Z. David Luo

Spinal Dorsal Horn Calcium Channel α2δ-1 Subunit Upregulation Contributes to Peripheral Nerve Injury-Induced Tactile Allodynia

Peripheral nerve injury induces upregulation of the calcium channel α2δ-1 structural subunit in dorsal root ganglia (DRG) and dorsal spinal cord of spinal nerve-ligated rats with neuropathic pain, suggesting a role of the calcium channel α2δ-1 subunit in central sensitization. To investigate whether spinal dorsal horn α2δ-1 subunit upregulation derives from increased DRG α2δ-1 subunit and plays a causal role in neuropathic pain development, we examined spinal dorsal hornα2δ-1 subunit expression with or without dorsal rhizotomy in spinal nerve-ligated rats and its correlation with tactile allodynia, a neuropathic pain state defined as reduced thresholds to non-noxious tactile stimulation. We also examined the effects of intrathecal α2δ-1 antisense oligonucleotides on α2δ-1 subunit expression and neuropathic allodynia in the nerve-ligated rats. Our data indicated that spinal nerve injury resulted in time-dependentα2δ-1 subunit upregulation in the spinal dorsal horn that correlated temporally with neuropathic allodynia development and maintenance. Dorsal rhizotomy diminished basal level expression and blocked injury-induced expression of the spinal dorsal hornα2δ-1 subunit and reversed injury-induced tactile allodynia. In addition, intrathecal α2δ-1 antisense oligonucleotides blocked injury-induced dorsal horn α2δ-1 subunit upregulation and diminished tactile allodynia. These findings indicate that α2δ-1 subunit basal expression occurs presynaptically and postsynaptically in spinal dorsal horn. Nerve injury induces mainly presynaptic α2δ-1 subunit expression that derives from increased α2δ-1 subunit in injured DRG neurons. Thus, changes in presynaptic α2δ-1 subunit expression contribute to injury-induced spinal neuroplasticity and central sensitization that underlies neuropathic pain development and maintenance.

Selective increase of tumour necrosis factor-alpha in injured and spared myelinated primary afferents after chronic constrictive injury of rat sciatic nerve

Chronic constriction of the sciatic nerve, leading to a hyperalgesic state, results in a partial lesion wherein some axons are injured and others remain intact. Here we sought to characterize reactive changes which occur in DRG cell bodies of injured and uninjured axons projecting to skin and muscle. Using immunohistochemistry combined with flurorogold and fluororuby retrograde labelling to define DRG cell bodies associated with injured and uninjured axons, we analysed the DRG immunoreactivity (IR) for tumour necrosis factor‐alpha (TNF), interleukin‐10 (IL‐10), the sensory neuron‐specific channel vanilloid receptor 1 (VR1), isolectin B4 (IB4) and calcitonin‐gene‐related peptide (CGRP) 4 days after a unilateral chronic constriction injury (CCI) of the rat sciatic nerve. TNF IR was predominantly localized in neuronal DRG cells. In DRG with an intact nerve, TNF IR was present in 45%, IL‐10 IR in 46%, VR1 IR in 44%, IB4 IR in 51% and CGRP IR in 40% of all neuronal profiles. Four days after CCI, TNF IR was increased in medium‐sized neurons, whereas IR for IL‐10, VR1 and IB4, predominantly present in small neurons, was reduced. Importantly, not only injured but also adjacent spared neurons contributed markedly to increased TNF IR. Neurons projecting to both muscle and skin displayed upregulated TNF IR after CCI. TNF in medium‐sized neurons colocalized with neurofilament and trkB, but not with IB4, trkA or RET, suggesting a selective phenotypic switch in presumably low‐threshold myelinated primary afferents. Spared myelinated fibres with intact sensory functions but upregulated TNF expression may contribute to behavioural changes observed after nerve injury.

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.

Regulation of expression of the sensory neuron-specific sodium channel SNS in inflammatory and neuropathic pain

Increased voltage-gated sodium channel activity may contribute to the hyperexcitability of sensory neurons in inflammatory and neuropathic pain states. We examined the levels of the transcript encoding the tetrodotoxin-resistant sodium channel SNS in dorsal root ganglion neurons in a range of inflammatory and neuropathic pain models in the rat. Local Freunds adjuvant or systemic nerve growth factor-induced inflammation did not substantially alter the total levels of SNS mRNA. When NGF-treated adult rat DRG neurons in vitro were compared with NGF-depleted control neurons, SNS total mRNA levels and the levels of membrane-associated immunoreactive SNS showed a small increase (17 and 25%, respectively), while CGRP levels increased fourfold. SNS expression is thus little dependent on NGF even though SNS transcript levels dropped by more than 60% 7-14 days after axotomy. In the streptozotocin diabetic rat SNS levels fell 25%, while in several manipulations of the L5/6 tight nerve ligation rat neuropathic pain model, SNS levels fell 40-80% in rat strains that are either susceptible or relatively resistant to the development of allodynia. Increased expression of SNS mRNA is thus unlikely to underlie sensory neuron hyperexcitability associated with inflammation, while lowered SNS transcript levels are associated with peripheral nerve damage.

Coupling gene chip analyses and rat genetic variances in identifying potential target genes that may contribute to neuropathic allodynia development

Genetic factors and nerve injury‐induced changes of gene expression in sensory neurons are potential contributors to tactile allodynia, a neuropathic pain state manifested as hypersensitivity to innocuous mechanical stimulation. To uncover genes relevant to neuropathic allodynia, we analyzed gene expression profiles in dorsal root ganglia (DRG) of spinal nerve‐ligated Harlan and Holtzman Sprague Dawley rats, strains with different susceptibilities to neuropathic allodynia. Using Affymetrix gene chips, we identified genes showing differential basal‐level expression in these strains without injury‐induced regulation. Of more than 8000 genes analyzed, less than 180 genes in each strain were regulated after injury, and 19–22% of that was regulated in a strain‐specific manner. Importantly, we identified functionally related genes that were co‐regulated post injury in one or both strains. In situ hybridization and real‐time PCR analyses of a subset of identified genes confirmed the patterns of the microarray data, and the former also demonstrated that injury‐induced changes occurred, not only in neurons, but also in non‐neuronal cells. Together, our studies provide a global view of injury plasticity in DRG of these rat stains and support a plasticity‐based mechanism mediating variations in allodynia susceptibility, thus providing a source for further characterization of neuropathic pain‐relevant genes and potential pathways.

Calcium channel alpha-2-delta-1 protein upregulation in dorsal spinal cord mediates spinal cord injury-induced neuropathic pain states.

&NA; Spinal cord injury (SCI) commonly results in the development of neuropathic pain, which can dramatically impair the quality of life for SCI patients. SCI‐induced neuropathic pain can be manifested as both tactile allodynia (a painful sensation to a non‐noxious stimulus) and hyperalgesia (an enhanced sensation to a painful stimulus). The mechanisms underlying these pain states are poorly understood. Clinical studies have shown that gabapentin, a drug that binds to the voltage‐gated calcium channel alpha‐2‐delta‐1 subunit (Cavα2δ‐1) proteins is effective in the management of SCI‐induced neuropathic pain. Accordingly, we hypothesized that tactile allodynia post SCI is mediated by an upregulation of Cavα2δ‐1 in dorsal spinal cord. To test this hypothesis, we examined whether SCI‐induced dysregulation of spinal Cavα2δ‐1 plays a contributory role in below‐level allodynia development in a rat spinal T9 contusion injury model. We found that Cavα2δ‐1 expression levels were significantly increased in L4–6 dorsal, but not ventral, spinal cord of SCI rats that correlated with tactile allodynia development in the hind paw plantar surface. Furthermore, both intrathecal gabapentin treatment and blocking SCI‐induced Cavα2δ‐1 protein upregulation by intrathecal Cavα2δ‐1 antisense oligodeoxynucleotides could reverse tactile allodynia in SCI rats. These findings support that SCI‐induced Cavα2δ‐1 upregulation in spinal dorsal horn is a key component in mediating below‐level neuropathic pain states, and selectively targeting this pathway may provide effective pain relief for SCI patients. Spinal cord contusion injury caused increased calcium channel Cavα2δ‐1 subunit expression in dorsal spinal cord that contributes to neuropathic pain states.

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.

Injury Discharges Regulate Calcium Channel Alpha-2-Delta-1 Subunit Upregulation in the Dorsal Horn that Contributes to Initiation of Neuropathic Pain

Abstract Previous studies have shown that peripheral nerve injury in rats induces increased expression of the voltage gated calcium channel (VGCC) alpha‐2‐delta‐1 subunit (Cavα2δ1) in spinal dorsal horn and sensory neurons in dorsal root ganglia (DRG) that correlates to established neuropathic pain states. To determine if injury discharges trigger Cavα2δ1 induction that contributes to neuropathic pain initiation, we examined allodynia onset and Cavα2δ1 levels in DRG and spinal dorsal horn of spinal nerve ligated rats after blocking injury induced neural activity with a local brief application of lidocaine on spinal nerves before the ligation. The lidocaine pretreatment blocked ligation‐induced discharges in a dose‐dependent manner. Similar pretreatment with the effective concentration of lidocaine diminished injury‐induced increases of the Cavα2δ1 in DRG and abolished that in spinal dorsal horn specifically, and resulted in a delayed onset of tactile allodynia post‐injury. Both dorsal horn Cavα2δ1 upregulation and tactile allodynia in the lidocaine pretreated rats returned to levels similar to that in saline pretreated controls 2 weeks post the ligation injury. In addition, preemptive intrathecal Cavα2δ1 antisense treatments blocked concurrently injury‐induced allodynia onset and Cavα2δ1 upregulation in dorsal spinal cord. These findings indicate that injury induced discharges regulate Cavα2δ1 expression in the spinal dorsal horn that is critical for neuropathic allodynia initiation. Thus, preemptive blockade of injury‐induced neural activity or Cavα2δ1 upregulation may be a beneficial option in neuropathic pain management.

Thrombospondin-4 Contributes to Spinal Sensitization and Neuropathic Pain States

Neuropathic pain is a common cause of pain after nerve injury, but its molecular basis is poorly understood. In a post-gene chip microarray effort to identify new target genes contributing to neuropathic pain development, we report here the characterization of a novel neuropathic pain contributor, thrombospondin-4 (TSP4), using a neuropathic pain model of spinal nerve ligation injury. TSP4 is mainly expressed in astrocytes and significantly upregulated in the injury side of dorsal spinal cord that correlates with the development of neuropathic pain states. TSP4 blockade by intrathecal antibodies, antisense oligodeoxynucleotides, or inactivation of the TSP4 gene reverses or prevents behavioral hypersensitivities. Intrathecal injection of TSP4 protein into naive rats is sufficient to enhance the frequency of EPSCs in spinal dorsal horn neurons, suggesting an increased excitatory presynaptic input, and to cause similar behavioral hypersensitivities. Together, these findings support that injury-induced spinal TSP4 may contribute to spinal presynaptic hypersensitivity and neuropathic pain states. Development of TSP4 antagonists has the therapeutic potential for target-specific neuropathic pain management.

Targeting voltage-gated calcium channels for neuropathic pain management.

SummaryVoltage-gated calcium channels (VGCC) play obligatory roles in diverse physiological functions. Pathological conditions leading to changes in their biophysical properties and expression levels may cause malfunctions of VGCC-mediated activities, resulting in disease states. It is believed that changes in VGCC properties under pain-inducing conditions may play a causal role in the development of chronic pain, including nerve injury-induced pain or neuropathic pain. For the past several decades, preclinical and clinical research in developing VGCC blockers or modulators for chronic pain management has been fruitful, leading to some U.S. Food and Drug Administration-approved drugs currently available for chronic pain management. However, their efficacy in pain relief is limited in some patients, and their long-term use is limited by their side-effect profiles. Certainly, there is room for improvement in developing more subtype-specific VGCC blockers or modulators for chronic pain conditions. In this review, we summarized the most recent preclinical and clinical studies related to chronic pain medications acting on the VGCC. We also included clinical trials aiming to expand the application of approved VGCC drugs to different pain states derived from various pathological conditions, as well as drug combination therapies trying to improve the efficacies and side-effect profiles of current pain medications.