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Characterization of gene expression in naturally occurring feline degenerative joint disease-associated pain.

Degenerative joint disease (DJD) associated-pain is a clinically relevant and common condition affecting domesticated cats and other species including humans. Identification of the neurobiological signature of pain is well developed in rodent pain models, however such information is lacking from animals or humans with naturally occurring painful conditions. In this study, identification of housekeeping genes (HKG) for neuronal tissue and expression levels of genes considered associated with chronic pain in rodent models were explored in cats with naturally occurring osteoarthritic pain. Fourteen adult cats were evaluated – seven without clinical signs of osteoarthritic pain, and seven with hind limb radiographic DJD and pain. Expression of an investigator-selected set of pain signaling genes (including ASIC3, ATF3, COX2, CX3CL1, NAV1.7, NAV1.8, NAV1.9, NGF, NK1R, TNFα, TRKA) in lumbar spinal cord dorsal horn and lumbar dorsal root ganglia tissues from clinically healthy cats and cats with DJD were studied using quantitative RT-PCR (qPCR). HKG identified as the most stable across all tissue samples were many of the ribosomal protein genes, such as RPL30 and RPS19. qPCR results showed ATF3 and CX3CL1 up-regulated in DJD-affected dorsal root ganglia compared to clinically healthy controls. In spinal cord, CX3CL1 was up-regulated and NGF was down-regulated when DJD-affected samples were compared to healthy samples. Further work is needed to understand the neurobiology of pain in naturally occurring disease and what rodent models are predictive of these changes in more heterogeneous populations such as domestic cats.

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A Bcr-Abl Inhibitor GNF-2 Attenuates Inflammatory Activation of Glia and Chronic Pain.

GNF-2 is an allosteric inhibitor of Bcr-Abl. It was developed as a new class of anti-cancer drug to treat resistant chronic myelogenous leukemia. Recent studies suggest that c-Abl inhibition would provide a neuroprotective effect in animal models of Parkinson's disease as well as in clinical trials. However, the role of c-Abl and effects of GNF-2 in glia-mediated neuroinflammation or pain hypersensitivity has not been investigated. Thus, in the present study, we tested the hypothesis that c-Abl inhibition by GNF-2 may attenuate the inflammatory activation of glia and the ensuing pain behaviors in animal models. Our results show that GNF-2 reduced lipopolysaccharide (LPS)-induced nitric oxide and pro-inflammatory cytokine production in cultured glial cells in a c-Abl-dependent manner. The small interfering ribonucleic acid (siRNA)-mediated knockdown of c-Abl attenuated LPS-induced nuclear factor kappa light chain enhancer of activated B cell (NF-κB) activation and the production of pro-inflammatory mediators in glial cell cultures. Moreover, GNF-2 administration significantly attenuated mechanical and thermal hypersensitivities in experimental models of diabetic and inflammatory pain. Together, our findings suggest the involvement of c-Abl in neuroinflammation and pain pathogenesis and that GNF-2 can be used for the management of chronic pain.

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Meningeal Mast Cells Contribute to ATP-Induced Nociceptive Firing in Trigeminal Nerve Terminals: Direct and Indirect Purinergic Mechanisms Triggering Migraine Pain.

Peripheral mechanisms of primary headaches such as a migraine remain unclear. Meningeal afferents surrounded by multiple mast cells have been suggested as a major source of migraine pain. Extracellular ATP released during migraine attacks is a likely candidate for activating meningeal afferents via neuronal P2X receptors. Recently, we showed that ATP also increased degranulation of resident meningeal mast cells (Nurkhametova et al., 2019). However, the contribution of ATP-induced mast cell degranulation in aggravating the migraine pain remains unknown. Here we explored the role of meningeal mast cells in the pro-nociceptive effects of extracellular ATP. The impact of mast cells on ATP mediated activation of peripheral branches of trigeminal nerves was measured electrophysiologically in the dura mater of adult wild type (WT) or mast cell deficient mice. We found that a spontaneous spiking activity in the meningeal afferents, at baseline level, did not differ in two groups. However, in WT mice, meningeal application of ATP dramatically (24.6-fold) increased nociceptive firing, peaking at frequencies around 10 Hz. In contrast, in mast cell deficient animals, ATP-induced excitation was significantly weaker (3.5-fold). Application of serotonin to meninges in WT induced strong spiking. Moreover, in WT mice, the 5-HT3 antagonist MDL-7222 inhibited not only serotonin but also the ATP induced nociceptive firing. Our data suggest that extracellular ATP activates nociceptive firing in meningeal trigeminal afferents via amplified degranulation of resident mast cells in addition to direct excitatory action on the nerve terminals. This highlights the importance of mast cell degranulation via extracellular ATP, in aggravating the migraine pain.

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Normalizing HDAC2 Levels in the Spinal Cord Alleviates Thermal and Mechanical Hyperalgesia After Peripheral Nerve Injury and Promotes GAD65 and KCC2 Expression.

Neuropathic pain is a worldwide health concern with poor treatment outcomes. Accumulating evidence suggests that histone hypoacetylation is involved in development and maintenance of neuropathic pain. Thus, many natural and synthetic histone deacetylase (HDACs) inhibitors were tested and exhibited a remarkable analgesic effect against neuropathic pain in animals. However, studies evaluating specific subtypes of HDACs contributing to neuropathic pain are limited. In this study, using the chronic constriction injury (CCI) rat model, we found that mRNA and protein levels of HDAC2 were increased in the lumbar spinal cord of rats after sciatic nerve injury. Intrathecal injection of TSA, a pan-HDAC inhibitor, suppressed the increase in HDAC2 protein but not mRNA, and showed a dose-dependent pain-relieving effect. By introducing HDAC2-specific shRNA into the spinal cord via a lentivirus vector, we confirmed that HDAC2 mediates mechanical and thermal hyperalgesia after nerve injury. Further examination found two essential participants in neuropathic pain in the inhibitory circuit of the central nervous system: GAD65 and KCC2 were increased in the spinal cord of CCI rats after HDAC2 knockdown. Thus, our research confirmed that HDAC2 was involved in mechanical and thermal hyperalgesia induced by peripheral nerve injury. Furthermore, GAD65 and KCC2 were the possible downstream targets of HDAC2 in pain modulation pathways.

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IFNγ-Stimulated Dendritic Cell Exosomes for Treatment of Migraine Modeled Using Spreading Depression.

Migraine is a common headache disorder characterized by unilateral, intense headaches. In migraine with aura, the painful headache is preceded by focal neurological symptoms that can be visual, sensory, or motor in nature. Spreading depression (the most likely cause of migraine with aura and perhaps related headache pain) results in increased neuronal excitability and related increases in inflammation and production of reactive oxygen species. This in turn can promote the transformation of low-frequency, episodic migraine into higher-frequency and eventually chronic migraine. Though migraine affects 11% of adults worldwide, with 3% experiencing chronic headache, existing therapies offer only modest benefits. Here, we focus on the mechanisms by which environmental enrichment (i.e., volitionally increased intellectual, social, and physical activity) mitigates spreading depression. In prior work, we have shown that exposure to environmental enrichment reduces susceptibility to spreading depression in rats. This protective effect is at least in part due to environmental enrichment-mediated changes in the character of serum exosomes produced by circulating immune cells. We went on to show that environmental enrichment-mimetic exosomes can be produced by stimulating dendritic cells with low levels of interferon gamma (a cytokine that is phasically increased during environmental enrichment). Interferon gamma-stimulated dendritic cell exosomes (IFNγ-DC-Exos) significantly improve myelination and reduce oxidative stress when applied to hippocampal slice cultures. Here, we propose that they may also be effective against spreading depression. We found that administration of IFNγ-DC-Exos reduced susceptibility to spreading depression and , suggesting that IFNγ-DC-Exos may be a potential therapeutic for migraine.

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Granulocyte-Colony Stimulating Factor-Induced Neutrophil Recruitment Provides Opioid-Mediated Endogenous Anti-nociception in Female Mice With Oral Squamous Cell Carcinoma.

Oral cancer patients report severe function-induced pain; severity is greater in females. We hypothesize that a neutrophil-mediated endogenous analgesic mechanism is responsible for sex differences in nociception secondary to oral squamous cell carcinoma (SCC). Neutrophils isolated from the cancer-induced inflammatory microenvironment contain β-endorphin protein and are identified by the Ly6G immune marker. We previously demonstrated that male mice with carcinogen-induced oral SCC exhibit less nociceptive behavior and a higher concentration of neutrophils in the cancer microenvironment compared to female mice with oral SCC. Oral cancer cells secrete granulocyte colony stimulating factor (G-CSF), a growth factor that recruits neutrophils from bone marrow to the cancer microenvironment. We found that recombinant G-CSF (rG-CSF, 5 μg/mouse, intraperitoneal) significantly increased circulating Ly6G neutrophils in the blood of male and female mice within 24 h of administration. In an oral cancer supernatant mouse model, rG-CSF treatment increased cancer-recruited Ly6G neutrophil infiltration and abolished orofacial nociceptive behavior evoked in response to oral cancer supernatant in both male and female mice. Local naloxone treatment restored the cancer mediator-induced nociceptive behavior. We infer that rG-CSF-induced Ly6G neutrophils drive an endogenous analgesic mechanism. We then evaluated the efficacy of chronic rG-CSF administration to attenuate oral cancer-induced nociception using a tongue xenograft cancer model with the HSC-3 human oral cancer cell line. Saline-treated male mice with HSC-3 tumors exhibited less oral cancer-induced nociceptive behavior and had more β-endorphin protein in the cancer microenvironment than saline-treated female mice with HSC-3 tumors. Chronic rG-CSF treatment (2.5 μg/mouse, every 72 h) increased the HSC-3 recruited Ly6G neutrophils, increased β-endorphin protein content in the tongue and attenuated nociceptive behavior in female mice with HSC-3 tumors. From these data, we conclude that neutrophil-mediated endogenous opioids warrant further investigation as a potential strategy for oral cancer pain treatment.

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Physical Activity Induces Nucleus Accumbens Genes Expression Changes Preventing Chronic Pain Susceptibility Promoted by High-Fat Diet and Sedentary Behavior in Mice.

Recent findings from rodent studies suggest that high-fat diet (HFD) increases hyperalgesia independent of obesity status. Furthermore, weight loss interventions such as voluntary physical activity (PA) for adults with obesity or overweight was reported to promote pain reduction in humans with chronic pain. However, regardless of obesity status, it is not known whether HFD intake and sedentary (SED) behavior is underlies chronic pain susceptibility. Moreover, differential gene expression in the nucleus accumbens (NAc) plays a crucial role in chronic pain susceptibility. Thus, the present study used an adapted model of the inflammatory prostaglandin E2 (PGE2)-induced persistent hyperalgesia short-term (PH-ST) protocol for mice, an HFD, and a voluntary PA paradigm to test these hypotheses. Therefore, we performed an analysis of differential gene expression using a transcriptome approach of the NAc. We also applied a gene ontology enrichment tools to identify biological processes associated with chronic pain susceptibility and to investigate the interaction between the factors studied: diet (standard diet vs. HFD), physical activity behavior (SED vs. PA) and PH-ST (PGE vs. saline). Our results demonstrated that HFD intake and sedentary behavior promoted chronic pain susceptibility, which in turn was prevented by voluntary physical activity, even when the animals were fed an HFD. The transcriptome of the NAc found 2,204 differential expression genes and gene ontology enrichment analysis revealed 41 biologic processes implicated in chronic pain susceptibility. Taking these biological processes together, our results suggest that genes related to metabolic and mitochondria stress were up-regulated in the chronic pain susceptibility group (SED-HFD-PGE), whereas genes related to neuroplasticity were up-regulated in the non-chronic pain susceptibility group (PA-HFD-PGE). These findings provide pieces of evidence that HFD intake and sedentary behavior provoked gene expression changes in the NAc related to promotion of chronic pain susceptibility, whereas voluntary physical activity provoked gene expression changes in the NAc related to prevention of chronic pain susceptibility. Finally, our findings confirmed previous literature supporting the crucial role of voluntary physical activity to prevent chronic pain and suggest that low levels of voluntary physical activity would be helpful and highly recommended as a complementary treatment for those with chronic pain.

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Spinal Interleukin-1β Inhibits Astrocyte Cytochrome P450c17 Expression Which Controls the Development of Mechanical Allodynia in a Mouse Model of Neuropathic Pain.

We have recently demonstrated that sciatic nerve injury increases the expression of spinal cytochrome P450c17, a key neurosteroidogenic enzyme, which plays a critical role in the development of peripheral neuropathic pain. However, the modulatory mechanisms responsible for the expression of spinal P450c17 have yet to be examined. Here we investigated the possible involvement of interleukin-1β (IL-1β) in altering P450c17 expression during the induction phase of neuropathic pain. Neuropathic pain was produced by chronic constriction injury (CCI) of the right sciatic nerve in mice and mechanical allodynia was evaluated in the hind paws using a von-Frey filament (0.16 g). Western blotting and immunohistochemistry were performed to assess the expression of spinal IL-1β, interleukin-1 receptor type 1 (IL-1R1), P450c17, and GFAP. Spinal IL-1β was significantly increased on day 1 post-surgery and its receptor, IL-1R1 was expressed in GFAP-positive astrocytes. Intrathecal administration of the recombinant interleukin-1 receptor antagonist (IL-1ra, 20 ng) on days 0 and 1 post-surgery enhanced GFAP expression on day 1 post-surgery and induced an early increase in P450c17 expression in astrocytes, but not in neurons. Administration of IL-1β (10 ng) on days 0 and 1 post-surgery blocked the enhancement of both spinal P450c17 and GFAP expression induced by IL-1ra (20 ng) administration. Intrathecal administration of IL-1ra (20 ng) on days 0 to 3 post-surgery also facilitated the CCI-induced development of mechanical allodynia, and this early developed pain was dose-dependently attenuated by the administration of the P450c17 inhibitor, ketoconazole (1, 3, or 10 nmol) or the astrocyte metabolic inhibitor, fluorocitrate (0.01, 0.03, or 0.1 nmol). These results demonstrate that early increases in spinal IL-1β temporally inhibit astrocyte P450c17 expression and astrocyte activation ultimately controlling the development of mechanical allodynia induced by peripheral nerve injury. These findings imply that spinal IL-1β plays an important role as an early, but transient, control mechanism in the development of peripheral neuropathic pain via the inhibition of astrocyte P450c17 expression and astrocyte activation.

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TRPV1 Channel Contributes to the Behavioral Hypersensitivity in a Rat Model of Complex Regional Pain Syndrome Type 1.

Complex regional pain syndrome type 1 (CRPS-I) is a debilitating pain condition that significantly affects life quality of patients. It remains a clinically challenging condition and the mechanisms of CRPS-I have not been fully elucidated. Here, we investigated the involvement of TRPV1, a non-selective cation channel important for integrating various painful stimuli, in an animal model of CRPS-I. A rat model of chronic post-ischemia pain (CPIP) was established to mimic CRPS-I. TRPV1 expression was significantly increased in hind paw tissue and small to medium-sized dorsal root ganglion (DRG) neurons of CPIP rats. CPIP rats showed increased TRPV1 current density and capsaicin responding rate in small-sized nociceptive DRG neurons. Local pharmacological blockage of TRPV1 with the specific antagonist AMG9810, at a dosage that does not produce hyperthermia or affect thermal perception or locomotor activity, effectively attenuated thermal and mechanical hypersensitivity in bilateral hind paws of CPIP rats and reduced the hyperexcitability of DRG neurons induced by CPIP. CPIP rats showed bilateral spinal astrocyte and microglia activations, which were significantly attenuated by AMG9810 treatment. These findings identified an important role of TRPV1 in mediating thermal and mechanical hypersensitivity in a CRPS-I animal model and further suggest local pharmacological blocking TRPV1 may represent an effective approach to ameliorate CRPS-I.

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Flexible and Lightweight Devices for Wireless Multi-Color Optogenetic Experiments Controllable via Commercial Cell Phones.

Optogenetics provide a potential alternative approach to the treatment of chronic pain, in which complex pathology often hampers efficacy of standard pharmacological approaches. Technological advancements in the development of thin, wireless, and mechanically flexible optoelectronic implants offer new routes to control the activity of subsets of neurons and nerve fibers . This study reports a novel and advanced design of battery-free, flexible, and lightweight devices equipped with one or two miniaturized LEDs, which can be individually controlled in real time. Two proof-of-concept experiments in mice demonstrate the feasibility of these devices. First, we show that blue-light devices implanted on top of the lumbar spinal cord can excite channelrhodopsin expressing nociceptors to induce place aversion. Second, we show that nocifensive withdrawal responses can be suppressed by green-light optogenetic (Archaerhodopsin-mediated) inhibition of action potential propagation along the sciatic nerve. One salient feature of these devices is that they can be operated via modern tablets and smartphones without bulky and complex lab instrumentation. In addition to the optical stimulation, the design enables the simultaneously wireless recording of the temperature in proximity of the stimulation area. As such, these devices are primed for translation to human patients with implications in the treatment of neurological and psychiatric conditions far beyond chronic pain syndromes.

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