A Narrative Review of Intestinal Microbiota's Impact on Migraine with Psychopathologies.

Migraine is a common and debilitating neurological disorder characterized by the recurrent attack of pulsating headaches typically localized on one side of the head associated with other disabling symptoms, such as nausea, increased sensitivity to light, sound and smell and mood changes. Various clinical factors, including the excessive use of migraine medication, inadequate acute treatment and stressful events, can contribute to the worsening of the condition, which may evolve to chronic migraine, that is, a headache present on >15 days/month for at least 3 months. Chronic migraine is frequently associated with various comorbidities, including anxiety and mood disorders, particularly depression, which complicate the prognosis, response to treatment and overall clinical outcomes. Emerging research indicates a connection between alterations in the composition of the gut microbiota and mental health conditions, particularly anxiety and depression, which are considered disorders of the gut-brain axis. This underscores the potential of modulating the gut microbiota as a new avenue for managing these conditions. In this context, it is interesting to investigate whether migraine, particularly in its chronic form, exhibits a dysbiosis profile similar to that observed in individuals with anxiety and depression. This could pave the way for interventions aimed at modulating the gut microbiota for treating difficult-to-manage migraines.

Effects of the Dual FAAH/MAGL Inhibitor AKU-005 on Trigeminal Hyperalgesia in Male Rats.

The inhibition of endocannabinoid hydrolysis by enzymatic inhibitors may interfere with mechanisms underlying migraine-related pain. The dual FAAH/MAGL inhibitor AKU-005 shows potent inhibitory activity in vitro. Here, we assessed the effect of AKU-005 in a migraine animal model based on nitroglycerin (NTG) administration. Male rats were treated with AKU-005 (0.5 mg/kg, i.p.) or vehicle 3 h after receiving NTG (10 mg/kg, i.p.) or NTG vehicle. One hour later, rats were subjected to the open field test followed by the orofacial formalin test. At the end of the test, we collected serum samples for assessing calcitonin gene-related peptide (CGRP) levels as well as meninges, trigeminal ganglia, and brain areas to assess mRNA levels of CGRP and pro-inflammatory cytokines, and endocannabinoid and related lipid levels. AKU-005 reduced NTG-induced hyperalgesia during the orofacial formalin test but did not influence NTG-induced changes in the open field test. It significantly reduced serum levels of CGRP, CGRP, and pro-inflammatory cytokine mRNA levels in the meninges, trigeminal ganglia, and central areas. Surprisingly, AKU-005 caused no change in endocannabinoids and related lipids in the regions evaluated. The present findings suggest that AKU-005 may have anti-migraine effects by reducing CGRP synthesis and release and the associated inflammatory events. This effect, however, does not seem mediated via an interference with the endocannabinoid pathway.

Peripheral Endocannabinoid Components and Lipid Plasma Levels in Patients with Resistant Migraine and Co-Morbid Personality and Psychological Disorders: A Cross-Sectional Study.

Resistant migraine characterizes those patients who have failed at least three classes of migraine prophylaxis. These difficult-to-treat patients are likely to be characterized by a high prevalence of psychological disturbances. A dysfunction of the endocannabinoid system (ECS), including alteration in the levels of endocannabinoid congeners, may underlie several psychiatric disorders and the pathogenesis of migraines. Here we explored whether the peripheral gene expression of major components of the ECS and the plasma levels of endocannabinoids and related lipids are associated with psychological disorders in resistant migraine. Fifty-one patients (age = 46.0 ± 11.7) with resistant migraine received a comprehensive psychological evaluation according to the DSM-5 criteria. Among the patients, 61% had personality disorders (PD) and 61% had mood disorders (MD). Several associations were found between these psychological disorders and peripheral ECS alterations. Lower plasma levels of palmitoiletanolamide (PEA) were found in the PD group compared with the non-PD group. The MD group was characterized by lower mRNA levels of diacylglycerol lipase α (DAGLα) and CB2 (cannabinoid-2) receptor. The results suggest the existence of peripheral dysfunction in some components of the ECS and an alteration in plasma levels of PEA in patients with resistant migraine and mood or personality disorders.

URB937 Prevents the Development of Mechanical Allodynia in Male Rats with Trigeminal Neuralgia.

Cannabinoids are proposed for alleviating neuropathic pain, but their use is limited by cannabimimetic side effects. The inhibition of the fatty acid amide hydrolase (FAAH), the degrading enzyme of the endocannabinoid anandamide, has received attention as an alternative to cannabinoids in the treatment of neuropathic pain. Here, we investigated the effect of URB937, a blood-brain barrier impermeant FAAH inhibitor, on experimentally induced mechanical allodynia in an animal model of trigeminal neuralgia. Male Sprague-Dawley rats were subjected to chronic constriction injury of the infraorbital nerve (IoN-CCI); operated animals were treated sub-chronically with URB937 (1 mg/kg, i.p.) or vehicle before or after trigeminal mechanical allodynia establishment. We also assayed mRNA expression levels of the pain neuropeptide calcitonin gene-related peptide (CGRP) and cytokines in the medulla, cervical spinal cord, and trigeminal ganglion ipsilateral to IoN-CCI using rt-PCR. URB937 treatment prevented the development of mechanical allodynia and IoN-CCI-induced changes in mRNA expression levels of CGRP and cytokines in the evaluated areas. When administered after allodynia development, URB937 prevented IoN-CCI-induced changes in CGRP and cytokine gene expression; this was not associated with a significant abrogation of the mechanical allodynia. These findings suggest that URB937 may counteract, but not reverse, the development of allodynia in trigeminal neuralgia. Further research is needed to elucidate the underlying mechanisms.

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.

Characterization of the biochemical and behavioral effects of cannabidiol: implications for migraine.

Cannabidiol (CBD) is the main pharmacologically active phytocannabinoid. CBD exerts an analgesic effect in several pain models, does not have side effects and has low toxicity. The data about CBD mechanisms of action in pain and its therapeutic potential in this area are limited. Here, we tested CBD effects in animal models specific for migraine. We assayed CBD distribution in plasma and in cranial areas related to migraine pain in male Sprague Dawley rats treated chronically (5 days). Successively, we tested CBD activity on the behavioral and biochemical effects induced in the acute and the chronic migraine animal models by nitroglycerin (NTG) administration. In the acute migraine model, rats received CBD (15 mg or 30 mg/kg, i.p) 3 h after NTG (10 mg/kg i.p.) or vehicle injection. In the chronic migraine model, rats were treated with CBD and NTG every other day over nine days with the following doses: CBD 30 mg/kg i.p., NTG 10 mg/kg i.p. We evaluated behavioral parameters with the open field and the orofacial formalin tests. We explored the fatty acid amide hydrolase gene expression, cytokines mRNA and protein levels in selected brain areas and CGRP serum level. CBD levels in the meninges, trigeminal ganglia, cervical spinal cord, medulla pons, and plasma were higher 1 h after the last treatment than after 24 h, suggesting that CBD penetrates but does not accumulate in these tissues. In the acute model, CBD significantly reduced NTG-induced trigeminal hyperalgesia and CGRP and cytokine mRNA levels in peripheral and central sites. In the chronic model, CBD caused a significant decrease in NTG-induced IL-6 protein levels in the medulla-pons, and trigeminal ganglion. It also reduced CGRP serum levels. By contrast, CBD did not modulate TNF-alpha protein levels and fatty acid amide hydrolase (FAAH) gene expression in any of investigated areas. In both experimental conditions, there was no modulation of anxiety, motor/exploratory behavior, or grooming. These findings show that CBD reaches brain areas involved in migraine pain after systemic administration. They also show for the first time that CBD modulates migraine-related nociceptive transmission, likely via a complex signaling mechanism involving different pathways.

Biomarkers of Migraine: An Integrated Evaluation of Preclinical and Clinical Findings.

In recent years, numerous efforts have been made to identify reliable biomarkers useful in migraine diagnosis and progression or associated with the response to a specific treatment. The purpose of this review is to summarize the alleged diagnostic and therapeutic migraine biomarkers found in biofluids and to discuss their role in the pathogenesis of the disease. We included the most informative data from clinical or preclinical studies, with a particular emphasis on calcitonin gene-related peptide (CGRP), cytokines, endocannabinoids, and other biomolecules, the majority of which are related to the inflammatory aspects and mechanisms of migraine, as well as other actors that play a role in the disease. The potential issues affecting biomarker analysis are also discussed, such as how to deal with bias and confounding data. CGRP and other biological factors associated with the trigeminovascular system may offer intriguing and novel precision medicine opportunities, although the biological stability of the samples used, as well as the effects of the confounding role of age, gender, diet, and metabolic factors should be considered.

Characterization of the Involvement of Tumour Necrosis Factor (TNF)-α-Stimulated Gene 6 (TSG-6) in Ischemic Brain Injury Caused by Middle Cerebral Artery Occlusion in Mouse.

The identification of novel targets to modulate the immune response triggered by cerebral ischemia is crucial to promote the development of effective stroke therapeutics. Since tumour necrosis factor (TNF)-α-stimulated gene 6 (TSG-6), a hyaluronate (HA)-binding protein, is involved in the regulation of immune and stromal cell functions in acute neurodegeneration, we aimed to characterize its involvement in ischemic stroke. Transient middle cerebral artery occlusion (1 h MCAo, followed by 6 to 48 of reperfusion) in mice resulted in a significant elevation in cerebral TSG-6 protein levels, mainly localized in neurons and myeloid cells of the lesioned hemisphere. These myeloid cells were clearly infiltrating from the blood, strongly suggesting that brain ischemia also affects TSG-6 in the periphery. Accordingly, TSG-6 mRNA expression was elevated in peripheral blood mononuclear cells (PBMCs) from patients 48 h after ischemic stroke onset, and TSG-6 protein expression was higher in the plasma of mice subjected to 1 h MCAo followed by 48 h of reperfusion. Surprisingly, plasma TSG-6 levels were reduced in the acute phase (i.e., within 24 h of reperfusion) when compared to sham-operated mice, supporting the hypothesis of a detrimental role of TSG-6 in the early reperfusion stage. Accordingly, systemic acute administration of recombinant mouse TSG-6 increased brain levels of the M2 marker Ym1, providing a significant reduction in the brain infarct volume and general neurological deficits in mice subjected to transient MCAo. These findings suggest a pivotal role of TSG-6 in ischemic stroke pathobiology and underscore the clinical relevance of further investigating the mechanisms underlying its immunoregulatory role.

Modulation of Glia Activation by TRPA1 Antagonism in Preclinical Models of Migraine.

Preclinical data point to the contribution of transient receptor potential ankyrin 1 (TRPA1) channels to the complex mechanisms underlying migraine pain. TRPA1 channels are expressed in primary sensory neurons, as well as in glial cells, and they can be activated/sensitized by inflammatory mediators. The aim of this study was to investigate the relationship between TRPA1 channels and glial activation in the modulation of trigeminal hyperalgesia in preclinical models of migraine based on acute and chronic nitroglycerin challenges. Rats were treated with ADM_12 (TRPA1 antagonist) and then underwent an orofacial formalin test to assess trigeminal hyperalgesia. mRNA levels of pro- and anti-inflammatory cytokines, calcitonin gene-related peptide (CGRP) and glia cell activation were evaluated in the Medulla oblongata and in the trigeminal ganglia. In the nitroglycerin-treated rats, ADM_12 showed an antihyperalgesic effect in both acute and chronic models, and it counteracted the changes in CGRP and cytokine gene expression. In the acute nitroglycerin model, ADM_12 reduced nitroglycerin-induced increase in microglial and astroglial activation in trigeminal nucleus caudalis area. In the chronic model, we detected a nitroglycerin-induced activation of satellite glial cells in the trigeminal ganglia that was inhibited by ADM_12. These findings show that TRPA1 antagonism reverts experimentally induced hyperalgesia in acute and chronic models of migraine and prevents multiple changes in inflammatory pathways by modulating glial activation.

Antagonism of CGRP Receptor: Central and Peripheral Mechanisms and Mediators in an Animal Model of Chronic Migraine.

Calcitonin-gene-related peptide (CGRP) plays a key role in migraine pathophysiology and more specifically in the mechanisms underlying peripheral and central sensitization. Here, we explored the interaction of CGRP with other pain mediators relevant for neuronal sensitization in an animal model of chronic migraine. Male Sprague-Dawley rats were exposed to nitroglycerin (NTG, 5 mg/kg, i.p.) or vehicle co-administered with the CGRP receptor antagonist olcegepant (2 mg/kg i.p.), or its vehicle, every other day over a 9-day period. Twenty-four hours after the last injection of NTG (or vehicle), behavioral test and ex vivo analysis were performed. Olcegepant attenuated NTG-induced trigeminal hyperalgesia in the second phase of the orofacial formalin test. Interestingly, it also reduced gene expression and protein levels of CGRP, pro-inflammatory cytokines, inflammatory-associated miRNAs (miR-155-5p, miR-382-5p, and miR-34a-5p), and transient receptor potential ankyrin channels in the medulla-pons area, cervical spinal cord, and trigeminal ganglia. Similarly, olcegepant reduced the NTG-induced increase in CGRP and inflammatory cytokines in serum. The findings show that the activation of the CGRP pathway in a migraine animal model was associated to the persistent activation of inflammatory pathways, which was paralleled by a condition of hyperalgesia. These molecular events are relevant for informing us about the mechanisms underlying chronic migraine.

Modelling migraine-related features in the nitroglycerin animal model: Trigeminal hyperalgesia is associated with affective status and motor behavior.

Migraine is a complex neurovascular disorder characterized by recurrent attacks of pain and other associated symptoms. Emotional-affective aspects are important components of pain, but so far they have been little explored in animal models of migraine. In this study, we aimed to explore the correlation between trigeminal hyperalgesia and affective status or behavioral components in a migraine-specific animal model. Male Sprague-Dawley rats were treated with nitroglycerin (10 mg/kg, i.p.) or its vehicle. Four hours later, anxiety, motor/exploratory behavior and grooming (a nociception index) were evaluated with the open field test. Rats were then exposed to formalin in the orofacial region to evaluate trigeminal hyperalgesia. The data analysis shows an inverse correlation between trigeminal hyperalgesia and motor or exploratory behavior, and a positive association with anxiety-like behavior or self-grooming. These findings further expand on the translational value of the migraine-specific model based on nitroglycerin administration and prompt additional parameters that can be investigated to explore migraine disease in its complexity.

Oculo-vestibular signs in experimentally induced migraine attacks: an exploratory analysis.

Vestibular symptoms accompanying headache are quite common in migraine patients. Based on the association of vertigo with migraine, vestibular migraine was included in the appendix of the 3rd edition of the International Classification of Headache Disorders as a possible migraine subtype worthy of further investigation. In this post hoc, exploratory analysis, we investigated the occurrence of oculo-vestibular signs (OVSs) during experimentally induced migraine attacks in 24 episodic migraine patients and 19 healthy controls exposed to sublingual nitroglycerin (NTG - 0.9 mg). A comprehensive clinical examination was performed at baseline, at the onset of the migraine-like attack, and immediately before hospital discharge (180 minutes after NTG administration). Three of the 13 migraine patients who developed a spontaneous-like migraine attack during the hospital observation period (23.1%) also developed OVSs during the induction test. Noteworthy, none of the patients with a negative induction test developed OVSs and no OVSs were reported in healthy subjects at any time point. The exploratory nature of our study does not allow to draw definite conclusions on the possible implications of a vestibular dysfunction in migraine pathophysiology. Our results however suggest that NTG administration may lend itself to investigate vestibular dysfunction in migraine, at least in a subset of patients. The present findings represent a starting point for designing future ad hoc and well-powered studies.

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.

The endocannabinoid system and related lipids as potential targets for the treatment of migraine-related pain.

BACKGROUND: Migraine is a complex and highly disabling neurological disease whose treatment remains challenging in many patients, even after the recent advent of the first specific-preventive drugs, namely monoclonal antibodies that target calcitonin gene-related peptide. For this reason, headache researchers are actively searching for new therapeutic targets. Cannabis has been proposed for migraine treatment, but controlled clinical studies are lacking. A major advance in cannabinoid research has been the discovery of the endocannabinoid system (ECS), which consists of receptors CB1 and CB2; their endogenous ligands, such as N-arachidonoylethanolamine; and the enzymes that catalyze endocannabinoid biosynthesis or degradation. Preclinical and clinical findings suggest a possible role for endocannabinoids and related lipids, such as palmitoylethanolamide (PEA), in migraine-related pain treatment. In animal models of migraine-related pain, endocannabinoid tone modulation via inhibition of endocannabinoid-catabolizing enzymes has been a particular focus of research. METHODS: To conduct a narrative review of available data on the possible effects of cannabis, endocannabinoids, and other lipids in migraine-related pain, relevant key words were used to search the PubMed/MEDLINE database for basic and clinical studies. RESULTS: Endocannabinoids and PEA seem to reduce trigeminal nociception by interacting with many pathways associated with migraine, suggesting a potential synergistic or similar effect. CONCLUSIONS: Modulation of the metabolic pathways of the ECS may be a basis for new migraine treatments. The multiplicity of options and the wealth of data already obtained in animal models underscore the importance of further advancing research in this area. Multiple molecules related to the ECS or to allosteric modulation of CB1 receptors have emerged as potential therapeutic targets in migraine-related pain. The complexity of the ECS calls for accurate biochemical and pharmacological characterization of any new compounds undergoing testing and development.

Dual Inhibition of FAAH and MAGL Counteracts Migraine-like Pain and Behavior in an Animal Model of Migraine.

The endocannabinoid system exerts an important role in pain processing and modulation. Modulation of the system with hydrolase inhibitors of anandamide (AEA) or 2-arachidonyl glycerol (2-AG) has proved effective in reducing migraine-like features in animal models of migraine. Here, we investigated the effect of dual inhibition of the AEA and 2-AG catabolic pathways in the nitroglycerin-based animal model of migraine. The dual inhibitor JZL195 was administered to rats 2 h after nitroglycerin or vehicle injection. Rats were then exposed to the open field test and the orofacial formalin test. At the end of the tests, they were sacrificed to evaluate calcitonin gene-related peptide (CGRP) serum levels and gene expression of CGRP and cytokines in the cervical spinal cord and the trigeminal ganglion. The dual inhibitor significantly reduced the nitroglycerin-induced trigeminal hyperalgesia and pain-associated behavior, possibly via cannabinoid 1 receptors-mediated action, but it did not change the hypomotility and the anxiety behaviors induced by nitroglycerin. The decreased hyperalgesia was associated with a reduction in CGRP and cytokine gene expression levels in central and peripheral structures and reduced CGRP serum levels. These data suggest an antinociceptive synergy of the endocannabinoid action in peripheral and central sites, confirming that this system participates in reduction of cephalic pain signals.

CD163 as a Potential Biomarker of Monocyte Activation in Ischemic Stroke Patients.

In ischemic stroke patients, a higher monocyte count is associated with disease severity and worse prognosis. The complex correlation between subset phenotypes and functions underscores the importance of clarifying the role of monocyte subpopulations. We examined the subtype-specific distribution of the CD163+ and CD80+ circulating monocytes and evaluated their association with the inflammatory status in 26 ischemic stroke patients and 16 healthy controls. An increased percentage of CD163+/CD16+ and CD163+/CD14++ events occurred 24 and 48 h after a stroke compared to the controls. CD163+ expression was more pronounced in CD16+ non-classical and intermediate monocytes, as compared to CD14+ classical subtype, 24 h after stroke. Conversely, the percentage of CD80+/CD16+ events was unaffected in patients; meanwhile, the percentage of CD80+/CD14+ events significantly increased only 24 h after stroke. Interleukin (IL)-1beta, TNF-alpha, and IL-4 mRNA levels were higher, while IL-10 mRNA levels were reduced in total monocytes from patients versus controls, at either 24 h or 48 h after stroke. The percentage of CD163+/CD16+ events 24 h after stroke was positively associated with NIHSS score and mRS at admission, suggesting that stroke severity and disability are relevant triggers for CD163+ expression in circulating CD16+ monocytes.

Spinal nociceptive sensitization and plasma palmitoylethanolamide levels during experimentally induced migraine attacks.

ABSTRACT: Migraine pathophysiology has been suggested to include dysregulation of the endocannabinoid system (ES). We simultaneously evaluated plasma anandamide (AEA) and palmitoylethanolamide (PEA) levels and spinal sensitization in a validated human model of migraine based on systemic nitroglycerin (NTG) administration. Twenty-four subjects with episodic migraine (MIG) and 19 healthy controls (HC) underwent blood sampling and investigation of nociceptive withdrawal reflex thresholds (RTh: single-stimulus threshold; TST: temporal summation threshold) before and 30 (T30), 60 (T60), and 120 (T120) minutes after sublingual NTG administration (0.9 mg). At baseline, the MIG and HC groups were comparable for plasma AEA (P = 0.822) and PEA (P = 0.182) levels, and for RTh (P = 0.142) and TST values (P = 0.150). Anandamide levels increased after NTG administration (P = 0.022) in both groups, without differences between them (P = 0.779). By contrast, after NTG administration, PEA levels increased in the MIG group at T120 (P = 0.004), while remaining stable in the HC group. Nitroglycerin administration induced central sensitization in the MIG group, which was recorded as reductions in RTh (P = 0.046) at T30 and T120, and in TST (P = 0.001) at all time points. In the HC group, we observed increases in RTh (P = 0.001) and TST (P = 0.008), which suggest the occurrence of habituation. We found no significant correlations between the ES and neurophysiological parameters. Our findings suggest a role for PEA in the ictal phase of episodic migraine. The ES does not seem to be directly involved in the modulation of NTG-induced central sensitization, which suggests that the observed PEA increase and spinal sensitization are parallel, probably unrelated, phenomena.

Characterization of CB2 Receptor Expression in Peripheral Blood Monocytes of Acute Ischemic Stroke Patients.

Both preclinical and clinical evidence supports the involvement of the endocannabinoid system in the pathobiology of cerebral ischemia. Selective cannabinoid-2 (CB2) receptor agonists exert significant neuroprotection in animal models of focal brain ischemia through a robust anti-inflammatory effect, involving both resident and peripheral immune cells. Nevertheless, no definitive studies demonstrating the relevance of CB2 receptors in human stroke exist.Using rtPCR and flow cytometry assays, we investigated CB2 receptor expression in circulating monocytes from 26 acute ischemic stroke patients and 16 age-matched healthy controls (CT). We also evaluated miR-665 expression, as potential CB2 receptor regulator. The median mRNA levels of CB2 were significantly (p < 0.0001) increased in total monocytes 24 h and 48 h after stroke as compared with CT. This was paralleled by elevation of miR-665 levels in monocytes collected from patients 24 h (p < 0.05 vs CT) and 48 h (p < 0.05 vs CT and p < 0.0001 vs 24 h) after ischemic stroke. Furthermore, an increased percentage of CB2+/CD16+ events, but not CB2+/CD14+ events, was found 24 h [20.17% (IQR, 17.22-23.58)] and 48 h [18.61% (IQR, 15.44-22.06)] after ischemic stroke when compared with CT [10.96% (IQR, 9.185-13.32)]. The percentage of CB2+/CD16+ events in monocytes was positively correlated with NIHSS score at entrance (r = 0.4327, p = 0.027). The potential beneficial functions of CD16+ intermediate and nonclassical monocytes in stroke and the elevated expression of CB2 receptor in these subsets strongly suggest that CB2 receptor agonists can be exploited for the treatment of ischemic stroke patients.

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.