Peripheral gating of mechanosensation by glial diazepam binding inhibitor.

We report that diazepam binding inhibitor (DBI) is a glial messenger mediating satellite glia-sensory neuron crosstalk in the dorsal root ganglion (DRG). DBI is highly expressed in satellite glia cells (SGCs) of mice, rat and human, but not in sensory neurons or most other DRG-resident cells. Knockdown of DBI results in a robust mechanical hypersensitivity without major effects on other sensory modalities. In vivo overexpression of DBI in SGCs reduces sensitivity to mechanical stimulation and alleviates mechanical allodynia in neuropathic and inflammatory pain models. We further show that DBI acts as an unconventional agonist and positive allosteric modulator at the neuronal GABAA receptors, particularly strongly effecting those with a high-affinity benzodiazepine binding site. Such receptors are selectively expressed by a subpopulation of mechanosensitive DRG neurons and these are also more enwrapped with DBI-expressing glia, as compared to other DRG neurons, suggesting a mechanism for specific effect of DBI on mechanosensation. These findings identified a new, peripheral neuron-glia communication mechanism modulating pain signalling, which can be targeted therapeutically.

Peripheral gating of pain by glial endozepine.

We report that diazepam binding inhibitor (DBI) is a glial messenger mediating satellite glia-sensory neuron crosstalk in the dorsal root ganglion (DRG). DBI is highly and specifically expressed in satellite glia cells (SGCs) of mice, rat and human, but not in sensory neurons or other DRG-resident cells. Knockdown of DBI results in a robust mechanical hypersensitivity without significant effects on other sensory modalities. In vivo overexpression of DBI in SGCs reduces sensitivity to mechanical stimulation and alleviates mechanical allodynia in neuropathic and inflammatory pain models. We further show that DBI acts as a partial agonist and positive allosteric modulator at the neuronal GABAA receptors, particularly strongly effecting those with a high-affinity benzodiazepine binding site. Such receptors are selectively expressed by a subpopulation of mechanosensitive DRG neurons and these are also more enwrapped with DBI-expressing glia, as compared to other DRG neurons, suggesting a mechanism for specific effect of DBI on mechanosensation. These findings identified a new, peripheral neuron-glia communication mechanism modulating pain signalling, which can be targeted therapeutically.

The Antinociceptive Effect of Sympathetic Block is Mediated by Transforming Growth Factor ß in a Mouse Model of Radiculopathy.

Although sympathetic blockade is clinically used to treat pain, the underlying mechanisms remain unclear. We developed a localized microsympathectomy (mSYMPX), by cutting the grey rami entering the spinal nerves near the rodent lumbar dorsal root ganglia (DRG). In a chemotherapy-induced peripheral neuropathy model, mSYMPX attenuated pain behaviors via DRG macrophages and the anti-inflammatory actions of transforming growth factor-ß (TGF-ß) and its receptor TGF-ßR1. Here, we examined the role of TGF-ß in sympathetic-mediated radiculopathy produced by local inflammation of the DRG (LID). Mice showed mechanical hypersensitivity and transcriptional and protein upregulation of TGF-ß1 and TGF-ßR1 three days after LID. Microsympathectomy prevented mechanical hypersensitivity and further upregulated Tgfb1 and Tgfbr1. Intrathecal delivery of TGF-ß1 rapidly relieved the LID-induced mechanical hypersensitivity, and TGF-ßR1 antagonists rapidly unmasked the mechanical hypersensitivity after LID+mSYMPX. In situ hybridization showed that Tgfb1 was largely expressed in DRG macrophages, and Tgfbr1 in neurons. We suggest that TGF-ß signaling is a general underlying mechanism of local sympathetic blockade.

Critical Pronociceptive Role of Family 2 Voltage-Gated Calcium Channels in a Novel Mouse Model of HIV-Associated Sensory Neuropathy.

Some people living with HIV present painful sensory neuropathy (HIV-SN) that is pharmacoresistant, sex-associated, and a major source of morbidity. Since the specific mechanisms underlying HIV-SN are not well understood, the aim of our study was to characterize a novel model of painful HIV-SN by combining the HIV-1 gp120 protein and the antiretroviral stavudine (d4T) in mice and to investigate the pronociceptive role of the family 2 voltage-gated calcium channel (VGCC) α1 subunit (Cav2.X channels) in such a model. HIV-SN was induced in male and female C57BL/6 mice by administration of gp120 and/or d4T and detected by a battery of behavior tests and by immunohistochemistry. The role of Cav2.X channels was assessed by the treatment with selective blockers and agonists as well as by mRNA detection. Repeated administration with gp120 and/or d4T produced long-lasting touch-evoked painful-like behaviors (starting at 6 days, reaching a maximum on day 13, and lasting up to 28 days after treatment started), with a greater intensity in female mice treated with the combination of gp120 + d4T. Moreover, gp120 + d4T treatment reduced the intraepidermal nerve fibers and well-being of female mice, without altering other behaviors. Mechanistically, gp120 + d4T treatment induced Cav2.1, 2.2, and 2.3 transcriptional increases in the dorsal root ganglion and the Cav2.X agonist-induced nociception. Accordingly, intrathecal selective Cav2.2 blockade presented longer and better efficacy in reversing the hyperalgesia induced by gp120 + d4T treatment compared with Cav2.1 or Cav2.3, but also presented the worst safety (inducing side effects at effective doses). We conclude that the family 2 calcium channels (Cav2.X) exert a critical pronociceptive role in a novel mouse model of HIV-SN.

Transient Receptor Potential Channel 4 Small-Molecule Inhibition Alleviates Migraine-Like Behavior in Mice.

Migraine is a common neurological disorder with few available treatment options. Recently, we have demonstrated the role of transient receptor potential cation channel subfamily C member 4 (TRPC4) in itch and the modulation of the calcitonin gene-related peptide (CGRP), a biomarker and emerging therapeutic target for migraine. In this study, we characterized the role of TRPC4 in pain and evaluated its inhibition as anti-migraine pain therapy in preclinical mouse models. First, we found that TRPC4 is highly expressed in trigeminal ganglia and its activation not only mediates itch but also pain. Second, we demonstrated that the small-molecule inhibitor ML204, a specific TRPC4 antagonist, significantly reduced episodic and chronic migraine-like behaviors in male and female mice after injection of nitroglycerin (NTG), a well-known migraine inducer in rodents and humans. Third, we found a significant decrease in CGRP protein levels in the plasma of both male and female mice treated with ML-204, which largely prevented the development of chronic migraine-like behavior. Using sensory neuron cultures, we confirmed that activation of TRPC4 elicited release of CGRP, which was significantly diminished by ML-204. Collectively, our findings identify TRPC4 in peripheral sensory neurons as a mediator of CGRP release and NTG-evoked migraine. Since a TRPC4 antagonist is already in clinical trials, we expect that this study will rapidly lead to novel and effective clinical treatments for migraineurs.

Sex-dependent Cav2.3 channel contribution to the secondary hyperalgesia in a mice model of central sensitization.

Central sensitization (CS) is characteristic of difficult to treat painful conditions, such as fibromyalgia and neuropathies and have sexual dimorphism involved. The calcium influx in nociceptive neurons is a key trigger for CS and the role of Cav2.1 and Cav2.2 voltage gated calcium channels (VGCC) in this role were evidenced with the use of ω-agatoxin IVA and ω-agatoxin MVIIA blockers, respectively. However, the participation of the α1 subunit of the voltage-gated channel Cav2.3, which conducts R-type currents, in CS is unknown. Furthermore, the role of sexual differences in painful conditions is still poorly understood. Thus, we investigated the role of Cav2.3 in capsaicin-induced secondary hyperalgesia in mice, which serve as a CS model predictive of the efficacy of novel analgesic drugs. Capsaicin injection in C57BL/6 mice caused secondary hyperalgesia from one to five hours after injection, and the effects were similar in male and female mice. In female but not male mice, intrathecal treatment with the Cav2.3 inhibitor SNX-482 partially and briefly reversed secondary hyperalgesia at a dose (300 pmol/site) that did not cause adverse effects. Moreover, Cav2.3 expression in the dorsal root ganglia (DRG) and spinal cord was reduced by intrathecal treatment with an antisense oligonucleotide (ASO) targeting Cav2.3 in female and male mice. However, ASO treatment was able to provide a robust and durable prevention of secondary hyperalgesia caused by capsaicin in female mice, but not in male mice. Thus, our results demonstrate that Cav2.3 inhibition, especially in female mice, has a relevant impact on a model of CS. Our results provide a proof of concept for Cav2.3 as a molecular target. In addition, the result associated to the role of differences in painful conditions linked to sex opens a range of possibilities to be explored and needs more attention. Thus, the relevance of testing Cav2.3 inhibition or knockdown in clinically relevant pain models is needed.

Experimental Sepsis Severity Score Associated to Mortality and Bacterial Spreading is Related to Bacterial Load and Inflammatory Profile of Different Tissues.

Pneumonia-induced sepsis is responsible for about 50% of cases in the world. Patients who develop severe sepsis and septic shock present organ dysfunction and elevated plasma cytokine levels, which may lead to death. Clinical scores are important to evaluate the framework of septic patients and are used to predict the syndrome progress, prognostics, and mortality. The objective of the present study was to verify the applicability of a murine clinical score system to experimental sepsis (pneumonia-induced sepsis in male mice) and to correlate it with mortality and bacterial dissemination in different organs. Results demonstrated that animals which present higher clinical scores (>3) are more likely to die. Animals presenting high clinical scores exhibited transient bacteremia and displayed bacterial spreading to different organs such as heart, kidney, liver, and brain. There is a correlation between clinical score and bacterial dissemination and consequently greater risk of death. In addition, animals which showed bacterial dissemination in more than three organs and high clinical scores presented high levels of cytokines (TNF-α, MCP-1, IL-6, and IL-10) in plasma, lung, heart, liver, kidney, and brain. Therefore, our study suggests that (1) severity scores have predictive power in experimental models of sepsis and (2) high concentrations of tissue cytokines may contribute to localized inflammation and be one of the factors responsible for the systemic inflammatory syndrome of sepsis.

Inhibitory effect of GB-2a (I3-naringenin-II8-eriodictyol) on melanogenesis.

ETHNOPHARMACOLOGY RELEVANCE: GB-2a is a I3-naringenin-II8-eriodictyol compound isolated from Garcinia gardneriana (Planchon & Triana) Zappi, a plant used in folk medicine for the treatment of skin disorders. AIM OF STUDY: In the search for new depigmenting agents, this study was carried out to investigate the in vitro effects of GB-2a isolated from G. gardneriana (Planchon & Triana) Zappi in B16F10 melanoma cells. MATERIALS AND METHODS: The effects of GB-2a were evaluated through determination of melanin biosynthesis in B16F10 melanoma cells in comparison with the reference drug kojic acid (500µM). In parallel, the GB-2a effect was assessed in a cell viability assay. Mushroom tyrosinase activity assays were conducted to verify the effect of this enzyme. In order to ascertain the nature of enzyme inhibition on tyrosinase, kinetics analysis of the GB-2a was performed with L-tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) substrates. RESULTS: The results showed that GB-2a biflavonoid significantly inhibited the melanin content, without reducing cell viability. GB-2a also showed a strong antityrosinase activity in the mushroom tyrosinase assay. GB-2a inhibited the tyrosinase activity, exerting a mixed inhibition. For the L-tyrosine substrate the inhibition was in non-competitive mode and for L-DOPA it was in uncompetitive mode. CONCLUSION: GB-2a biflavonoid promoted inhibition on tyrosinase activity and reduced melanin biosynthesis in B16F10 cells, which suggests great potential for medical and cosmetic uses as a depigmenting agent.