Decoy peptide targeted to Toll-IL-1R domain inhibits LPS and TLR4-active metabolite morphine-3 glucuronide sensitization of sensory neurons

Accumulating evidence indicates that Toll-like receptor (TLR) signaling adapter protein interactions with Toll/Interleukin-1 Receptor (TIR) domains present in sensory neurons may modulate neuropathic pain states. Following ligand interaction with TLRs, TIR serves to both initiate intracellular signaling and facilitate recruitment of signaling adapter proteins to the intracytoplasmic domain. Although TLR TIR is central to a number of TLR signaling cascades, its role in sensory neurons is poorly understood. In this study we investigated the degree to which TLR TIR decoy peptide modified to include a TAT sequence (Trans-Activator of Transcription gene in HIV; TAT-4BB) affected LPS-induced intracellular calcium flux and excitation in sensory neurons, and behavioral changes due to TLR4 active metabolite, morphine-3-glucuronide (M3G) exposure in vivo. TAT-4BB inhibited LPS-induced calcium changes in a majority of sensory neurons and decreased LPS-dependent neuronal excitability in small diameter neurons. Acute systemic administration of the TAT-4BB reversed M3G-induced tactile allodynia in a dose-dependent manner but did not affect motor activity, anxiety or responses to noxious thermal stimulus. These data suggest that targeting TLR TIR domains may provide novel pharmacological targets to reduce or reverse TLR4-dependent pain behavior in the rodent

Acrolein involvement in sensory and behavioral hypersensitivity following spinal cord injury in the rat

Growing evidence suggests that oxidative stress, as associated with spinal cord injury (SCI), may play a critical role in both neuroinflammation and neuropathic pain conditions. The production of the endogenous aldehyde acrolein, following lipid peroxidation during the inflammatory response, may contribute to peripheral sensitization and hyperreflexia following SCI via the TRPA1-dependent mechanism. Here we report that there are enhanced levels of acrolein and increased neuronal sensitivity to the aldehyde for at least 14 days after SCI. Concurrent with injury-induced increases in acrolein concentration is an increased expression of TRPA1 in the lumbar (L3-L6) sensory ganglia. As proof of the potential pronociceptive role for acrolein, intrathecal injections of acrolein revealed enhanced sensitivity to both tactile and thermal stimuli for up to 10 days, supporting the compound’s pro-nociceptive functionality. Treatment of SCI animals with the acrolein scavenger hydralazine produced moderate improvement in tactile responses as well as robust changes in thermal sensitivity for up to 49 days. Taken together, these data suggests that acrolein directly modulates SCI-associated pain behavior, making it a novel therapeutic target for preclinical and clinical SCI as an analgesic

Identification of a functional interaction of HMGB1 with Receptor for Advanced Glycation End-products in a model of neuropathic pain

Recent studies indicate that the release of high mobility group box 1 (HMGB1) following nerve injury may play a central role in the pathogenesis of neuropathic pain. HMGB1 is known to influence cellular responses within the nervous system via two distinct receptor families; the Receptor for Advanced Glycation End-products (RAGE) and Toll-like receptors (TLRs). The degree to which HMGB1 activates a receptor is thought to be dependent upon the oxidative state of the ligand, resulting in the functional isoforms of all-thiol HMGB1 (at-HMGB1) acting through RAGE, and disufide HMGB1 (ds-HMGB1) interacting with TLR4. Though it is known that dorsal root ganglia (DRG) sensory neurons exposed to HMGB1 and TLR4 agonists can influence excitation, the degree to which at-HMGB1 signaling through neuronal RAGE contributes to neuropathic pain is unknown. Here we demonstrate that at-HMGB1 activation of nociceptive neurons is dependent on RAGE and not TLR4. To distinguish the possible role of RAGE on neuropathic pain, we characterized the changes in RAGE mRNA expression up to one month after tibial nerve injury (TNI). RAGE mRNA expression in lumbar dorsal root ganglion (DRG) is substantially increased by post-injury day (PID) 28 when compared with sham injured rodents. Protein expression at PID28 confirms this injury-induced event in the DRG. Moreover, a single exposure to monoclonal antibody to RAGE (RAGE Ab) failed to abrogate pain behavior at PID 7, 14 and 21. However, RAGE Ab administration produced reversal of mechanical hyperalgesia on PID28. Thus, at-HMGB1 activation through RAGE may be responsible for sensory neuron sensitization and mechanical hyperalgesia associated with chronic neuropathic pain states

Bone pain induced by multiple myeloma is reduced by targeting V-ATPase and ASIC3

Multiple myeloma (MM) patients experience severe bone pain (MMBP) that is undertreated and poorly understood. In this study, we studied MMBP in an intratibial mouse xenograft model which employs JJN3 human MM cells. In this model, mice develop MMBP associated in bone with increased sprouting of calcitonin gene-related peptide-positive (CGRP+) sensory nerves and in dorsal root ganglia (DRG) with upregulation of phosphorylated ERK1/2 (pERK1/2) and pCREB, two molecular indicators of neuron excitation. We found that JJN3 cells expressed a vacuolar proton pump (V-ATPase) that induced an acidic bone microenvironment. Inhibition of JJN3-colonized bone acidification by a single injection of the selective V-ATPase inhibitor, bafilomycin A1, decreased MMBP, CGRP+ SN sprouting, and pERK1/2 and pCREB expression in DRG. CGRP+ sensory nerves also expressed increased levels of the acid-sensing nociceptor ASIC3. Notably, a single injection of the selective ASIC3 antagonist APETx2 dramatically reduced MMBP in the model. Mechanistic investigations in primary DRG neurons co-cultured with JJN3 cells showed increased neurite outgrowth and excitation inhibited by bafilomycin A1 or APETx2. Further, combining APETx2 with bafilomycin A1 reduced MMBP to a greater extent than either agent alone. Lastly, combining bafilomycin A1 with the osteoclast inhibitor zoledronic acid was sufficient to ameliorate MMBP which was refractory to zoledronic acid. Overall, our results show that osteoclasts and MM cooperate to induce an acidic bone microenvironment that evokes MMBP as a result of the excitation of ASIC3-activated sensory neurons. Further, they present a mechanistic rationale for targeting ASIC3 on neurons along with the MM-induced acidic bone microenvironment as a strategy to relieve MMBP in patients

The HMGB1-RAGE Inflammatory Pathway: Implications for Brain Injury-Induced Pulmonary Dysfunction

SIGNIFICANCE: Deceased patients who have suffered severe traumatic brain injury (TBI) are the largest source of organs for lung transplantation. However, due to severely compromised pulmonary lung function, only one-third of these patients are eligible organ donors, with far fewer capable of donating lungs (∼ 20%). As a result of this organ scarcity, understanding and controlling the pulmonary pathophysiology of potential donors are key to improving the health and long-term success of transplanted lungs. RECENT ADVANCES: Although the exact mechanism by which TBI produces pulmonary pathophysiology remains unclear, it may be related to the release of damage-associated molecular patterns (DAMPs) from the injured tissue. These heterogeneous, endogenous host molecules can be rapidly released from damaged or dying cells and mediate sterile inflammation following trauma. In this review, we highlight the interaction of the DAMP, high-mobility group box protein 1 (HMGB1) with the receptor for advanced glycation end-products (RAGE), and toll-like receptor 4 (TLR4). CRITICAL ISSUES: Recently published studies are reviewed, implicating the release of HMGB1 as producing marked changes in pulmonary inflammation and physiology following trauma, followed by an overview of the experimental evidence demonstrating the benefits of blocking the HMGB1-RAGE axis. FUTURE DIRECTIONS: Targeting the HMGB1 signaling axis may increase the number of lungs available for transplantation and improve long-term benefits for organ recipient patient outcomes

Carbamazepine potentiates the effectiveness of morphine in a rodent model of neuropathic pain.

Approximately 60% of morphine is glucuronidated to morphine-3-glucuronide (M3G) which may aggravate preexisting pain conditions. Accumulating evidence indicates that M3G signaling through neuronal Toll-like receptor 4 (TLR4) may be central to this proalgesic signaling event. These events are known to include elevated neuronal excitability, increased voltage-gated sodium (NaV) current, tactile allodynia and decreased opioid analgesic efficacy. Using an in vitro ratiometric-based calcium influx analysis of acutely dissociated small and medium-diameter neurons derived from lumbar dorsal root ganglion (DRG), we observed that M3G-sensitive neurons responded to lipopolysaccharide (LPS) and over 35% of these M3G/LPS-responsive cells exhibited sensitivity to capsaicin. In addition, M3G-exposed sensory neurons significantly increased excitatory activity and potentiated NaV current as measured by current and voltage clamp, when compared to baseline level measurements. The M3G-dependent excitability and potentiation of NaV current in these sensory neurons could be reversed by the addition of carbamazepine (CBZ), a known inhibitor of several NaV currents. We then compared the efficacy between CBZ and morphine as independent agents, to the combined treatment of both drugs simultaneously, in the tibial nerve injury (TNI) model of neuropathic pain. The potent anti-nociceptive effects of morphine (5 mg/kg, i.p.) were observed in TNI rodents at post-injury day (PID) 7-14 and absent at PID21-28, while administration of CBZ (10 mg/kg, i.p.) alone failed to produce anti-nociceptive effects at any time following TNI (PID 7-28). In contrast to either drug alone at PID28, the combination of morphine and CBZ completely attenuated tactile hyperalgesia in the rodent TNI model. The basis for the potentiation of morphine in combination with CBZ may be due to the effects of a latent upregulation of NaV1.7 in the DRG following TNI. Taken together, our observations demonstrate a potential therapeutic use of morphine and CBZ as a combinational treatment for neuropathic pain

Morphine-3-glucuronide (M3G) potentiation of sodium currents is blocked by carbamazepine (CBZ).

<p>A, currents were elicited by performing voltage steps from a holding potential of −80 mV, in 5 mV increments, from −70 mV to +50 mV. B, representative traces from dorsal root ganglia cells treated with either 0.1% DMSO (Vehicle), 500 ug CBZ, 3 µM M3G, or both 3 µM M3G and 500 ug CBZ. C, peak currents (pA) were normalized to cell capacitance (pF) to measure current density (pA/pF). Asterisks indicate statistically significant differences between treatment groups and vehicle group (*P<0.05, one way ANOVA with Tukey's post-hoc test). Bars represent mean ± S.E.</p

Morphine-3-glucuronide (M3G) increased excitability of nociceptive dorsal root ganglion neurons is reversed in the presence of carbamazepine (CBZ).

<p>(A) Current clamp recordings were performed on small (≥30 µm) to medium (≥40 µm) dorsal root ganglion (DRG) neurons (L1-6) from naïve rats. Firing of two to four action potentials (APs) was elicited by a 1 second depolarizing current injection (ranging from 0.1 to 0.6 nA depending on the cell) every 30 seconds. Representative recordings demonstrating that application of 3 µg/mL M3G increases the number of elicited APs. Bath applied CBZ reversed the effect of M3G-increased excitability. (B) Group data showing that M3G caused a significant increase in DRG AP firing that is reversed by CBZ (*P<0.05).</p

The effect of intraperitoneally administered morphine (MOR), carbamazepine (CBZ) or combination of MOR/CBZ on tactile allodynia in the tibial nerve injury (TNI) model at post-injury day (PID) 28.

<p>(A) Neither MOR or CBZ affect tactile allodynia induced by TNI. Each line represents the groups mean and SEM of 6–10 female rats. Drug group behavior at 60 minute or 120 minute vs TNI baseline (BL) behavior. (B) Effect of co-administered MOR and CBZ on tactile allodynia in the tibial nerve injury (TNI) model at post-injury day 28. The ability of MOR/CBZ to attenuate tactile allodynia induced by TNI was dose-dependent. Each line represents the groups mean and SEM of 6–10 rats. (*P<0.05; combination therapy group vs TNI baseline (BL) behavior).</p