Yong-Yong Li

Tumor necrosis factor-α induces long-term potentiation of C-fiber evoked field potentials in spinal dorsal horn in rats with nerve injury: The role of NF-kappa B, JNK and p38 MAPK

Compelling evidence has shown that in hippocampus tumor necrosis factor alpha (TNF-alpha) at pathological concentration inhibits long-term potentiation (LTP), a synaptic model of learning and memory. In the present work we investigated the role of TNF-alpha in LTP of C-fiber evoked field potentials in spinal dorsal horn, which is relevant to pathological pain. We showed that spinal application of TNF-alpha affected neither basal synaptic transmission mediated by C-fibers nor spinal LTP of C-fiber evoked field potentials induced by tetanic stimulation in intact rats. However, in rats with neuropathic pain, produced by either lumbar 5 ventral root transection (L5 VRT) or spared nerve injury (SNI), spinal application of TNF-alpha induced LTP of C-fiber evoked field potentials. Spinal application of JNK inhibitor (SP600125) or p38 MAPK inhibitor (SB203580) did not affect the spinal LTP induced by tetanic stimulation in intact rats, but completely blocked LTP induced by TNF-alpha in L5 VRT rats. NF-kappa B (NF-kappaB) inhibitor (PDTC) also blocked LTP induced by TNF-alpha. These results suggest that TNF-alpha and its downstream molecules may have no acute effect on spinal synaptic transmission in intact animals and induce LTP in rats with neuropathic pain produced by nerve injury.

p38 activation in uninjured primary afferent neurons and in spinal microglia contributes to the development of neuropathic pain induced by selective motor fiber injury

Compelling evidence shows that the adjacent uninjured primary afferents play an important role in the development of neuropathic pain after nerve injury. The underlying mechanisms, however, are largely unknown. In the present study, the selective motor fiber injury was performed by L5 ventral root transection (L5 VRT), and p38 activation in dorsal root ganglia (DRG) and L5 spinal dorsal horn was examined. The results showed that phospho-p38 immunoreactivity (p-p38-IR) was increased in both L4 and L5 DRGs, starting on day 1 and persisting for nearly 3 weeks (P<0.05) following L5 VRT and that the activated p38 was confined in neurons, especially in IB4 positive C-type neurons. L5 VRT also induced p38 activation in L5 spinal dorsal horn, occurred at the first day after the lesion and lasted for 2 weeks (P<0.05). The activated p38 is restricted entirely in spinal microglia. In contrast, selective injury of sensory neurons by L5 dorsal root transection (L5 DRT) failed to induce behavioral signs of neuropathic pain and activated p38 only in L5 DRG but not in L4 DRG and L5 spinal dorsal horn. Intraperitoneal injection of thalidomide, an inhibitor of TNF-alpha synthesis, prevented p38 activation in DRG and spinal cord. Intrathecal injection of p38 inhibitor SB203580, starting before L5 VRT, inhibited the abnormal pain behaviors. Post-treatment with SB203580 performed at the 1st day or at the 8th day after surgery also reduced established neuropathic pain. These data suggest that p38 activation in uninjured DRGs neurons and in spinal microglia is necessary for the initiation and maintenance of neuropathic pain induced by L5 VRT.

TNF-α contributes to up-regulation of Nav1.3 and Nav1.8 in DRG neurons following motor fiber injury

&NA; A large body of evidence has demonstrated that the ectopic discharges of action potentials in primary afferents, resulted from the abnormal expression of voltage gated sodium channels (VGSCs) in dorsal root ganglion (DRG) neurons following peripheral nerve injury are important for the development of neuropathic pain. However, how nerve injury affects the expression of VGSCs is largely unknown. Here, we reported that selective injury of motor fibers by L5 ventral root transection (L5‐VRT) up‐regulated Nav1.3 and Nav1.8 at both mRNA and protein level and increased current densities of TTX‐S and TTX‐R channels in DRG neurons, suggesting that nerve injury may up‐regulate functional VGSCs in sensory neurons indirectly. As the up‐regulated Nav1.3 and Nav1.8 were highly co‐localized with TNF‐&agr;, we tested the hypothesis that the increased TNF‐&agr; may lead to over‐expression of the sodium channels. Indeed, we found that peri‐sciatic administration of recombinant rat TNF‐&agr; (rrTNF) without any nerve injury, which produced lasting mechanical allodynia, also up‐regulated Nav1.3 and Nav1.8 in DRG neurons in vivo and that rrTNF enhanced the expression of Nav1.3 and Nav1.8 in cultured adult rat DRG neurons in a dose‐dependent manner. Furthermore, inhibition of TNF‐&agr; synthesis, which prevented neuropathic pain, strongly inhibited the up‐regulation of Nav1.3 and Nav1.8. The up‐regulation of the both channels following L5‐VRT was significantly lower in TNF receptor 1 knockout mice than that in wild type mice. These data suggest that increased TNF‐&agr; may be responsible for up‐regulation of Nav1.3 and Nav1.8 in uninjured DRG neurons following nerve injury.

Brain-derived neurotrophic factor contributes to spinal long-term potentiation and mechanical hypersensitivity by activation of spinal microglia in rat

It has been shown that following peripheral nerve injury brain-derived neurotrophic factor (BDNF) released by activated microglia contributes to neuropathic pain, but whether BDNF affects the function of microglia is still unknown. In the present work we found that spinal application of BDNF, which induced long-term potentiation (LTP) of C-fiber evoked field potentials, activated spinal microglia in naïve animals, while pretreatment with microglia inhibitor minocycline blocked BDNF-induced LTP. In addition, following LTP induction by BDNF, both phosphorylated Src-family kinases (p-SFKs) and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) were up-regulated only in spinal microglia but not in neurons and astrocytes, whilst spinal application of SFKs inhibitor (PP2 or SU6656) or p38 MAPK inhibitor (SB203580) blocked BDNF-induced LTP and suppressed microglial activation. As spinal LTP at C-fiber synapses is considered to underlie neuropathic pain, we subsequently examined whether BDNF may contribute to mechanical hypersensitivity by activation of spinal microglia using spared nerve injury (SNI) model. Following SNI BDNF and TrkB receptor were up-regulated mainly in dorsal horn neurons and in activated microglia, and p-SFKs and p-p38 MAPK were increased exclusively in microglia. Intrathecal injection of BDNF scavenger TrkB-Fc starting before SNI, which prevented the behavioral sign of neuropathic pain, suppressed both microglial activation and the up-regulation of p-SFKs and p-p38 MAPK produced by SNI. Thus, the increased BDNF/TrkB signaling in spinal dorsal horn may contribute to neuropathic pain by activation of microglia following peripheral nerve injury and inhibition of SFKs or p38 MAPK may selectively inhibit microglia in spinal dorsal horn.

BDNF induces late-phase LTP of C-fiber evoked field potentials in rat spinal dorsal horn

Several lines of evidence have shown that in some brain regions brain-derived neurotrophic factor (BDNF) is important for long-term potentiation (LTP), a synaptic model of memory storage. In the present work we evaluate the role of BDNF in LTP of C-fiber evoked field potentials in spinal dorsal horn, a synaptic model of pain memory. We found that spinal application of BDNF-induced LTP of C-fiber evoked field potentials with a long latency, lasting for >8 h, and the effect was blocked by either tyrosine kinase inhibitor (K252a) or BNDF scavenger (TrkB-Fc). The potentiation produced by BDNF was occluded by late-phase LTP (L-LTP) but not by early-phase LTP (E-LTP) induced by electrical stimulation. Pretreatment of K252a or TrkB-Fc selectively blocked spinal L-LTP induced by low-frequency stimulation (LFS) but not E-LTP. BDNF-induced LTP was completely abolished by the protein synthesis inhibitor (anisomycin), by N-methyl-D-aspartate (NMDA) receptor blocker (MK-801), by extracellular signal-regulated protein kinase (ERK) inhibitor (PD98059) or by p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580) but not by c-Jun N-terminal kinase (JNK) inhibitor (SP600125). Nuclear factor-kappaB (NF-kappaB) inhibitor (PDTC) also suppressed spinal BDNF-LTP. The results suggest that BDNF play a crucial role in protein synthesis-dependent L-LTP in spinal dorsal horn via activation of ERK, p38 MAPK and NF-kappaB signal pathways.

ATP induces long-term potentiation of C-fiber-evoked field potentials in spinal dorsal horn: The roles of P2X4 receptors and p38 MAPK in microglia

Many studies have shown that adenosine triphosphate (ATP), as a neurotransmitter, is involved in plastic changes of synaptic transmission in central nervous system. In the present study, we tested whether extracellular ATP can induce long‐term potentiation (LTP) of C‐fiber‐evoked field potentials in spinal dorsal horn. The results showed the following: (1) ATP at a concentration of 0.3 mM induced spinal LTP of C‐fiber‐evoked field potentials, lasting for at least 5 h; (2) spinal application of 2′,3′‐O‐(2,4,6‐trinitrophenyl)adenosine‐5‐triphosphate (TNP‐ATP; an antagonist of P2X1–4 receptors), but not pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS; an antagonist of P2X1,2,3,5,7 receptors), 30 min before ATP blocked ATP‐induced LTP, indicating that ATP may induce spinal LTP by activation of P2X4 receptors; (3) at 60 min after LTP induction the level of phospho‐p38 mitogen‐activated protein kinase (p‐p38 MAPK) was significantly elevated and at 180 min after LTP the number of P2X4 receptors increased significantly; both p‐p38 and P2X4 receptors were exclusively co‐located with the microglia marker, but not with neuronal or astrocyte marker; (4) spinal application of TNP‐ATP but not PPADS prevented p38 activation; (5) spinal application of SB203580, a p38 MAPK inhibitor, prevented both spinal LTP and the upregulation of P2X4 receptors. The results suggested that ATP may activate p38 MAPK by binding to intrinsic P2X4 receptors in microglia, and subsequently enhance the expression of P2X4 receptors, contributing to spinal LTP.

The direction of synaptic plasticity mediated by C-fibers in spinal dorsal horn is decided by Src-family kinases in microglia: The role of tumor necrosis factor-α

Previous studies have shown that Src-family kinases (SFKs) are selectively activated in spinal microglia following peripheral nerve injury and the activated SFKs play a key role for the development of neuropathic pain. To investigate the underlying mechanism, in the present study the effect of SFKs on long-term potentiation (LTP) at C-fiber synapses in spinal dorsal horn, which is believed as central mechanism of neuropathic pain, was investigated in adult rats. Electrophysiological data revealed that pretreatment with either microglia inhibitor (minocycline, 200 microM) or SFKs inhibitors (PP2, 100 microM and SU6656, 200 microM) reversed the effect of high frequency stimulation (HFS), that is, HFS, which induces long-term potentiation (LTP) normally, induced long-term depression (LTD) after inhibition of either microglia or SFKs. Western blotting analysis showed that the level of phosphorylated SFKs (p-SFKs) in ipsilateral spinal dorsal horn was transiently increased after LTP induced by HFS, starting at 15 min and returning to control level at 60 min after HFS. Double-labeled immunofluorescence staining demonstrated that p-SFKs were highly restricted to microglia. Furthermore, we found that the inhibitory effects of minocycline or SU6656 on spinal LTP were reversed by spinal application of rat recombinant tumor necrosis factor-alpha (TNF-alpha 0.5 ng/ml, 200 microl). HFS failed to induce LTP of C-fiber evoked field potentials in TNF receptor-1 knockout mice and in rats pretreated with TNF-alpha neutralization antibody (0.6 microg/ml, 200 microl). The results suggested that in spinal dorsal horn activation of SFKs in microglia might control the direction of plastic changes at C-fiber synapses and TNF-alpha might be involved in the process.

TNF-α enhances the currents of voltage gated sodium channels in uninjured dorsal root ganglion neurons following motor nerve injury.

The ectopic discharges observed in uninjured dorsal root ganglion (DRG) neurons following various lesions of spinal nerves have been attributed to functional alterations of voltage-gated sodium channels (VGSCs). Such mechanisms may be important for the development of neuropathic pain. However, the pathophysiology underlying the functional modulation of VGSCs following nerve injury is largely unknown. Here, we studied this issue with use of a selective lumbar 5 ventral root transection (L5-VRT) model, in which dorsal root ganglion (DRG) neurons remain intact. We found that the L5-VRT increased the current densities of TTX-sensitive Na channels as well as currents in Nav1.8, but not Nav1.9 channels in uninjured DRG neurons. The thresholds of action potentials decreased and firing rates increased in DRG neurons following L5-VRT. As we found that levels of tumor necrosis factor-alpha (TNF-α) increased in cerebrospinal fluid (CSF) and in DRG tissue after L5-VRT, we tested whether the increased TNF-α might result in the changes in sodium channels. Indeed, recombinant rat TNF (rrTNF) enhanced the current densities of TTX-S and Nav1.8 in cultured DRG neurons dose-dependently. Furthermore, genetic deletion of TNF receptor 1 (TNFR-1) in mice attenuated the mechanical allodynia and prevented the increase in sodium currents in DRG neurons induced by L5-VRT. These data suggest that the increase in sodium currents in uninjured DRG neurons following nerve injury might be mediated by over-production of TNF-α.

Role of phosphorylation of ERK in induction and maintenance of LTP of the C‐fiber evoked field potentials in spinal dorsal horn

Previous works have shown that activation of extracellular signal‐regulated kinase (ERK)/cAMP response element binding protein (CREB) pathway is essential for long‐term potentiation (LTP) in hippocampus. In the present study, the role of the ERK/CREB pathway in LTP of C‐fiber evoked field potentials in spinal dorsal horn, which is relevant to pathologic pain, was investigated in adult rats. Western blotting analysis showed that the protein level of phosphorylated ERK (p‐ERK) in ipsilateral spinal dorsal horn was transiently increased after LTP induction, starting at 15 min and returning to control at 60 min after tetanic stimulation and that the protein level of p‐CREB increased at 30 min, persisting for at least 3 hr after LTP induction. Double immunofluorescence staining showed that p‐ERK and p‐CREB were only located in neurons but not in glial cells in the spinal dorsal horn after LTP induction. More importantly, we found that spinal application of PD 98059 (100 μM), a selective MEK inhibitor, at 30 min before tetanic stimulation blocked LTP induction and prevented the increase in p‐ERK and p‐CREB in spinal dorsal horn. When applied 15 min after LTP induction, PD98059 reversed established LTP. The drug, however, did not affect the spinal LTP, when applied at 30 min after LTP. Our results suggested that activation of ERK/CREB pathway in spinal dorsal neurons is necessary for induction and maintenance of long‐term potentiation of the C‐fiber evoked field potentials.

The up-regulation of IL-6 in DRG and spinal dorsal horn contributes to neuropathic pain following L5 ventral root transection.

Our previous works have shown that pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) plays an important role in neuropathic pain produced by lumber 5 ventral root transection (L5-VRT). In the present work we evaluate the role of interleukin-6 (IL-6), another key inflammatory cytokine, in the L5-VRT model. We found that IL-6 was up-regulated in the ipsilateral L4 and L5 dorsal root ganglian (DRG) neurons and in bilateral lumbar spinal cord following L5-VRT. Double immunofluorescence stainings revealed that in DRGs the increased immunoreactivity (IR) of IL-6 was almost restricted in neuronal cells, while in the spinal dorsal horn IL-6-IR up-regulated in both glial cells (astrocyte and microglia) and neurons. Intrathecal administration of IL-6 neutralizing antibody significantly delayed the induction of mechanical allodynia in bilateral hindpaws after L5-VRT. Furthermore, inhibition of TNF-α synthesis by intraperitoneal thalidomide prevented both mechanical allodynia and the up-regulation of IL-6 in DRGs following L5-VRT. These data suggested that the increased IL-6 in afferent neurons and spinal cord contribute to the development of neuropathic pain following motor fiber injury, and that TNF-α is responsible for the up-regulation of IL-6.