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Background Mutations in gene encoding the Na,K-ATPase α isoform are associated with familial hemiplegic migraine type 2. Migraine with aura is a known risk factor for heart disease. The Na,K-ATPase is important for cardiac function, but its role for heart disease remains unknown. We hypothesized that is a susceptibility gene for heart disease and aimed to assess the underlying disease mechanism. Methods and Results Mice heterozygous for the familial hemiplegic migraine type 2-associated G301R mutation in the gene (α mice) and matching wild-type controls were compared. Reduced expression of the Na,K-ATPase α isoform and increased expression of the α isoform were observed in hearts from α mice (Western blot). Left ventricular dilation and reduced ejection fraction were shown in hearts from 8-month-old α mice (cardiac magnetic resonance imaging), and this was associated with reduced nocturnal blood pressure (radiotelemetry). Cardiac function and blood pressure of 3-month-old α mice were similar to wild-type mice. Amplified Na,K-ATPase-dependent Src kinase/Ras/Erk1/2 (p44/42 mitogen-activated protein kinase) signaling was observed in hearts from 8-month-old α mice, and this was associated with mitochondrial uncoupling (respirometry), increased oxidative stress (malondialdehyde measurements), and a heart failure-associated metabolic shift (hyperpolarized magnetic resonance). Mitochondrial membrane potential (5,5´,6,6´-tetrachloro-1,1´,3,3´-tetraethylbenzimidazolocarbocyanine iodide dye assay) and mitochondrial ultrastructure (transmission electron microscopy) were similar between the groups. Proteomics of heart tissue further suggested amplified Src/Ras/Erk1/2 signaling and increased oxidative stress and provided the molecular basis for systolic dysfunction in 8-month-old α mice. Conclusions Our findings suggest that mutation leads to disturbed cardiac metabolism and reduced cardiac function mediated via Na,K-ATPase-dependent reactive oxygen species signaling through the Src/Ras/Erk1/2 pathway.
Learn More >Originally characterized as an oncoprotein overexpressed in many forms of cancer that participates in numerous cellular pathways, DEK has since been well described regarding the regulation of epigenetic markers and transcription factors in neurons. However, its role in neuropathic allodynia processes remain elusive and intriguingly complex. Here, we show that DEK, which is induced in spinal dorsal horn neurons after spinal nerve ligation (SNL), is regulated by miR-489-3p. Moreover, SNL-induced decrease in miR-489-3p expression increased the expression of DEK, which recruited TET1 to the promoter fragments of the Bdnf, Grm5, and Stat3 genes, thereby enhancing their transcription in the dorsal horn. Remarkably, these effects were also induced by intrathecally administering naïve animals with miR-489-3p inhibitor, which could be inhibited by knockdown of TET1 siRNA or DEK siRNA. Conversely, delivery of intrathecal miR-489-3p-mimic into SNL rats attenuated allodynia behavior and reversed protein expression coupled to the promoter segments in the dorsal horn. Thus, a spinal miR-489-3p/DEK/TET1 transcriptional axis may contribute to neuropathic allodynia. These results may provide a new target for treating neuropathic allodynia.
Learn More >Chronic inflammatory pain represents one of the largest subsets of chronic pain diagnoses, which affect nearly a quarter of individuals in the United States and cost nearly $600 billion dollars annually. Chronic pain leads to persistent sensory hypersensitivities, as well as emotional and cognitive disturbances. Evidence suggests that melanocortin 4 receptors (MC4Rs) mediate pain-signaling and pain-like behaviors via actions at various nodes in the pain-neural axis, but the field lacks a complete understanding of the potential role of MC4Rs in chronic inflammatory pain in males and females. The central amygdala (CeA) expresses high quantities of MC4R and receives pain-related information from the periphery, and in vivo CeA manipulations alter nociceptive behavior in pain-naïve and in animals with chronic pain. Here, we tested the hypothesis that MC4Rs in the CeA modulate thermal nociception and mechanical sensitivity, as well as pain avoidance, in male and female Wistar rats, using a model of chronic inflammatory pain (Complete Freud's Adjuvant; CFA). First, we report that CFA produces long-lasting hyperalgesia in adult male and female Wistar rats, and long-lasting pain avoidance in male Wistar rats. Second, we report that MC4R antagonism in the CeA reduces thermal nociception and mechanical sensitivity in male and female Wistar rats treated with CFA. Finally, we report that MC4R antagonism in the CeA reduces pain avoidance in male, and that this effect is not due to drug effects on locomotor activity. Our results indicate that a model of chronic inflammatory pain produces long-lasting increases in pain-like behaviors in adult male and female Wistar rats, and that antagonism of MC4Rs in the CeA reverses those effects.
Learn More >Paclitaxel-induced downregulation of two-pore domain K+ channel 1.1 (K1.1) caused by increasing DNA methylation within its gene promoter in the dorsal root ganglion (DRG) contributes to neuropathic pain. Given that ten-eleven translocation methylcytosine dioxygenase 1 (TET1) promotes DNA demethylation and gene transcription, the present study investigated whether DRG overexpression of TET1 produces an antinociceptive effect on the paclitaxel-induced nociceptive hypersensitivity.
Learn More >Certain constituents in migraine food triggers and non-steroidal anti-inflammatory drugs (NSAIDs) inhibit sulfotransferases (SULTs) that detoxify drugs/chemicals and play role in the metabolism of neurotransmitters. We aimed to dissect SULT1A1 modulation of CSD susceptibility and behavior in an in vivo experimental model using hesperidin, a SULT1A1 inhibitor found in citrus fruits (known migraine triggers) and mefenamic acid (SULT1A1 inhibitor), an NSAID to simulate medication overuse.
Learn More >Chronic pain development is a frequent outcome of severe stressor exposure, with or without tissue injury. Enduring stress-induced hyperalgesia (ESIH) is believed to play a central role, but the precise mechanisms mediating the development of chronic posttraumatic pain, and the time-dependency of these mechanisms, remain poorly understood. Clinical and pre-clinical data suggest that the inhibition of FK506-binding protein 51 (FKBP51), a key stress system regulator, might prevent ESIH. We evaluated whether peritraumatic inhibition of FKBP51 in an animal model of traumatic stress exposure, the single prolonged stress (SPS) model, reversed ESIH evaluated via daily mechanical von Frey testing. FKBP51 inhibition was achieved using SAFit2, a potent and specific small molecule inhibitor of FKBP51, administered to male and female Sprague-Dawley rats via intraperitoneal injection. To assess timing effects, FKBP51 was administered at different times relative to stress (SPS) exposure. SAFit2 administration immediately after SPS produced a complete reversal in ESIH lasting >7d. In contrast, SAFit2 administration 72h following SPS produced only temporary hyperalgesia reversal, and administration 120h following SPS had no effect. Similarly, animals undergoing SPS together with tissue injury (plantar incision) receiving SAFit2 immediately post-surgery developed acute hyperalgesia but recovered by 4d and did not develop ESIH. These data suggest that (1) FKBP51 plays an important, time-dependent role in ESIH pathogenesis, (2) time windows of opportunity may exist to prevent ESIH via FKBP51 inhibition after traumatic stress, with or without tissue injury, and (3) the use of inhibitors of specific pathways may provide new insights into chronic post-traumatic pain development.
Learn More >Neuropathic pain in rodents can be driven by ectopic spontaneous activity (SA) generated by sensory neurons in dorsal root ganglia (DRG). The recent demonstration that SA in dissociated human DRG neurons is associated with reported neuropathic pain in patients enables detailed comparison of pain-linked electrophysiological alterations driving SA in human DRG neurons to alterations that distinguish SA in nociceptors from SA in low-threshold mechanoreceptors (LTMRs) in rodent neuropathy models. Analysis of recordings from dissociated somata of patient-derived DRG neurons showed that SA and corresponding pain in both sexes were significantly associated with the three functional electrophysiological alterations sufficient to generate SA in the absence of extrinsic depolarizing inputs. These include enhancement of depolarizing spontaneous fluctuations of membrane potential (DSFs), which were analyzed quantitatively for the first time in human DRG neurons. The functional alterations were indistinguishable from SA-driving alterations reported for nociceptors in rodent chronic pain models. Irregular, low-frequency DSFs in human DRG neurons closely resemble DSFs described in rodent nociceptors while differing substantially from the high-frequency sinusoidal oscillations described in rodent LTMRs. These findings suggest that conserved physiological mechanisms of SA in human nociceptor somata can drive neuropathic pain despite documented cellular differences between human and rodent DRG neurons. Perspective: Electrophysiological alterations in human sensory neurons associated with patient-reported neuropathic pain include all three of the functional alterations that logically can promote spontaneous activity. The similarity of distinctively altered spontaneous depolarizations in human DRG neurons and rodent nociceptors suggests that spontaneously active human nociceptors can persistently promote neuropathic pain in patients.
Learn More >Fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) are promising targets for neuropathic pain and other CNS disorders. Based on our previous lead compound SIH 3, we designed and synthesized a series of 4-methylsulfonylphenyl semicarbazones and evaluated for FAAH and MAGL inhibition properties. Most of the compounds showed potency towards both enzymes with leading FAAH selectivity. Compound (Z)-2-(2,6-dichlorobenzylidene)-N-(4-(methylsulfonyl)phenyl)hydrazine-1-carboxamide emerged as the lead inhibitor against both FAAH (IC = 11 nM) and MAGL (IC = 36 nM). The lead inhibitor inhibited FAAH by non-competitive mode, but showed a mixed-type inhibition against MAGL. Molecular docking study unveiled that the docked ligands bind favorably to the active sites of FAAH and MAGL. The lead inhibitor interacted with FAAH and MAGL via π-π stacking via phenyl ring and hydrogen bonding through sulfonyl oxygen atoms or amide NH. Moreover, the stability of docked complexes was rationalized by molecular simulation studies. PAMPA assay revealed that the lead compound is suitable for blood-brain penetration. The lead compound showed better cell viability in lipopolysaccharide-induced neurotoxicity assay in SH-SY5Y cell lines. Further, in-vivo experiments unveiled that dual inhibitor was safe up to 2000 mg/kg with no hepatotoxicity. The dual FAAH-MAGL inhibitor produced significant anti-nociceptive effect in the CCI model of neuropathic pain without altering locomotion activity. Lastly, the lead compound exhibited promising ex-vivo FAAH/MAGL inhibition activity at the dose of 10 mg/kg and 20 mg/kg. Thus, these findings suggest that the semicarbazone-based lead compound can be a potential template for the development of agents for neuropathic pain.
Learn More >The mechanisms underlying the transition from acute to chronic pain are unclear but may involve the persistence or strengthening of pain memories acquired in part through associative learning. Contextual cues, which comprise the environment in which events occur, were recently described as a critical regulator of pain memory; both male rodents and humans exhibit increased pain sensitivity in environments recently associated with a single painful experience. It is unknown, however, how repeated exposure to an acute painful unconditioned stimulus in a distinct context modifies pain sensitivity or the expectation of pain in that environment. To answer this question, we conditioned mice to associate distinct contexts with either repeated administration of a mild visceral pain stimulus (intraperitoneal injection of acetic acid) or vehicle injection over the course of three days. On the final day of experiments animals received either an acid injection or vehicle injection prior to being placed into both contexts. In this way, contextual control of pain sensitivity and pain expectation could be tested respectively. When re-exposed to the noxious stimulus in a familiar environment, both male and female mice exhibited context-dependent conditioned analgesia, a phenomenon mediated by endogenous opioid signaling. However, when expecting the presentation of a painful stimulus in a given context, males exhibited conditioned hypersensitivity whereas females exhibited endogenous opioid-mediated conditioned analgesia. These results are evidence that pain perception and engagement of endogenous opioid systems can be modified through their psychological association with environmental cues. Successful determination of the brain circuits involved in this sexually dimorphic anticipatory response may allow for the manipulation of pain memories, which may contribute to the development of chronic pain states.
Learn More >Ca2.3 channels are subthreshold voltage-gated calcium channels that play crucial roles in neurotransmitter release and regulation of membrane excitability, yet modulation of these channels with endogenous molecules and their role in pain processing is not well studied. Here, we hypothesized that an endogenous amino acid l-cysteine could be a modulator of these channels and may affect pain processing in mice. To test this hypothesis, we employed conventional patch-clamp technique in the whole-cell configuration using recombinant Ca2.3 subunit stably expressed in human embryonic kidney (HEK-293) cells. We found in our in vitro experiments that l-cysteine facilitated gating and increased the amplitudes of recombinant Ca2.3 currents likely by chelating trace metals that tonically inhibit the channel. In addition, we took advantage of mouse genetics in vivo using the acetic acid visceral pain model that was performed on wildtype and homozygous Cacna1e knockout male littermates. In ensuing in vivo experiments, we found that l-cysteine administered both subcutaneously and intraperitoneally evoked more prominent pain responses in the wildtype mice, while the effect was completely abolished in knockout mice. Conversely, intrathecal administration of l-cysteine lowered visceral pain response in the wildtype mice, and again the effect was completely abolished in the knockout mice. Our study strongly suggests that l-cysteine-mediated modulation of Ca2.3 channels plays an important role in visceral pain processing. Furthermore, our data are consistent with the contrasting roles of Ca2.3 channels in mediating visceral nociception in the peripheral and central pain pathways.
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