 0.1μg propentofylline and EA elongated analgesic time of EA treatment for at least 2 hours. The co-application of 1μg propentofylline and EA produced more potent antiallodynia than the effect of 1μg propentofylline or EA alone in magnitude. The analgesic ratio was 60%(n= 10) in EA group. However, the ratio increased respectively up to 91.7%(n=12) and 100%(n=6) when EA combined with 0.1μg or 1μg propentofylline. The activation of microglia and astrocytes induced by TSS was inhibited significantly by single intrathecal injection of propentofylline. These results indicate that propentofylline and EA have a synergetic analgesia by interrupting spinal glial function.