Faculty Bibliography

Peripheral nerve injury-induced mechanical allodynia is often accompanied by abnormalities in the higher cortical regions, yet the mechanisms underlying such maladaptive cortical plasticity remain unclear. Here, we show that in male mice, structural and functional changes in the primary somatosensory cortex (S1) caused by peripheral nerve injury require neuron-microglial signaling within the local circuit. Following peripheral nerve injury, microglia in the S1 maintain ramified morphology and normal density but up-regulate the mRNA expression of brain-derived neurotrophic factor (BDNF). Using in vivo two-photon imaging and Cx3cr1CreER;Bdnfflox mice, we show that conditional knockout of BDNF from microglia prevents nerve injury-induced synaptic remodeling and pyramidal neuron hyperactivity in the S1, as well as pain hypersensitivity in mice. Importantly, S1-targeted removal of microglial BDNF largely recapitulates the beneficial effects of systemic BDNF depletion on cortical plasticity and allodynia. Together, these findings reveal a pivotal role of cerebral microglial BDNF in somatosensory cortical plasticity and pain hypersensitivity.

In DRG an increase in miR-133b-3p, miR-143-3p, and miR-1-3p correlates with the lack of development of neuropathic pain following a peripheral nerve injury. Using lentiviral (LV) vectors we found that a single injection of LV-miR-133b-3p or LV-miR-143-3p immediately after a peripheral nerve injury prevented the development of sustained mechanical and cold allodynia. Injection of LV-miR-133b-3p or LV-miR-143-3p by themselves or in combination, on day 3 post-injury produced a partial and transient reduction in mechanical allodynia and a sustained decrease in cold allodynia. Injection of LV-miR-1-3p has no effect. Co-injection of LV-miR-1a with miR-133b-3p or miR-143-3p on day 3 post-injury produced a sustained decrease in mechanical and cold allodynia. In DRG cultures, miR-133b-3p and miR-143-3p but not miR-1-3p, enhanced the depolarization-evoked cytoplasmic calcium increase. Using 3'UTR target clones containing a Gaussian luciferase reporter gene we found that with the 3'UTR-Scn2b, miR-133-3p and miR-143-3p reduced the expression while miR-1-3p enhanced the expression of the reporter gene. With the 3'UTR-TRPM8, miR-133-3p and miR-143-3p reduced the expression and miR-1-3p had no effect. With the 3'UTR-Piezo2, miR-133-3p increased the expression while miR-143-3p and miR-1-3p had no effect. LV-miR133b-3p, LV-miR-143-3p and LV-miR1a-3p reduced Scn2b-mRNA and Piezo2-mRNA. LV-miR133b-3p and LV-miR-143-3p reduced TRPM8-mRNA. LV-miR-133b-3p and LV-miR-143-3p prevent the development of chronic pain when injected immediately after the injury, but are only partially effective when injected at later times. LV-miR-1a-3p had no effect on pain, but complemented the actions of LV-miR-133b-3p or LV-miR-143-3p resulting in a sustained reversal of pain when co-injected 3 days following nerve injury.

Previous studies have shown that following peripheral nerve injury there was a downregulation of the gap junction protein connexin 36 (Cx36) in the spinal cord; however, it is not known whether Cx36 protein is expressed in the dorsal root ganglia (DRGs), nor if its levels are altered following peripheral nerve injuries. Here we address these aspects in the adult rat lumbar DRG. Cx36 mRNA was detected using qRT-PCR, and Cx36 protein was identified in DRG sections using immunohistochemistry (IHC) and immunofluorescence (IF). Double staining revealed that Cx36 co-localizes with both anti-β-III tubulin, a neuronal marker, and anti-glutamine synthetase, a satellite glial cell (SGC) marker. In neurons, Cx36 staining was mostly uniform in somata and fibers of all sizes and its intensity increased at the cell membranes. This labeling pattern was in contrast with Cx36 IF dots mainly found at junctional membranes in islet beta cells used as a control tissue. Co-staining with anti-Cx43 and anti-Cx36 showed that whereas mostly uniform staining of Cx36 was found throughout neurons and SGCs, Cx43 IF puncta were localized to SGCs. Cx36 mRNA was expressed in normal lumbar DRG, and it was significantly down-regulated in L4 DRG of rats that underwent sciatic nerve injury resulting in persistent hypersensitivity. Collectively, these findings demonstrated that neurons and SGCs express Cx36 protein in normal DRG, and suggested that perturbation of Cx36 levels may contribute to chronic neuropathic pain resulting from a peripheral nerve injury.

BACKGROUND: AMPAkines augment the function of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the brain to increase excitatory outputs. These drugs are known to relieve persistent pain. However, their role in acute pain is unknown. Furthermore, a specific molecular and anatomic target for these novel analgesics remains elusive. METHODS: The authors studied the analgesic role of an AMPAkine, CX546, in a rat paw incision (PI) model of acute postoperative pain. The authors measured the effect of AMPAkines on sensory and depressive symptoms of pain using mechanical hypersensitivity and forced swim tests. The authors asked whether AMPA receptors in the nucleus accumbens (NAc), a key node in the brain's reward and pain circuitry, can be a target for AMPAkine analgesia. RESULTS: Systemic administration of CX546 (n = 13), compared with control (n = 13), reduced mechanical hypersensitivity (50% withdrawal threshold of 6.05 +/- 1.30 g [mean +/- SEM] vs. 0.62 +/- 0.13 g), and it reduced depressive features of pain by decreasing immobility on the forced swim test in PI-treated rats (89.0 +/- 15.5 vs. 156.7 +/- 18.5 s). Meanwhile, CX546 delivered locally into the NAc provided pain-relieving effects in both PI (50% withdrawal threshold of 6.81 +/- 1.91 vs. 0.50 +/- 0.03 g; control, n = 6; CX546, n = 8) and persistent postoperative pain (spared nerve injury) models (50% withdrawal threshold of 3.85 +/- 1.23 vs. 0.45 +/- 0.00 g; control, n = 7; CX546, n = 11). Blocking AMPA receptors in the NAc with 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione inhibited these pain-relieving effects (50% withdrawal threshold of 7.18 +/- 1.52 vs. 1.59 +/- 0.66 g; n = 8 for PI groups; 10.70 +/- 3.45 vs. 1.39 +/- 0.88 g; n = 4 for spared nerve injury groups). CONCLUSIONS: AMPAkines relieve postoperative pain by acting through AMPA receptors in the NAc.

BACKGROUND: The function of the Cannabinoid 1 receptor (CB1R) in the development of neuropathic pain is not clear. Mounting evidence suggest that CB1R expression and activation may contribute to pain. Cannabinoid 1 receptor knockout mice (CB1R-/-) generated on a C57Bl/6 background exhibit hypoalgesia in the hotplate assay and formalin test. These findings suggest that Cannabinoid 1 receptor expression mediates the responses to at least some types of painful stimuli. By using this mouse line, we sought to determine if the lack of Cannabinoid 1 receptor unveils a general hypoalgesic phenotype, including protection against the development of neuropathic pain. The acetone test was used to measure cold sensitivity, the electronic von Frey was used to measure mechanical thresholds before and after spared-nerve injury, and analysis of footprint patterns was conducted to determine if motor function is differentially affected after nerve-injury in mice with varying levels of Cannabinoid 1 receptor. RESULTS: At baseline, CB1R-/- mice were hypersensitive in the acetone test, and this phenotype was maintained after spared-nerve injury. Using calcium imaging of lumbar dorsal root ganglion (DRG) cultures, a higher percentage of neurons isolated from CB1R-/- mice were menthol sensitive relative to DRG isolated from wild-type (CB1R+/+) mice. Baseline mechanical thresholds did not differ among genotypes, and mechanical hypersensitivity developed similarly in the first two weeks following spared-nerve injury (SNI). At two weeks post-SNI, CB1R-/- mice recovered significantly from mechanical hypersensitivity, while the CB1R+/+ mice did not. Heterozygous knockouts (CB1R+/-) transiently developed cold allodynia only after injury, but recovered mechanical thresholds to a similar extent as the CB1R-/- mice. Sciatic functional indices, which reflect overall nerve health, and alternation coefficients, which indicate uniformity of strides, were not significantly different among genotypes. CONCLUSION: Cold allodynia and significant recovery from spared-nerve injury-induced mechanical hypersensitivity are two novel phenotypes which characterize the global CB1R-/- mice. An increase in transient receptor potential channel of melastatin 8 channel function in DRG neurons may underlie the cold phenotype. Recovery of mechanical thresholds in the CB1R knockouts was independent of motor function. These results indicate that CB1R expression contributes to the development of persistent mechanical hypersensitivity, protects against the development of robust cold allodynia but is not involved in motor impairment following spared-nerve injury in mice.

Following injury, primary sensory neurons undergo changes that drive central sensitization and contribute to the maintenance of persistent hypersensitivity. NR2B expression in the dorsal root ganglia (DRG) has not been previously examined in neuropathic pain models. Here, we investigated if changes in NR2B expression within the DRG are associated with hypersensitivities that result from peripheral nerve injuries. This was done by comparing the NR2B expression in the DRG derived from two modalities of the spared nerve injury (SNI) model, since each variant produces different neuropathic pain phenotypes. Using the electronic von Frey to stimulate the spared and non-spared regions of the hindpaws, we demonstrated that sural-SNI animals develop sustained neuropathic pain in both regions while the tibial-SNI animals recover. NR2B expression was measured at Day 23 and Day 86 post-injury. At Day 23 and 86 post-injury, sural-SNI animals display strong hypersensitivity, whereas tibial-SNI animals display 50 and 100% recovery from post-injury-induced hypersensitivity, respectively. In tibial-SNI at Day 86, but not at Day 23 the perinuclear region of the neuronal somata displayed an increase in NR2B protein. This retention of NR2B protein within the perinuclear region, which will render them non-functional, correlates with the recovery observed in tibial-SNI. In sural-SNI at Day 86, DRG displayed an increase in NR2B mRNA which correlates with the development of sustained hypersensitivity in this model. The increase in NR2B mRNA was not associated with an increase in NR2B protein within the neuronal somata. The latter may result from a decrease in kinesin Kif17, since Kif17 mediates NR2B transport to the soma's plasma membrane. In both SNIs, microglia/macrophages showed a transient increase in NR2B protein detected at Day 23 but not at Day 86, which correlates with the initial post-injury induced hypersensitivity in both SNIs. In tibial-SNI at Day 86, but not at Day 23, satellite glia cells (SGCs) displayed an increase in NR2B protein. This study is the first to characterize of cell-specific changes in NR2B expression within the DRG following peripheral nerve injury. We discuss how the observed NR2B changes in DRG can contribute to the different neuropathic pain phenotypes displayed by each SNI variant.

Peripheral nerve injury alters the expression of hundreds of proteins in dorsal root ganglia (DRG). Targeting some of these proteins has led to successful treatments for acute pain, but not for sustained post-operative neuropathic pain. The latter may require targeting multiple proteins. Since a single microRNA (miR) can affect the expression of multiple proteins, here, we describe an approach to identify chronic neuropathic pain-relevant miRs. We used two variants of the spared nerve injury (SNI): Sural-SNI and Tibial-SNI and found distinct pain phenotypes between the two. Both models induced strong mechanical allodynia, but only Sural-SNI rats maintained strong mechanical and cold allodynia, as previously reported. In contrast, we found that Tibial-SNI rats recovered from mechanical allodynia and never developed cold allodynia. Since both models involve nerve injury, we increased the probability of identifying differentially regulated miRs that correlated with the quality and magnitude of neuropathic pain and decreased the probability of detecting miRs that are solely involved in neuronal regeneration. We found seven such miRs in L3-L5 DRG. The expression of these miRs increased in Tibial-SNI. These miRs displayed a lower level of expression in Sural-SNI, with four having levels lower than those in sham animals. Bioinformatic analysis of how these miRs could affect the expression of some ion channels supports the view that, following a peripheral nerve injury, the increase of the seven miRs may contribute to the recovery from neuropathic pain while the decrease of four of them may contribute to the development of chronic neuropathic pain. The approach used resulted in the identification of a small number of potentially neuropathic pain relevant miRs. Additional studies are required to investigate whether manipulating the expression of the identified miRs in primary sensory neurons can prevent or ameliorate chronic neuropathic pain following peripheral nerve injuries.

Satellite glia cells (SGCs), within the dorsal root ganglia (DRG), surround the somata of most sensory neurons. SGCs have been shown to interact with sensory neurons and appear to be involved in the processing of afferent information. We found that in rat DRG various N-methyl-d-aspartate receptor (NMDAr) subunits were expressed in SGCs in intact ganglia and in vitro. In culture, when SGCs were exposed to brief pulses of NMDA they evoked transient increases in cytoplasmic calcium that were inhibited by specific NMDA blockers (MK-801, AP5) while they were Mg2+ insensitive indicating that SGCs express functional NMDAr. The percentage of NMDA responsive SGCs was similar in mixed- (SGCs plus neurons) and SGC-enriched cultures. The pattern of the magnitude changes of the NMDA-evoked response was similar in SGCs and DRG neurons when they were in close proximity, suggesting that the NMDA response of SGCs and DRG neurons is modulated by their interactions. Treating the cultures with nerve growth factor, and/or prostaglandin E2 did not alter the percentage of SGCs that responded to NMDA. Since glutamate appears to be released within the DRG, the detection of functional NMDAr in SGCs suggests that their NMDAr activity could contribute to the interactions between neurons and SGCs. In summary we demonstrated for the first time that SGCs express functional NMDAr.

OBJECTIVES/OBJECTIVE:This study reports on the rapid isolation of verbascoside from Lippia citriodora H.B.K. (Verbenaceae), an inexpensive and widespread source, and the evaluation of its antihyperalgesic activity. METHODS:Isolation of verbascoside was achieved by size exclusion chromatography with Sephadex LH-20 eluting with 50% EtOH, which is proposed as a fast and efficient method of separation. KEY FINDINGS/RESULTS:The antihyperalgesic activity of verbascoside was tested by in-vivo assay using the paw-pressure test in two animal models of neuropathic pain: a peripheral mononeuropathy produced either by a chronic constriction injury of the sciatic nerve (CCI) or by an intra-articular injection of sodium monoiodoacetate (MIA). CONCLUSIONS:Verbascoside administered intraperitoneally at a dose of 100 mg/kg reverted the mechanical hyperalgesia in both CCI and MIA treated rats, as evaluated in the paw-pressure test. Verbascoside was also effective against mechanical hyperalgesia after oral administration at doses of 300 and 600 mg/kg.

The involvement of substance P (SP) in neuronal sensitization through the activation of the neurokinin-1-receptor (NK1r) in postsynaptic dorsal horn neurons has been well established. In contrast, the role of SP and NK1r in primary sensory dorsal root ganglion (DRG) neurons, in particular in the soma, is not well understood. In this study, we evaluated whether SP modulated the NMDA-evoked transient increase in cytoplasmic Ca(2+) ([Ca(2+)](cyt)) in the soma of dissociated adult DRG neurons. Cultures were treated with nerve growth factor (NGF), prostaglandin E(2) (PGE(2)) or both NGF+PGE(2). Treatment with NGF+PGE(2) increased the percentage of N-methyl-d-aspartate (NMDA) responsive neurons. There was no correlation between the percentage of NMDA responsive neurons and the level of expression of the NR1 and NR2B subunits of the NMDA receptor or of the NK1r. Pretreatment with SP did not alter the percentage of NMDA responsive neurons; while it potentiated the NMDA-evoked [Ca(2+)](cyt) transient by increasing its magnitude and by prolonging the period during which small- and some medium-sized neurons remained NMDA responsive. The SP-mediated potentiation was blocked by the SP-antagonist ([D-Pro(4), D-Trp(7,9)]-SP (4-11)) and by the protein kinase C (PKC) blocker bisindolylmaleimide I (BIM); and correlated with the phosphorylation of PKCepsilon. The Nk1r agonist [Sar(9), Met(O(2))(11)]-SP (SarMet-SP) also potentiated the NMDA-evoked [Ca(2+)](cyt) transient. Exposure to SP or SarMet-SP produced a rapid increase in the labeling of phosphorylated-PKCepsilon. In none of the conditions we detected phosphorylation of the NR2B subunit at Ser-1303. Phosphorylation of the NR2B subunit at Tyr1472 was enhanced to a similar extent in cells exposed to NMDA, SP or NMDA+SP, and that enhancement was blocked by BIM. Our findings suggest that NGF and PGE(2) may contribute to the injury-evoked sensitization of DRG neurons in part by enhancing their NMDA-evoked [Ca(2+)](cyt) transient in all sized DRG neurons; and that SP may further contribute to the DRG sensitization by enhancing and prolonging the NMDA-evoked increase in [Ca(2+)](cyt) in small- and medium-sized DRG neurons