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Human carbonic anhydrase (CA; EC 4.2.1.1) isoforms II and VII are implicated in neuronal excitation, seizures and neuropathic pain (NP). Their selective inhibition over off-target CAs is expected to produce an anti-NP action devoid of side effects due to promiscuous CA modulation. Here a drug-design strategy based on the observation of (dis)similarities between the target CA active sites was planned with benzenesulfonamide derivatives and, for the first time, a phosphorus-based linker. Potent and selective CA II-VII inhibitors were identified among the synthesized phenyl(thio)phosphon(amid)ates 3-22. X-ray crystallography depicted the binding mode of phosphonic acid 3 to both CA II and VII. The most promising derivatives, after evaluation of their stability in acidic media, were tested in a mouse model of oxaliplatin-induced neuropathy. The most potent compound racemic mixture was subjected to HPLC enantioseparation and the identification of the eutomer, the (S)-enantiomer, allowed to halve the dose totally relieving allodynia in mice.
Learn More >Prior studies have shown that patients suffering from chronic Low Back Pain (cLBP) have impaired somatosensory processing including reduced tactile acuity, i.e. reduced ability to resolve fine spatial details with the perception of touch. The central mechanism(s) underlying reduced tactile acuity are unknown but may include changes in specific brain circuitries (e.g. neuroplasticity in primary somatosensory cortex, S1). Furthermore, little is known about the linkage between changes in tactile acuity and the amelioration of cLBP by somatically-directed therapeutic interventions, such as acupuncture. In this longitudinal neuroimaging study, we evaluated healthy control adults (HC, N=50) and a large sample of cLBP patients (N=102) with structural brain imaging (T1-weighted MRI for Voxel Based Morphometry, VBM; Diffusion Tensor Imaging, DTI) and tactile acuity testing using two-point discrimination threshold (2PDT) over the lower back (site of pain) and finger (control) locations. Patients were evaluated at baseline and following a 4-week course of acupuncture, with patients randomized to either verum acupuncture, two different forms of sham acupuncture (designed with or without somatosensory afference), or no-intervention usual care control. At baseline, cLBP patients demonstrated reduced acuity (greater 2PDT, P=0.01) over the low back, but not finger (P=0.29) locations compared to HC, suggesting that chronic pain affects tactile acuity specifically at body regions encoding the experience of clinical pain. At baseline, Gray Matter Volume (GMV) was elevated and Fractional Anisotropy (FA) was reduced, respectively, in the S1-back region of cLBP patients compared to controls (P<0.05). GMV in cLBP correlated with greater 2PDT-back scores (ρ=0.27, P=0.02). Following verum acupuncture, tactile acuity over the back was improved (reduced 2PDT) and greater improvements were associated with reduced S1-back GMV (ρ=0.52, P=0.03) and increased S1-back adjacent white matter FA (ρ=-0.56, P=0.01). These associations were not seen for non-verum control interventions. Thus, S1 neuroplasticity in cLBP is linked with deficits in tactile acuity and, following acupuncture therapy, may represent early mechanistic changes in somatosensory processing that track with improved tactile acuity.
Learn More >Single cell sequencing has provided unprecedented information about the transcriptomic diversity of somatosensory systems. Here we describe a simple and versatile in situ hybridization (ISH) based approach for mapping this information back to the tissue. We illustrate the power of this approach by demonstrating that ISH localization with just eight probes is sufficient to distinguish all major classes of neurons in sections of the trigeminal ganglion. To further simplify the approach and make transcriptomic class assignment and cell segmentation automatic we developed a machine learning approach for analyzing images from multiprobe ISH experiments. We demonstrate the power of in situ class assignment by examining the expression patterns of voltage gated sodium channels that play roles in distinct somatosensory processes and pain. Specifically, this analysis resolves intrinsic problems with single cell sequencing related to the sparseness of data leading to ambiguity about gene expression patterns. We also used the multiplex in situ approach to study the projection fields of the different neuronal classes. Our results demonstrate that the surface of the eye and meninges are targeted by broad arrays of neural classes despite their very different sensory properties but exhibit idiotypic patterns of innervation at a quantitative level. Very surprisingly, itch related neurons extensively innervated the meninges, indicating that these transcriptomic cell classes are not simply labeled lines for triggering itch. Together, these results substantiate the importance of a sensory neuron's peripheral and central connections as well as its transcriptomic class in determining its role in sensation.
Learn More >Recent studies have drawn the attention to the link between Alcohol Use Disorder (AUD) and the presence of pain. Indeed, the correct management of pain in patients with a previous history of AUD has been reported to decrease the risk of relapse in alcohol drinking, suggesting that in this prone population, pain may increase the vulnerability to relapse. Previous data in male rats revealed that inflammatory pain desensitizes mu opioid receptors (MORs) in the ventral tegmental area (VTA) and increases intake of high doses of heroine. Due to the relevant role of MORs in alcohol effects, we hypothesize that pain may also alter alcohol reinforcing properties and therefore affect alcohol relapse in male rats. Our microdialysis studies show that the presence of inflammatory pain blunted the increase of extracellular dopamine levels in the Nucleus Accumbens induced by 1.5g/kg of ethanol (s.c.). Moreover, we also revealed that the administration of 52 nmol of ethanol into the VTA failed to induce place preference only in inflammatory pain-suffering animals, and a higher dose (70nmol) was necessary to reverse this effect. Finally, we evaluated the effect of inflammatory pain on the alcohol deprivation effect (ADE) in long-term ethanol-experienced male rats. After four cycles of free ethanol intake and abstinence periods, inflammatory pain induced ADE without affecting its magnitude. These intriguing data reveals the impact of pain on neurochemical and behavioral effects following alcohol administration but also underscore the necessity of finding an appropriate paradigm to determine the long-term behavioral consequences.
Learn More >Pharmacological tools for chronic visceral pain management are still limited and inadequate. A3 adenosine receptor (A3AR) agonists are effective in different models of persistent pain. Recently their activity has been related to the block of N-type voltage-gated Ca channels (Cav2.2) in dorsal root ganglia (DRG) neurons. The present work aimed to evaluate the efficacy of A3AR agonists in reducing post-inflammatory visceral hypersensitivity in both male and female rats. Colitis was induced by the intra-colonic instillation of 2,4-dinitrobenzenesulfonic acid (DNBS; 30 mg in 0.25 ml 50% EtOH). Visceral hypersensitivity was assessed by measuring the viscero-motor response and the abdominal withdrawal reflex to colorectal distension. The effects of A3AR agonists (MRS5980 and Cl-IB-MECA) were evaluated over time after DNBS injection and compared to that of the selective Cav2.2 blocker PD173212, and the clinically used drug linaclotide. A3AR agonists significantly reduced DNBS-evoked visceral pain both in the post-inflammatory (14 and 21 days after DNBS injection) and persistence (28 and 35 days after DNBS) phases. Efficacy was comparable to effects induced by linaclotide. PD173212 fully reduced abdominal hypersensitivity to control values, highlighting the role of Cav2.2. The effects of MRS5980 and Cl-IB-MECA were completely abolished by the selective A3AR antagonist MRS1523. Furthermore, patch-clamp recordings showed that A3AR agonists inhibited Cav2.2 in DRG neurons isolated from either control or DNBS-treated rats. The effect on Ca current was PD173212-sensitive and prevented by MRS1523. A3AR agonists are effective in relieving visceral hypersensitivity induced by DNBS, suggesting a potential therapeutic role against abdominal pain.
Learn More >Rheumatoid arthritis-associated pain is poorly managed, often persisting when joint inflammation is pharmacologically controlled. Comparably, in the mouse K/BxN serum-transfer model of inflammatory arthritis, hind-paw nociceptive hypersensitivity occurs with ankle joint swelling (5 days post-immunisation) persisting after swelling has resolved (25 days post-immunisation). In this study, lipid mediator profiling of lumbar dorsal root ganglia (DRG), the site of sensory neuron cell bodies innervating the ankle joints, 5 days and 25 days after serum transfer demonstrated a shift in specialised pro-resolving lipid mediator (SPM) profiles. Persistent nociception without joint swelling was associated with low concentrations of the SPM Maresin-1 (MaR1) and high macrophage numbers in DRG. MaR1 application to cultured DRG neurons inhibited both capsaicin-induced increase of intracellular calcium ions and release of calcitonin gene-related peptide (CGRP) in a dose-dependent manner. Furthermore, in peritoneal macrophages challenged with lipopolysaccharide, MaR1 reduced pro-inflammatory cytokine expression. Systemic MaR1 administration caused sustained reversal of nociceptive hypersensitivity and reduced inflammatory macrophage numbers in DRG. Unlike gabapentin, which was used as positive control, systemic MaR1 did not display acute anti-hyperalgesic action. Therefore, these data suggest that MaR1 effects observed following K/BxN serum transfer relate to modulation of macrophage recruitment, more likely than to direct actions on sensory neurons. Our study highlights that, in DRG, aberrant pro-resolution mechanisms play a key role in arthritis joint pain dissociated from joint swelling, opening novel approaches for RA pain treatment.
Learn More >High-threshold mechanosensitive and mechano-insensitive ("silent") nociceptors have similar electrical thresholds for transcutaneous sine wave stimulation at 4 Hz that selectively activates cutaneous C-nociceptors in human skin. Their fundamentally different functions particularly in chronic pain warrant differential stimulation protocols. We used transcutaneously delivered slow depolarizing stimuli (half-sine, 500 ms duration, 0.01 – 1 mA) in humans to assess intensity-response relations for the induction of pain psycho-physically and recorded activation of mechanosensitive and silent nociceptors in healthy volunteers by microneurography. Differential C-fiber activation was confirmed in single fiber recordings in pig allowing stimulation amplitudes up to 10 mA. Perception and pain thresholds to half-sine wave pulses were 0.06 ± 0.03 mA and 0.18 ± 0.1 mA, respectively, and caused pain in an amplitude-dependent manner (n=24). When matched for pain intensity, only sine wave stimulation induced an instant widespread axon reflex erythema (n=10). In human microneurography, half-sine stimulation activated mechanosensitive nociceptors (n=13), but only one of 11 silent nociceptors. In pig skin, the amplitude-dependent activation of mechanosensitive nociceptors was confirmed (0.2 – 1 mA, n=28) and activation thresholds for most silent nociceptors (n=13) were found above 10 mA. Non-nociceptive low threshold mechanosensitive C-fibers (n=14) displayed lower activation thresholds for half-sine wave stimuli with an amplitude-dependent discharge increase between 0.01 and 0.1 mA. We conclude that transcutaneous electrical stimulation with 500 ms half-sine wave pulses between 0.2 and 1 mA causes amplitude-dependent pain by preferential activation of mechanosensitive C-nociceptors.
Learn More >Over the last 3 years and for the first time in 60 years, life expectancy in the United States has declined across all racial groups primarily because of drug overdoses, alcohol abuse, and suicide. A public health response to the opioid crisis must expand its focus to more broadly include children, adolescents, and young adults while increasing efforts toward preventing new cases of opioid addiction, early identification of individuals with opioid-abuse disorder, and ensuring access to effective opioid addiction treatment, while simultaneously continuing to safely meet the needs of patients experiencing pain.
Learn More >Neuropathic pain is an intractable medical condition with few or no options for effective treatment. Emerging evidence shows a strong structure-function relationship between dendritic spine dysgenesis and the presence of neuropathic pain. Post-mortem tissue analyses can only imply dynamic structural changes associated with injury-induced pain. Here, we profiled the dynamics of dendritic spines over time on the same superficial dorsal horn (lamina II) neurons before and after peripheral nerve injury-induced pain. We employed a two-photon, whole-animal imaging paradigm that permitted repeat imaging of the same dendritic branches of these neurons in C57/Bl6 Thy1-YFP male mice. Our study demonstrates for the first time the ongoing, steady-state changes in dendritic spine dynamics in the dorsal horn associated with peripheral nerve injury and pain. Ultimately, the relationship between altered dendritic spine dynamics and neuropathic pain may serve as a structure-based opportunity to investigate mechanisms of pain following injury and disease.This work is important because it demonstrates for the first time: a)The powerful utility of intravital study of dendritic spine dynamics in the superficial dorsal horn.b)That nerve injury-induced pain triggers changes in dendritic spine steady-state behavior in the spinal cord dorsal horn.c)This work opens the door to further investigations of spinal cord dendritic spine dynamics in the context of injury and disease.
Learn More >The development of chronic pain is a complex mechanism that is still not fully understood. Multiple somatic and visceral afferent pain signals, when experienced over time, cause a strengthening of certain neural circuitry through peripheral and central sensitization, resulting in the physical and emotional perceptual chronic pain experience. The mind-altering qualities of psychedelics have been attributed, through serotonin 2A (5-HT) receptor agonism, to 'reset' areas of functional connectivity (FC) in the brain that play prominent roles in many central neuropathic states. Psychedelic substances have a generally favorable safety profile, especially when compared with opioid analgesics. Clinical evidence to date for their use for chronic pain is limited; however, several studies and reports over the past 50 years have shown potential analgesic benefit in cancer pain, phantom limb pain and cluster headache. While the mechanisms by which the classic psychedelics may provide analgesia are not clear, several possibilities exist given the similarity between 5-HT activation pathways of psychedelics and the nociceptive modulation pathways in humans. Additionally, the alterations in FC seen with psychedelic use suggest a way that these agents could help reverse the changes in neural connections seen in chronic pain states. Given the current state of the opioid epidemic and limited efficacy of non-opioid analgesics, it is time to consider further research on psychedelics as analgesics in order to improve the lives of patients with chronic pain conditions.
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