Julia Borges Paes Lemes

The excitability of afferents involved in nociceptive signaling reflects the interaction of several co-expressed membrane receptors. Current studies have shown that Toll-like receptor-4 (TLR-4) signaling can exacerbate excitation evoked by transient receptor potential vanilloid type 1 (TRPV1) activity, and this interaction plays a key role in driving and sustaining facilitated pain states. The mechanism by which this potentiated TRPV1 activity secondary to TLR-4 agonism occurs in sensory neurons remains unknown, although intracellular kinase activity is a strong candidate. To address this hypothesized linkage, neuronal cell cultures prepared from dorsal root ganglia (DRG) of male wildtype (WT) and Tlr4 ^-/- mice were used to evaluate calcium transients of neurons after capsaicin administration in culture, pre-treated for 30 minutes with the TLR-4 agonist, lipopolysaccharide (LPS). TRPV1 protein expression at the neuron surface in cultured DRG cells with or without LPS treatment was quantified by flow cytometry assay. The roles of protein kinase A (PKA) and C were assessed using selective inhibitors (KT5720 for PKA and Chelerythrine chloride for PKC) applied to WT-DRG neurons or administered in vivo by intraplantar or intrathecal injection, prior to LPS and capsaicin administration. Behavioral effects of in vivo TRPV1 activation were assessed through paw flinch responses evoked by intraplantar capsaicin injection and by hind paw tactile thresholds measured by von Frey filaments. LPS incubation in cultured DRG neurons enhances the intensity of calcium influx following TRPV1 activation in WT but not Tlr4 ^-/ cells. The augmented calcium influx evoked by capsaicin was prevented by the inhibition of PKA but not PKC. Similarly, mice treated with LPS in the hind paw displayed greater nociceptive responding after capsaicin and increased tactile allodynia. The facilitated component was prevented by the local pre-treatment with the PKA inhibitor. Correspondingly, lumbar spinal blockade of PKA resulted in temporary reversal of hyperalgesia induced by intrathecal LPS injection in mice. Together, these results demonstrate the relevance of TLR-4 in modulating the excitability of nociceptor signaling by regulating TRPV1, thereby influencing pain transmission through PKA signaling.

Nociceptive afferent neurons within the dorsal root ganglion (DRG) detect and relay painful peripheral stimuli, and the malfunctioning of this process leads to sustained pain states. Animal model studies have been invaluable for demonstrating the importance of the DRG nociceptor in pain sensation and the development of related analgesic targets. However, a human in vitro model of nociception is essential to confirming the relevance of preclinical findings for therapeutic drug development. We characterized the nociceptive properties of differentiated cells from the human DRG‐derived immortalized cell line HD10.6 and developed their use into an in vitro model of human nociceptive signaling and therapy. Within differentiated HD10.6 cells, we confirmed the abundance and function of machinery linked with pain sensation, including key ion channels (TRPV1, NaV1.7) and afferent peptides (CGRP, Substance P), by immunofluorescence and calcium influx assays. Through whole‐cell patch clamp, including current clamp and voltage clamp, we recorded the baseline electrophysiological parameters of differentiated HD10.6 cells. We further found that differentiated HD10.6 cells express the mu opioid receptor 1 protein, and that mu agonist DAMGO blocks depolarization‐evoked calcium influx in a naloxone‐reversible fashion. Importantly, excitation and peripheral sensitization were induced within HD10.6 cells in response to an inflammatory cocktail, mirroring nociceptors in a pain state during and after tissue damage or inflammation. HD10.6 cells were also cultured into dual‐chambered microfluidic devices to mirror the physiological anatomy of the nociceptor. Within this system, genetic therapy adeno‐associated‐virus was successfully taken up by the peripheral terminals and transported to the soma.

Nociceptive afferent neurons within the dorsal root ganglion (DRG) detect and relay painful stimuli from the periphery to the brain, and the malfunctioning of this process leads to sustained pain states. Animal model studies have been invaluable for demonstrating the importance of the DRG nociceptor in pain sensation and the development of related analgesic targets. However, there are functional biological differences between human and animal model nociceptors. Therefore, a complementary in vitro model of human nociception is critical to confirming the relevance of preclinical findings for therapeutic drug development. We characterized the nociceptive properties of differentiated cells from the human DRG-derived immortalized cell line HD10.6. Within differentiated HD10.6 cells, we documented the abundance and localization of nociceptive machinery central to regulating excitability and linked with pain sensation including ion channels TRPV1 and NaV1.7 and afferent peptides CGRP and Substance P. Using calcium influx imaging assays, we confirmed the electrical functionality of TRPV1 and NaV1.7 in HD10.6 cells, and through whole-cell patch clamp, we found similar baseline electrophysiological parameters of HD10.6 cells to those previously observed in human patient DRGs. Further, we found that differentiated HD10.6 cells express the mu opioid receptor 1 protein, and DAMGO, a mu agonist, blocks depolarization-evoked calcium influx in a naloxone-reversible fashion. Importantly, using an inflammatory cocktail, excitation and peripheral sensitization are induced within HD10.6 cells, mirroring nociceptors in a pain state during or after tissue damage or inflammation. Finally, HD10.6 cells were also cultured into dual-chambered microfluidic devices to mirror the biological anatomy of the nociceptor. Within this system, we demonstrated the uptake of adeno-associated-virus (AAV) by the peripheral terminals and AAV transport to the soma. Altogether, we have developed the use of HD10.6 cells to create a system of human nociceptive signaling on a chip to study human nociceptor physiology and intervention. Perspective: There are essential differences between human and animal model nociceptors. Here, we develop a physiological model of “nociceptive signaling on a chip” using human-derived nociceptors to ultimately enhance the translatability of preclinical afferent signaling research to the human patient.

Background: Assessing knee joint pain in experimental OA (EOA) models remains a significant challenge. Our study demonstrates that the use of an adapted electronic von Frey (aVF) device, featuring a modified tip, surpasses the standard von Frey (sVF) in detecting knee joint pain behavior and evaluating the efficacy of analgesic treatments in OA model induced by monoiodine acetate (MIA). Results: The sVF was able to induce a behavior profile in naïve animals characterized by a hind paw flinching and withdrawal reflex. This behavioral response was affected by intraplantar lidocaine, which prone to the increase in mechanical thresholds related to sVF and validate it as pain related behavior. On the aVF method, the animals displayed no alterations at their mechanical thresholds in presence or absence of lidocaine, suggesting minimal stimulation of hind paw by the modified methodology. In animals where an osteoarthritic phenotype was induced by MIA, the aVF was able to detect a significant reduction on joint mechanical thresholds. The behavior linked to aVF was significantly affected by systemic delivery of morphine, confirming a nociceptive-like phenotype and suggesting a pain behavior predominantly triggered by joint flexion. The aVF was also able to detect an analgesic profile in MIA-OA rats treated with dexamethasone, LPS-RS, fucoidan, and morphine, indicating effectiveness in measure different the response profile triggered by analgesic drugs that affect joint pain perception. Conclusions: Our results suggest aVF as a more appropriate method to evaluate joint pain in rats.

Background: Genome targeting strategies to address NaV 1.7 mediated signaling in nociceptive afferents produce highly selective and persistent analgesic outcomes. Here, we analyze the concentration-dependent effects of the reduction of primary afferent NaV 1.7 channel expression by intrathecal delivery of an antisense oligonucleotide on pain behaviors and the covariance of Scn9a knock-down on NaV 1.7 message expression and channel binding. Methods: Male Sprague-Dawley rats were implanted with lumbar intrathecal catheters and dosed with different NaV 1.7 ASO concentrations (100 to 3000 μg;10 μL). Pain behavior assays were conducted 0-28 days after ASO injections. Brain, spinal cords (SC) and dorsal root ganglia (DRGs) were collected. Quantification of ASO knock-down was assessed through RT-qPCR. NaV 1.7 expression was assessed by binding of NaV 1.7 fluorescent labeled antagonist (ATTO488PTx-II). Distribution studies were performed using anti-ASO antibody staining in brain, SCs and DRGs. Results: NaV 1.7 message was detected in nerve, DRG and SC. Intrathecal ASO induced a concentration dependent gradient of knock down in DRGs (lumbar to cervical) of Scn9a mRNA and ATTO488PTx-II binding in small DRG neurons, and in spinal parenchyma, and a suppression of pain behaviors initiated by mechanical compression, inflammation and following intraplantar NaV1.7 agonist (OD1) or formalin. At 1000 µg, there was a 47% reduction in phase 2 flinching, a 60% reduction in DRG mRNA and a 36% reduction in ATTO488PTx-II DRG binding in comparison with mismatch controls. Although marked changes were seen at the sensory ganglia level and spinal dorsal horn, no changes in NaV 1.7 binding or mRNA were detected in sciatic nerves. Reduction in DRG message displayed a rostrocaudal gradient that corresponded with ASO distribution. Conclusions: The study presents how NaV 1.7 ASOs reduce primary afferent channel binding through an effective knock-down on Scn9a mRNA, and channel binding leading to a covariate reduction in pain behavior.

A significant portion of adolescents suffer from mental illnesses and persistent pain due to repeated stress. The components of the nervous system that link stress and pain in early life remain unclear. Prior studies in adult mice implicated the innate immune system, specifically Toll-like receptors (TLRs), as critical for inducing long-term anxiety and pain-like behaviors in social defeat stress (SDS) models. In this work, we investigated the pain and anxiety behavioral phenotypes of wild-type and TLR4-deficient juvenile mice subjected to repeated SDS and evaluated the engagement of TLR4 by measuring dimerization in the spinal cord, dorsal root ganglia, and prefrontal cortex. Male juvenile (4-week-old) mice (C57BL/6J or Tlr4^-/-) underwent six social defeat sessions with adult aggressor (CD1) mice. In WT mice, SDS promotes chronic mechanical allodynia and thermal hyperalgesia assessed via von Frey testing and the Hargreaves test, respectively. In parallel, the stressed WT mice exhibited transient anxiety-like behavior and long-lasting locomotor activity reduction in the open-field test. Tlr4^-/--stressed animals were resistant to the induction of pain-like behavior but had a remnant of anxious behavior, spending less time in the center of the arena. In WT SDS, there were concordant robust increases in TLR4 dimerization in dorsal root ganglia macrophages and spinal cord microglia, indicating TLR4 activation. These results suggest that the chronic pain phenotype and locomotor impairment induced by SDS in juvenile mice depends on TLR4 engagement evidenced by dimerization in immune cells of the dorsal root ganglia and spinal cord.

A significant portion of adolescents suffer from mental illnesses and persistent pain due to repeated stress. It remains unclear which components of the nervous system are involved in the linkage between stress and pain at an early age. Prior studies in adult mice implicated the innate immune system, specifically Toll-like receptors (TLR), as critical for inducing long-term anxiety and pain-like behavior in social defeat stress (SDS) model. In this work, we investigated the pain and anxiety behavioral phenotypes of wild-type and TLR4-deficient juvenile mice subjected to repeated SDS and evaluated the engagement of TLR4 by measuring dimerization in the spinal cord, dorsal root ganglia, and prefrontal cortex. Male juvenile (4-week-old) mice (C57BL6/J or Tlr4-/-) underwent six social defeat sessions with adult CD1 aggressors. In WT mice SDS promotes chronic mechanical allodynia by von Frey testing and thermal hyperalgesia by Hargreaves test. In parallel, the stressed WT mice exhibited transient anxiety-like behavior and long-lasting locomotor activity reduction in the open-field test. Tlr4-/- stressed animals were resistant to the induction of pain-like behavior but had a remnant of anxious behavior, spending less time in the center of the arena. In WT SDS, there were concordant robust increases of TLR4 dimerization in dorsal root ganglia macrophages and spinal cord microglia, indicating activation. These results suggest that the chronic pain phenotype and locomotor impairment induced by SDS in juvenile mice depends on TLR4 engagement evidenced by dimerization in immune cells of the dorsal root ganglia and spinal cord.

Inflammation is a vital process for the injured tissue restoration and one of its hallmarks is inflammatory hyperalgesia. The cyclooxygenase (COX) pathway is strongly related to the inflammatory and painful process. Usually, the COX-1 isoform is described as homeostatic, while COX-2 is characterized as inducible in inflammatory conditions. Although it is well known that neutrophil cells are the first to arrive at the inflamed site and the major source of COX-2 is still unknown, the specific role of neutrophil-derived COX-2 in the pain process is. Thus, in the present study, we demonstrate for the first time that neutrophil-derived COX-2 plays a key role in peripheral inflammatory hyperalgesia. Conditional knockout mice for COX-2 in neutrophils (COX-2 ^{fl/fl: Mrp8cre±}) exhibited higher pain sensitivity after carrageenan (CG) injection and long-lasting IL-1β-induced hyperalgesia compared with the control group (COX-2 ^fl/fl). Also, CG-induced inflammation in COX-2 ^{fl/fl: Mrp8cre±} mice showed COX-1 overexpression, and increased neutrophil migration and pro-inflammatory cytokines (e.g., IL-1β and CXCL1). These findings revealed that neutrophil COX-2 has an important role in the regulation of inflammatory hyperalgesia.

Background and Purpose: While dipyrone is a widely used analgesic, its mechanism of action is not completely understood. Recently, we have reported that the dipyrone metabolite 4‐aminoantipyrine (4‐AA) reduces PGE₂‐induced pain‐related behaviour through cannabinoid CB₁ receptors. Here, we ascertained, in naive and PGE₂‐induced “inflamed” conditions, both in vivo and in vitro, the molecular mechanisms involved in the 4‐AA‐induced analgesic effects.Experimental Approach: The effect of local administration of 4‐AA (160 μg per paw) on capsaicin (0.12 μg per paw) injection‐induced pain‐related behaviour and 4‐AA's effect on 500‐nM capsaicin‐induced changes in intracellular calcium concentration ([Ca²⁺]_i) in cultured primary sensory neurons were assessed in vivo and in vitro, respectively.Key Results: 4‐AA reduced capsaicin‐induced nociceptive behaviour in naive and inflamed conditions through CB₁ receptors. 4‐AA (100 μM) reduced capsaicin‐induced increase in [Ca²⁺]_i in a CB₁ receptor‐dependent manner, when PGE₂ was not present. Following PGE₂ application, 4‐AA (1–50 μM) increased the [Ca²⁺]_i. Although 4‐AA activated both TRPV1 and TRPA1 channels, increased [Ca²⁺]_i was mediated through TRPV1 channels. Activation of TRPV1 channels resulted in their desensitisation. Blocking CB₁ receptors reduced both the excitatory and desensitising effects of 4‐AA.Conclusion and Implications: CB₁ receptor‐mediated inhibition of TRPV1 channels and TRPV1‐mediated Ca²⁺‐influx‐ and CB₁ receptor‐dependent desensitisation of TRPV1 channels contribute to the anti‐nociceptive effect of 4‐AA in naive and inflamed conditions respectively. Agonists active at both CB₁ receptors and TRPV1 channels might be useful as analgesics, particularly in inflammatory conditions.