Expression of miR-155 in monocytes of people with migraine: association with phenotype, disease severity and inflammatory profile.

BACKGROUND: miR-155 is involved in the generation and maintenance of inflammation and pain, endothelial function and immune system homeostasis, all functions that are relevant for migraine. The present study aims to assess the levels of miR-155 in migraine subtypes (episodic and chronic) in comparison to age- and sex-matched healthy controls. METHODS: This is a cross-sectional, controlled, study involving three study groups: I) episodic migraine (n = 52, EM), II) chronic migraine with medication overuse (n = 44, CM-MO), and III) healthy controls (n = 32, HCs). We assessed the interictal gene expression levels of miR-155, IL-1ß, TNF-α, and IL-10 in peripheral blood monocytes using rtPCR. The monocytic differentiation toward the M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes was assessed in circulating monocytes with flow cytometry analysis and cell sorting. RESULTS: miR-155 gene expression was higher in CM-MO group (2.68 ± 2.47 Relative Quantification - RQ) when compared to EM group (1.46 ± 0.85 RQ, p = 0.006) and HCs (0.44 ± 0.18 RQ, p = 0.001). In addition, miR-155 gene expression was higher in EM group when compared to HCs (p = 0.001). A multivariate analysis confirmed the difference between EM and CM-MO groups after correction for age, sex, smoking habit, preventive treatment, aura, presence of psychiatric or other pain conditions. We found higher gene expression of IL-1ß, TNF-α, and lower gene expression of IL-10 in migraine participants when compared to HCs (p = 0.001 for all comparisons). TNF-α and IL-10 genes alterations were more prominent in CM-MO when compared to EM participants (p = 0.001). miR-155 positively correlated with IL-1ß (p = 0.001) and TNF-α (p = 0.001) expression levels. Finally, in people with CM-MO, we described an up-regulated percentage of events in both M1 and M2 monocytic profiles. CONCLUSIONS: Our study shows for the first time a specific profile of activation of miR-155 gene expression levels in monocytes of selected migraine subpopulations, more pronounced in subjects with CM-MO. Interestingly, mir-155 expression correlated with markers of activation of the inflammatory and immune systems. The CM-MO subpopulation showed a peculiar increase of both pro-inflammatory and anti-inflammatory monocytes which worths further investigation. TRIAL REGISTRATION: www. CLINICALTRIALS: gov . (NCT05891808).

Activity of FAAH-Inhibitor JZP327A in an Experimental Rat Model of Migraine.

Increased anandamide levels via fatty acid amide hydrolase (FAAH) inhibition can decrease the pronociceptive responses and inflammatory mediators in animal models of migraine. Here, we profile the pharmacological activity of the FAAH inhibitor JZP327A, a chiral 1,3,4-oxadiazol-2(3H)-one compound, in the mediation of spontaneous and nocifensive behaviour in the animal models of migraine based on nitroglycerin (NTG) administration. JZP327A (0.5 mg/kg, i.p.) or vehicle was administered to male rats 3 h after NTG (10 mg/kg, i.p.) or NTG vehicle injection. The rats were then exposed to the open field test and an orofacial formalin test 1 h later. The levels of endocannabinoids and lipid-related substances, and the expression of pain and inflammatory mediators were evaluated in cranial tissues and serum. The findings show that JZP327A did not affect NTG-induced changes in the spontaneous behaviour of rats, while it inhibited NTG-induced hyperalgesia at the orofacial formalin test. Furthermore, JZP327A dramatically decreased the gene expression of calcitonin gene-related peptide (CGRP), tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) in the trigeminal ganglia and medulla-pons, while it did not change endocannabinoids or lipids levels nor CGRP serum levels in the same tissues. These data suggest an anti-hyperalgesic role for JZP327A in the NTG model, which is mediated by the inhibition of the inflammatory cascade of events. This activity does not seem mediated by a change in the levels of endocannabinoids and lipid amides.

Potentiation of endocannabinoids and other lipid amides prevents hyperalgesia and inflammation in a pre-clinical model of migraine.

Targeting fatty acid amide hydrolase (FAAH) is a promising therapeutic strategy to combat certain forms of pain, including migraine headache. FAAH inhibitors, such as the O-biphenyl-3-yl carbamate URB597, have been shown to produce anti-hyperalgesic effects in animal models of migraine. The objective of this study was to investigate the behavioral and biochemical effects of compounds ARN14633 and ARN14280, two URB597 analogs with improved solubility and bioavailability, in a migraine-specific rat model in which trigeminal hyperalgesia is induced by nitroglycerin (NTG) administration. ARN14633 (1 mg/kg, i.p.) and ARN14280 (3 mg/kg, i.p.) were administered to adult male Sprague-Dawley rats 3 hours after NTG injection. One hour after the administration of either compound, rats were subjected to the orofacial formalin test. ARN14633 and ARN14280 attenuated NTG-induced nocifensive behavior and reduced transcription of genes encoding neuronal nitric oxide synthase, pain mediators peptides (calcitonin gene-related peptide, substance P) and pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta and 6) in the trigeminal ganglion, cervical spinal cord and medulla. Finally, both compounds strongly elevated levels of endocannabinoids and/or other FAAH substrates in cervical spinal cord and medulla, and, to a lesser extent, in the trigeminal ganglia. The results indicate that the novel global FAAH inhibitors ARN14633 and ARN14280 elicit significant anti-hyperalgesic effects in a migraine-specific animal model and inhibit the associated peptidergic-inflammatory response. Although the precise mechanism underlying these effects remains to be elucidated, our results support further investigational studies of FAAH blockade as a potential therapeutic strategy to treat migraine conditions.

Double-Binding Botulinum Molecule with Reduced Muscle Paralysis: Evaluation in In Vitro and In Vivo Models of Migraine.

With a prevalence of 15%, migraine is the most common neurological disorder and among the most disabling diseases, taking into account years lived with disability. Current oral medications for migraine show variable effects and are frequently associated with intolerable side effects, leading to the dissatisfaction of both patients and doctors. Injectable therapeutics, which include calcitonin gene-related peptide-targeting monoclonal antibodies and botulinum neurotoxin A (BoNT/A), provide a new paradigm for treatment of chronic migraine but are effective only in approximately 50% of subjects. Here, we investigated a novel engineered botulinum molecule with markedly reduced muscle paralyzing properties which could be beneficial for the treatment of migraine. This stapled botulinum molecule with duplicated binding domain-binary toxin-AA (BiTox/AA)-cleaves synaptosomal-associated protein 25 with a similar efficacy to BoNT/A in neurons; however, the paralyzing effect of BiTox/AA was 100 times less when compared to native BoNT/A following muscle injection. The performance of BiTox/AA was evaluated in cellular and animal models of migraine. BiTox/AA inhibited electrical nerve fiber activity in rat meningeal preparations while, in the trigeminovascular model, BiTox/AA raised electrical and mechanical stimulation thresholds in Aδ- and C-fiber nociceptors. In the rat glyceryl trinitrate (GTN) model, BiTox/AA proved effective in inhibiting GTN-induced hyperalgesia in the orofacial formalin test. We conclude that the engineered botulinum molecule provides a useful prototype for designing advanced future therapeutics for an improved efficacy in the treatment of migraine.

Chronic and intermittent administration of systemic nitroglycerin in the rat induces an increase in the gene expression of CGRP in central areas: potential contribution to pain processing.

BACKGROUND: Calcitonin gene related peptide (CGRP) is a key neuropeptide involved in the activation of the trigeminovascular system and it is likely related to migraine chronification. Here, we investigated the role of CGRP in an animal model that mimics the chronic migraine condition via repeated and intermittent nitroglycerin (NTG) administration. We also evaluated the modulatory effect of topiramate on this experimental paradigm. Male Sprague-Dawley rats were injected with NTG (5 mg/kg, i.p.) or vehicle, every 2 days over a 9-day period (5 total injections). A group of animals was injected with topiramate (30 mg/kg, i.p.) or saline every day for 9 days. Twenty-four hours after the last administration of NTG or vehicle, animals underwent tail flick test and orofacial Von Frey test. Rats were subsequently sacrificed to evaluate c-Fos and CGRP gene expression in medulla-pons region, cervical spinal cord and trigeminal ganglia. RESULTS: NTG administration induced spinal hyperalgesia and orofacial allodynia, together with a significant increase in the expression of CGRP and c-Fos genes in trigeminal ganglia and central areas. Topiramate treatment prevented NTG-induced changes by reversing NTG-induced hyperalgesia and allodynia, and inhibiting CGRP and c-Fos gene expression in all areas evaluated. CONCLUSIONS: These findings point to the role of CGRP in the processes underlying migraine chronification and suggest a possible interaction with gamma-aminobutyrate (GABA) and glutamate transmission to induce/maintain central sensitization and to contribute to the dysregulation of descending pain system involved in chronic migraine.

Endothelial nitric oxide synthase inhibition triggers inflammatory responses in the brain of male rats exposed to ischemia-reperfusion injury.

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) plays a role in preserving and maintaining the brain's microcirculation, inhibiting platelet aggregation, leukocyte adhesion, and migration. Inhibition of eNOS activity results in exacerbation of neuronal injury after ischemia by triggering diverse cellular mechanisms, including inflammatory responses. To examine the relative contribution of eNOS in stroke-induced neuroinflammation, we analyzed the effects of systemic treatment with l-N-(1-iminoethyl)ornithine (L-NIO), a relatively selective eNOS inhibitor, on the expression of MiR-155-5p, a key mediator of innate immunity regulation and endothelial dysfunction, in the cortex of male rats subjected to transient middle cerebral artery occlusion (tMCAo) followed by 24 hr of reperfusion. Inducible NO synthase (iNOS) and interleukin-10 (IL-10) mRNA expression were evaluated by real-time polymerase chain reaction in cortical homogenates and in resident and infiltrating immune cells isolated from ischemic cortex. These latter cells were also analyzed for their expression of CD40, a marker of M1 polarization of microglia/macrophages.tMCAo produced a significant elevation of miR155-5p and iNOS expression in the ischemic cortex as compared with sham surgery. eNOS inhibition by L-NIO treatment further elevated the cortical expression of these inflammatory mediators, while not affecting IL-10 mRNA levels. Interestingly, modulation of iNOS occurred in resident and infiltrating immune cells of the ischemic hemisphere. Accordingly, L-NIO induced a significant increase in the percentage of CD40+ events in CD68+ microglia/macrophages of the ischemic cortex as compared with vehicle-injected animals. These findings demonstrate that inflammatory responses may underlie the detrimental effects due to pharmacological inhibition of eNOS in cerebral ischemia.

The role of the transient receptor potential ankyrin type-1 (TRPA1) channel in migraine pain: evaluation in an animal model.

BACKGROUND: Clinical and experimental studies have pointed to the possible involvement of the transient receptor potential ankyrin type-1 (TRPA1) channels in migraine pain. In this study, we aimed to further investigate the role of these channels in an animal model of migraine using a novel TRPA1 antagonist, ADM_12, as a probe. METHODS: The effects of ADM_12 on nitroglycerin-induced hyperalgesia at the trigeminal level were investigated in male rats using the quantification of nocifensive behavior in the orofacial formalin test. The expression levels of the genes coding for c-Fos, TRPA1, calcitonin gene-related peptide (CGRP) and substance P (SP) in peripheral and central areas relevant for migraine pain were analyzed. CGRP and SP protein immunoreactivity was also evaluated in trigeminal nucleus caudalis (TNC). RESULTS: In rats bearing nitroglycerin-induced hyperalgesia, ADM_12 showed an anti-hyperalgesic effect in the second phase of the orofacial formalin test. This effect was associated to a significant inhibition of nitroglycerin-induced increase in c-Fos, TRPA1 and neuropeptides mRNA levels in medulla-pons area, in the cervical spinal cord and in the trigeminal ganglion. No differences between groups were seen as regards CGRP and SP protein expression in the TNC. CONCLUSIONS: These findings support a critical involvement of TRPA1 channels in the pathophysiology of migraine, and show their active role in counteracting hyperalgesia at the trigeminal level.

Influence of Estrogen Modulation on Glia Activation in a Murine Model of Parkinson's Disease.

Epidemiological data suggest a sexual dimorphism in Parkinson disease (PD), with women showing lower risk of developing PD. Vulnerability of the nigrostriatal pathway may be influenced by exposure to estrogenic stimulation throughout fertile life. To further address this issue, we analyzed the progression of nigrostriatal damage, microglia and astrocyte activation and microglia polarization triggered by intrastriatal injection of dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA) in male, female and ovariectomized (OVX) mice, as well as in OVX mice supplemented with 17ßestradiol (OVX+E). Animals were sacrificed at different time points following 6-OHDA injection and brain sections containing striatum and substantia nigra pars compacta (SNc) underwent immunohistochemistry for tyrosine hydroxylase (TH) (dopaminergic marker), immunofluorescence for IBA1 and GFAP (markers of microglia and astrocyte activation, respectively) and triple immunoflorescent to identify polarization of microglia toward the cytotoxic M1 (DAPI/IBA1/TNFα) or cytoprotective M2 (DAPI/IBA1/CD206) phenotype. SNc damage induced by 6-OHDA was significantly higher in OVX mice, as compared to all other experimental groups, at 7 and 14 days after surgery. Astrocyte activation was higher in OVX mice with respect the other experimental groups, at all time points. Microglial activation in the SNc was detected at earlier time points in male, female and OVX+E, while in OVX mice was detected at all time-points. Microglia polarization toward the M2, but not the M1, phenotype was detected in female and OVX+E mice, while the M1 phenotype was observed only in male and OVX mice. Our results support the protective effects of estrogens against nigrostriatal degeneration, suggesting that such effects may be mediated by an interaction with microglia, which tend to polarize preferentially toward an M2, cytoprotective phenotype in the presence of intense estrogenic stimulation.

Modulation of cerebral RAGE expression following nitric oxide synthase inhibition in rats subjected to focal cerebral ischemia.

The receptor for advanced glycation endproducts (RAGE) is a key mediator of neuroinflammation following cerebral ischemia. Nitric oxide (NO) plays a dualistic role in cerebral ischemia, depending on whether it originates from neuronal, inducible or endothelial synthase. Although a dynamic interplay between RAGE and NO pathways exists, its relevance in ischemic stroke has not been investigated. The aim of this study is to evaluate the effect of the NO synthase (NOS) inhibition on RAGE expression in rats subjected to transient middle cerebral artery occlusion (tMCAo). Full-length (fl-RAGE) gene expression was elevated in the striatum and, to a lesser extent, in the cortex of rats undergone tMCAo. The exacerbation of cortical damage caused by systemic administration of L-N-(1-iminoethyl)ornithine (L-NIO), a relatively selective inhibitor of endothelial NOS (eNOS), was associated with elevated mRNA levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α and fl-RAGE in both the cortex and the striatum. Conversely, NG-nitro-l-arginine methyl ester (L-NAME), a non-selective NOS inhibitor, decreased cortical damage, did not affect cerebral cytokine mRNA levels, while it increased fl-RAGE mRNA expression only in the striatum. Fl-RAGE striatal protein levels varied accordingly with observed mRNA changes in the striatum, while in the cortex, RAGE protein levels were reduced by tMCAo and further decreased following L-NIO treatment. Modulation of RAGE expression by different inhibitors of NOS may have opposite effects on transient cortical ischemia: the non selective inhibition of NOS activity is protective, while the selective inhibition of eNOS is harmful, probably via the activation of inflammatory pathways.

Andrographis Paniculata shows anti-nociceptive effects in an animal model of sensory hypersensitivity associated with migraine.

Administration of nitroglycerin (NTG) to rats induces a hyperalgesic condition and neuronal activation of central structures involved in migraine pain. In order to identify therapeutic strategies for migraine pain, we evaluated the anti-nociceptive activity of Andrographis Paniculata (AP), a herbaceous plant, in the hyperalgesia induced by NTG administration in the formalin test. We also analyzed mRNA expression of cytokines in specific brain areas after AP treatment. Male Sprague-Dawley rats were pre-treated with AP extract 30 minutes before NTG or vehicle injection. The data show that AP extract significantly reduced NTG-induced hyperalgesia in phase II of the test, 4 hours after NTG injection. In addition, AP extract reduced IL-6 mRNA expression in the medulla and mesencephalon and also mRNA levels of TNFalpha in the mesencephalic region. These findings suggest that AP extract may be a potential therapeutic approach in the treatment of general pain, and possibly of migraine.

Incremental Value of Platelet Reactivity Over a Risk Score of Clinical and Procedural Variables in Predicting Bleeding After Percutaneous Coronary Intervention via the Femoral Approach: Development and Validation of a New Bleeding Risk Score.

BACKGROUND: Growing evidence suggests that platelet reactivity (PR) may predict bleeding. We investigate the incremental value of PR in predicting bleeding after percutaneous coronary intervention (PCI) via the femoral approach over a validated bleeding risk score (BRS) of clinical and procedural variables. METHODS AND RESULTS: A total of 800 patients undergoing elective PCI via the femoral approach were included. PR was measured before PCI with the VerifyNow P2Y12 assay and low PR was defined as a P2Y12 reaction unit value ≤ 178. Calculation of the BRS included the following: age, sex, intra-aortic balloon pump, glycoprotein IIb/IIIa inhibitors, chronic kidney disease, anemia, and low-molecular-weight heparin within 48-hour pre-PCI. A new risk score including low PR (BRS-PR) was developed and validated in an independent cohort of patients (n = 310). Bleeding events at 30 days after PCI were defined according to the thrombolysis in myocardial infarction, Randomized Evaluation in PCI Linking Angiomax to Reduced Clinical Events (REPLACE)-2, and Bleeding Academic Research Consortium criteria. Both BRS and PR showed high discriminatory power for bleeding (area under the curve [AUC] > 0.7 for all definitions). Discriminatory power of BRS-PR (AUC = 0.809 for thrombolysis in myocardial infarction bleeding; AUC = 0.814 for Bleeding Academic Research Consortium class ≥ 2 bleeding; AUC = 0.708 for Bleeding Academic Research Consortium class ≥ 3 bleeding; and AUC = 0.813 for REPLACE-2 bleeding) was significantly higher than that of BRS alone (P < 0.001 for all bleeding definitions). In the validation set, BRS-PR showed higher discriminatory power for thrombolysis in myocardial infarction bleeding than BRS alone (AUC = 0.788 versus 0.709; P = 0.036). CONCLUSIONS: PR has incremental predictive value on bleeding events after elective PCI via the femoral approach over a validated risk score of clinical and procedural variables. A risk score including PR yields significantly better prognostic performance compared with the original BRS.