Kang-Wu Li

Calcium Channel α2δ1 Subunit Mediates Spinal Hyperexcitability in Pain Modulation

&NA; Mechanisms of chronic pain, including neuropathic pain, are poorly understood. Upregulation of voltage‐gated calcium channel (VGCC) &agr;2&dgr;1 subunit (Cav&agr;2&dgr;1) in sensory neurons and dorsal spinal cord by peripheral nerve injury has been suggested to contribute to neuropathic pain. To investigate the mechanisms without the influence of other injury factors, we have created transgenic mice that constitutively overexpress Cav&agr;2&dgr;1 in neuronal tissues. Cav&agr;2&dgr;1 overexpression resulted in enhanced currents, altered kinetics and voltage‐dependence of VGCC activation in sensory neurons; exaggerated and prolonged dorsal horn neuronal responses to mechanical and thermal stimulations at the periphery; and pain behaviors. However, the transgenic mice showed normal dorsal horn neuronal responses to windup stimulation, and behavioral responses to tissue‐injury/inflammatory stimuli. The pain behaviors in the transgenic mice had a pharmacological profile suggesting a selective contribution of elevated Cav&agr;2&dgr;1 to the abnormal sensations, at least at the spinal cord level. In addition, gabapentin blocked VGCC currents concentration‐dependently in transgenic, but not wild‐type, sensory neurons. Thus, elevated neuronal Cav&agr;2&dgr;1 contributes to specific pain states through a mechanism mediated at least partially by enhanced VGCC activity in sensory neurons and hyperexcitability in dorsal horn neurons in response to peripheral stimulation. Modulation of enhanced VGCC activity by gabapentin may underlie at least partially its antihyperalgesic actions.

Profiling of Dynamically Changed Gene Expression in Dorsal Root Ganglia Post Peripheral Nerve injury and A Critical Role of Injury-Induced Glial Fibrillary Acetic Protein in Maintenance of Pain Behaviors

ABSTRACT To explore cellular changes in sensory neurons after nerve injury and to identify potential target genes contributing to different stages of neuropathic pain development, we used Affymetrix oligo arrays to profile gene expression patterns in L5/6 dorsal root ganglia (DRG) from the neuropathic pain model of left L5/6 spinal nerve ligation at different stages of neuropathic pain development. Our data indicated that nerve injury induced changes in expression of genes with similar biological functions in a temporal specific manner that correlates with particular stages of neuropathic pain development, indicating dynamic neuroplasticity in the DRG in response to peripheral nerve injury and during neuropathic pain development. Data from post‐array validation indicated that there was a temporal correlation between injury‐induced expression of the glial fibrillary acidic protein (GFAP), a marker for activated astrocytes, and neuropathic pain development. Spinal nerve ligation injury in GFAP knockout mice resulted in neuropathic pain states with similar onset, but a shortened duration compared with that in age, and gender‐matched wild‐type littermates. Intrathecal GFAP antisense oligonucleotide treatment in injured rats with neuropathic pain states reversed injury‐induced behavioral hypersensitivity and GFAP upregulation in DRG and spinal cord. Together, these findings indicate that injury‐induced GFAP upregulation not only serves as a marker for astrocyte activation, but it may also play a critical, but yet identified, role in the maintenance of neuropathic pain states.

Calcium channel α2δ1 proteins mediate trigeminal neuropathic pain states associated with aberrant excitatory synaptogenesis.

Background: Factors mediating orofacial neuropathic pain are not well defined. Results: Trigeminal nerve injury-induced calcium channel α2δ1 protein up-regulation in trigeminal ganglia and spinal complex correlated with enhanced spinal presynaptic neurotransmission, excitatory synaptogenesis, and orofacial pain states. Conclusion: This neuroplasticity may mediate orofacial neuropathic pain states by enhancing dorsal horn excitatory synaptic neurotransmission. Significance: This reveals a mechanism underlying orofacial neuropathic pain states. To investigate a potential mechanism underlying trigeminal nerve injury-induced orofacial hypersensitivity, we used a rat model of chronic constriction injury to the infraorbital nerve (CCI-ION) to study whether CCI-ION caused calcium channel α2δ1 (Cavα2δ1) protein dysregulation in trigeminal ganglia and associated spinal subnucleus caudalis and C1/C2 cervical dorsal spinal cord (Vc/C2). Furthermore, we studied whether this neuroplasticity contributed to spinal neuron sensitization and neuropathic pain states. CCI-ION caused orofacial hypersensitivity that correlated with Cavα2δ1 up-regulation in trigeminal ganglion neurons and Vc/C2. Blocking Cavα2δ1 with gabapentin, a ligand for the Cavα2δ1 proteins, or Cavα2δ1 antisense oligodeoxynucleotides led to a reversal of orofacial hypersensitivity, supporting an important role of Cavα2δ1 in orofacial pain processing. Importantly, increased Cavα2δ1 in Vc/C2 superficial dorsal horn was associated with increased excitatory synaptogenesis and increased frequency, but not the amplitude, of miniature excitatory postsynaptic currents in dorsal horn neurons that could be blocked by gabapentin. Thus, CCI-ION-induced Cavα2δ1 up-regulation may contribute to orofacial neuropathic pain states through abnormal excitatory synapse formation and enhanced presynaptic excitatory neurotransmitter release in Vc/C2.

Application of pulsed radiofrequency currents to rat dorsal root ganglia modulates nerve injury-induced tactile allodynia.

BACKGROUND: Application of pulsed radiofrequency (PRF) currents to the dorsal root ganglia (DRG) has been reported to produce relief from certain pain states without causing thermal ablation. In this study, we examined the direct correlation between PRF application to DRG associated with spinal nerve injury and reversal of injury-induced behavioral hypersensitivity in a rat neuropathic pain model. METHODS: Neuropathic lesioning was performed via left L5 spinal nerve ligation on male adult Sprague-Dawley rats. Once the injured rats had developed tactile allodynia, one group was then assigned to PRF treatment of the L5 DRG and another group was assigned to the sham treatment to the DRG. Behavioral testing was performed on both the control and treated paws using the von Frey filament test before the surgery and at indicated days. The resulting data were analyzed using a linear mixed model to assess the overall difference between the treatment groups and the overall difference among the study days. Cohens d statistic was computed from paired difference-from-baseline scores for each of the 14 study days after treatment and these measures of effect size were then used to descriptively compare the recovery patterns over time for each study group. RESULTS: Spinal nerve injury resulted in the development of behavioral hypersensitivity to von Frey filament stimulation (allodynia) in the hindpaw of the left (injury) side. Mixed linear modeling showed a significant difference between the treatment groups (P = 0.0079) and a significant change of paw withdrawal threshold means over time (P = 0.0006) for all 12 animals. Evaluation of Cohens d (effect size) revealed that the PRF-treated animals exhibited better recovery and recorded larger effect sizes than the sham-treated animals on 10 of the 14 post-PRF treatment days and exhibited moderate-to-strong effects posttreatment at days 8 to 10 and at and beyond day 32. CONCLUSIONS: Findings from this study support that PRF of the DRG causes reversal of nerve injury (spinal nerve ligation)-induced tactile allodynia in rats. This allodynia reversal indicates that nonablative PRF acting via modulation of the DRG can speed recovery in nerve injury–induced pain.

Trigeminal nerve injury-induced thrombospondin-4 up-regulation contributes to orofacial neuropathic pain states in a rat model

Injury to the trigeminal nerve often results in the development of chronic pain states including tactile allodynia, or hypersensitivity to light touch, in orofacial area, but its underlying mechanisms are poorly understood. Peripheral nerve injury has been shown to cause up‐regulation of thrombospondin‐4 (TSP4) in dorsal spinal cord that correlates with neuropathic pain development. In this study, we examined whether injury‐induced TSP4 is critical in mediating orofacial pain development in a rat model of chronic constriction injury to the infraorbital nerve.

Central Mechanisms Mediating Thrombospondin-4-induced Pain States.

Peripheral nerve injury induces increased expression of thrombospondin-4 (TSP4) in spinal cord and dorsal root ganglia that contributes to neuropathic pain states through unknown mechanisms. Here, we test the hypothesis that TSP4 activates its receptor, the voltage-gated calcium channel Cavα2δ1 subunit (Cavα2δ1), on sensory afferent terminals in dorsal spinal cord to promote excitatory synaptogenesis and central sensitization that contribute to neuropathic pain states. We show that there is a direct molecular interaction between TSP4 and Cavα2δ1 in the spinal cord in vivo and that TSP4/Cavα2δ1-dependent processes lead to increased behavioral sensitivities to stimuli. In dorsal spinal cord, TSP4/Cavα2δ1-dependent processes lead to increased frequency of miniature and amplitude of evoked excitatory post-synaptic currents in second-order neurons as well as increased VGlut2- and PSD95-positive puncta, indicative of increased excitatory synapses. Blockade of TSP4/Cavα2δ1-dependent processes with Cavα2δ1 ligand gabapentin or genetic Cavα2δ1 knockdown blocks TSP4 induced nociception and its pathological correlates. Conversely, TSP4 antibodies or genetic ablation blocks nociception and changes in synaptic transmission in mice overexpressing Cavα2δ1. Importantly, TSP4/Cavα2δ1-dependent processes also lead to similar behavioral and pathological changes in a neuropathic pain model of peripheral nerve injury. Thus, a TSP4/Cavα2δ1-dependent pathway activated by TSP4 or peripheral nerve injury promotes exaggerated presynaptic excitatory input and evoked sensory neuron hyperexcitability and excitatory synaptogenesis, which together lead to central sensitization and pain state development.

Thrombospondin-4 contributes to spinal cord injury-induced changes in nociception.

Our previous data have indicated that nerve injury‐induced up‐regulation of thrombospondin‐4 (TSP4) proteins in dorsal spinal cord plays a causal role in neuropathic pain state development in a spinal nerve ligation model. To investigate whether TSP4 proteins also contribute to the development of centrally mediated changes in nociception after spinal cord injury (SCI), we investigated whether SCI induced TSP4 dysregulation, and if so, whether this change correlated with changes in nociception in a T9 spinal cord contusion injury model.

Synaptic ultrastructure changes in trigeminocervical complex posttrigeminal nerve injury.

Trigeminal nerves collecting sensory information from the orofacial area synapse on second‐order neurons in the dorsal horn of subnucleus caudalis and cervical C1/C2 spinal cord (Vc/C2, or trigeminocervical complex), which is critical for sensory information processing. Injury to the trigeminal nerves may cause maladaptive changes in synaptic connectivity that plays an important role in chronic pain development. Here we examined whether injury to the infraorbital nerve, a branch of the trigeminal nerves, led to synaptic ultrastructural changes when the injured animals have developed neuropathic pain states. Transmission electron microscopy was used to examine synaptic profiles in Vc/C2 at 3 weeks postinjury, corresponding to the time of peak behavioral hypersensitivity following chronic constriction injury to the infraorbital nerve (CCI‐ION). Using established criteria, synaptic profiles were classified as associated with excitatory (R‐), inhibitory (F‐), and primary afferent (C‐) terminals. Each type was counted within the superficial dorsal horn of the Vc/C2 and the means from each rat were compared between sham and injured animals; synaptic contact length was also measured. The overall analysis indicates that rats with orofacial pain states had increased numbers and decreased mean synaptic length of R‐profiles within the Vc/C2 superficial dorsal horn (lamina I) 3 weeks post‐CCI‐ION. Increases in the number of excitatory synapses in the superficial dorsal horn of Vc/C2 could lead to enhanced activation of nociceptive pathways, contributing to the development of orofacial pain states. J. Comp. Neurol. 524:309–322, 2016.

Exposure of the dorsal root ganglion to pulsed radiofrequency current in a neuropathic pain model of peripheral nerve injury.

The spinal nerve ligation model of neuropathic pain in rats, as originally described by Kim and Chung (Pain 50:355-363, 1992), provides an excellent venue to study the antinociception and modulation effects of pulsed radiofrequency (PRF) current in pain processing. We describe the procedure of application of PRF current near the exposed L5 dorsal root ganglion (DRG) in rats with L5 spinal nerve ligation injury-induced behavioral hypersensitivity. This method employs the direct visualization of the L5 DRG, allowing for confirmation of the location of the PRF probe adjacent to the DRG.