Hericenone C attenuates the second phase of formalin-induced nociceptive behavior by suppressing the accumulation of CD11c-positive cells in the paw epidermis via phosphorylated P65.

Hericenone C is one of the most abundant secondary metabolites derived from Hericium erinaceus, under investigation for medicinal properties. Here, we report that Hericenone C inhibits the second phase of formalin-induced nociceptive behavior in mice. As the second phase is involved in inflammation, in a mechanistic analysis on cultured cells targeting NF-κB response element (NRE): luciferase (Luc)-expressing cells, lipopolysaccharide (LPS)-induced NRE::Luc luciferase activity was found to be significantly inhibited by Hericenone C. Phosphorylation of p65, which is involved in the inflammatory responses of the NF-κB signaling pathway, was also induced by LPS and significantly reduced by Hericenone C. Additionally, in mice, the number of CD11c-positive cells increased in the paw during the peak of the second phase of the formalin test, which decreased upon Hericenone C intake. Our findings confirm the possibility of Hericenone C as a novel therapeutic target for pain-associated inflammation.

Motor behavior induced by bergamot essential oil in experimental tasks is differentially modulated by pretreatment with metabotropic glutamate receptor 2/3 or 5 antagonists.

Bergamot essential oil shows anxiolytic-relaxant effects devoid of sedative action and motor impairment typical of benzodiazepines. Considering the potential for clinical of these effects, it is important to understand the underlying mechanisms of the phytocomplex. Modulation of glutamate group I and II metabotropic receptors is involved in stress and anxiety disorders, in cognition and emotions and increases locomotor activity and wakefulness. Interestingly, early data indicate that bergamot essential oil modulates glutamatergic transmission in specific manifestations of the central nervous system. The aim of this work is to investigate if selective antagonists of metabotropic glutamate 2/3 and 5 receptors affect behavioral parameters modulated by the phytocomplex. Male Wistar rats were used to measure behavioral parameters to correlate anxiety and motor activity using elevated plus maze (EPM), open field (OF), and rotarod tasks. Bergamot essential oil increases in EPM the time spent in open/closed arms and reduces total number of entries. The essential oil also increases immobility in EPM and OF and not affect motor coordination in rotarod. Pretreatment with the metabotropic glutamate antagonists does not affect the time spent in open/close arms, however, differently affects motor behavior measured after administration of phytocomplex. Particularly, glutamate 2/3 antagonist reverts immobility and glutamate 5 antagonist potentiates this parameter induced by the phytocomplex. Our data show that modulation of both metabotropic glutamate receptors is likely involved in some of behavioral effects of bergamot essential oil.

Exploitation of Autophagy Inducers in the Management of Dementia: A Systematic Review.

The social burden of dementia is remarkable since it affects some 57.4 million people all over the world. Impairment of autophagy in age-related diseases, such as dementia, deserves deep investigation for the detection of novel disease-modifying approaches. Several drugs belonging to different classes were suggested to be effective in managing Alzheimer's disease (AD) by means of autophagy induction. Useful autophagy inducers in AD should be endowed with a direct, measurable effect on autophagy, have a safe tolerability profile, and have the capability to cross the blood-brain barrier, at least with poor penetration. According to the PRISMA 2020 recommendations, we propose here a systematic review to appraise the measurable effectiveness of autophagy inducers in the improvement of cognitive decline and neuropsychiatric symptoms in clinical trials and retrospective studies. The systematic search retrieved 3067 records, 10 of which met the eligibility criteria. The outcomes most influenced by the treatment were cognition and executive functioning, pointing at a role for metformin, resveratrol, masitinib and TPI-287, with an overall tolerable safety profile. Differences in sample power, intervention, patients enrolled, assessment, and measure of outcomes prevents generalization of results. Moreover, the domain of behavioral symptoms was found to be less investigated, thus prompting new prospective studies with homogeneous design. PROSPERO registration: CRD42023393456.

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.

Tumor Necrosis Factor (TNF)-α-Stimulated Gene 6 (TSG-6): A Promising Immunomodulatory Target in Acute Neurodegenerative Diseases.

Tumor necrosis factor (TNF)-α-stimulated gene 6 (TSG-6), the first soluble chemokine-binding protein to be identified in mammals, inhibits chemotaxis and transendothelial migration of neutrophils and attenuates the inflammatory response of dendritic cells, macrophages, monocytes, and T cells. This immunoregulatory protein is a pivotal mediator of the therapeutic efficacy of mesenchymal stem/stromal cells (MSC) in diverse pathological conditions, including neuroinflammation. However, TSG-6 is also constitutively expressed in some tissues, such as the brain and spinal cord, and is generally upregulated in response to inflammation in monocytes/macrophages, dendritic cells, astrocytes, vascular smooth muscle cells and fibroblasts. Due to its ability to modulate sterile inflammation, TSG-6 exerts protective effects in diverse degenerative and inflammatory diseases, including brain disorders. Emerging evidence provides insights into the potential use of TSG-6 as a peripheral diagnostic and/or prognostic biomarker, especially in the context of ischemic stroke, whereby the pathobiological relevance of this protein has also been demonstrated in patients. Thus, in this review, we will discuss the most recent data on the involvement of TSG-6 in neurodegenerative diseases, particularly focusing on relevant anti-inflammatory and immunomodulatory functions. Furthermore, we will examine evidence suggesting novel therapeutic opportunities that can be afforded by modulating TSG-6-related pathways in neuropathological contexts and, most notably, in stroke.

Efficacy of Essential Oils in Relieving Cancer Pain: A Systematic Review and Meta-Analysis.

Over 80% of patients affected by cancer develops cancer-related pain, one of the most feared consequences because of its intractable nature, particularly in the terminal stage of the disease. Recent evidence-based recommendations on integrative medicine for the management of cancer pain underline the role of natural products. The present systematic review and meta-analysis aims at appraising for the first time the efficacy of aromatherapy in cancer pain in clinical studies with different design according to the most updated Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2020 recommendations. The search retrieves 1002 total records. Twelve studies are included and six are eligible for meta-analysis. The present study demonstrates significant efficacy of the use of essential oils in the reduction of the intensity of pain associated with cancer (p < 0.00001), highlighting the need for earlier, more homogeneous, and appropriately designed clinical trials. Good certainty body of evidence is needed for effective and safe management of cancer-related pain using essential oils by establishment of a step-by-step preclinical-to-clinical pathway to provide a rational basis for clinical use in integrative oncology. PROSPERO registration: CRD42023393182.

Scopolamine-Induced Memory Impairment in Mice: Effects of PEA-OXA on Memory Retrieval and Hippocampal LTP.

Transient global amnesia, both persistent and transient, is a very common neuropsychiatric syndrome. Among animal models for amnesia and testing new drugs, the scopolamine test is the most widely used for transient global amnesia (TGA). This study examined the scopolamine-induced deficits in working memory, discriminative memory, anxiety, and motor activity in the presence of intranasal PEA-OXA, a dual antagonist of presynaptic α2 and H3 receptors. Male C57BL/6 mice were treated with intraperitoneal scopolamine (1 mg/kg) with or without pre-treatment (15 min) or post-treatment (15 min) with intranasal PEA-OXA (10 mg/kg). It was seen that scopolamine induced deficits of discriminative and spatial memory and motor deficit. These changes were associated with a loss of synaptic plasticity in the hippocampal dentate gyrus: impaired LTP after lateral entorhinal cortex/perforant pathway tetanization. Furthermore, hippocampal Ach levels were increased while ChA-T expression was reduced following scopolamine administration. PEA-OXA either prevented or restored the scopolamine-induced cognitive deficits (discriminative and spatial memory). However, the same treatment did not affect the altered motor activity or anxiety-like behavior induced by scopolamine. Consistently, electrophysiological analysis showed LTP recovery in the DG of the hippocampus, while the Ach level and ChoA-T were normalized. This study confirms the neuroprotective and pro-cognitive activity of PEA-OXA (probably through an increase in the extracellular levels of biogenic amines) in improving transient memory disorders for which the available pharmacological tools are obsolete or inadequate and not directed on specific pathophysiological targets.

Transgenic Mice for the Translational Study of Neuropathic Pain and Dystonia.

Murine models are fundamental in the study of clinical conditions and the development of new drugs and treatments. Transgenic technology has started to offer advantages in oncology, encompassing all research fields related to the study of painful syndromes. Knockout mice or mice overexpressing genes encoding for proteins linked to pain development and maintenance can be produced and pain models can be applied to transgenic mice to model the most disabling neurological conditions. Due to the association of movement disorders with sensitivity and pain processing, our group focused for the first time on the role of the torsinA gene GAG deletion-responsible for DYT1 dystonia-in baseline sensitivity and neuropathic responses. The aim of the present report are to review the complex network that exists between the chaperonine-like protein torsinA and the baseline sensitivity pattern-which are fundamental in neuropathic pain-and to point at its possible role in neurodegenerative diseases.

Analgesic Characteristics of NanoBEO Released by an Airless Dispenser for the Control of Agitation in Severe Dementia.

Chronic pain is one of the most common causes of the need for clinical evaluation, acquiring more importance in the elderly with cognitive impairment. Reduced self-reporting capabilities cause unrelieved pain contributing to the development of agitation. Safe and effective pain treatment can afford the management of agitation without the serious increase in death risk associated with neuroleptics. To this aim, the essential oil of bergamot (BEO), proven by rigorous evidence to have strong preclinical anti-nociceptive and anti-allodynic properties, has been engineered (NanoBEO, patent EP 4003294) to allow randomized, double-blind, placebo-controlled trials (BRAINAID, NCT04321889). The present study: (1) assesses the analgesic effects of a single therapeutic dose of NanoBEO, as supplied by an airless dispenser for clinical translation, in models of inflammatory, neuropathic, and sensitization types of pain relevant to clinic; (2) provides a dose-response analysis of the efficacy of NanoBEO on scratching behavior, a typical behavioral disturbance occurring in dementia. A single therapeutic dose of NanoBEO confirms efficacy following thirty minutes pre-treatment with capsaicin and on the central sensitization phase induced by formalin. Moreover, it has an ID50 of 0.6312 mg and it is efficacious on static and dynamic mechanical allodynia. Altogether, the gathered results strengthen the potential of NanoBEO for clinical management of pain and agitation.

Pattern of treatment of behavioural and psychological symptoms of dementia and pain: evidence on pharmacoutilization from a large real-world sample and from a centre for cognitive disturbances and dementia.

PURPOSE: Data concerning the number of diagnoses and of the drugs prescribed to patients affected by dementia are still scarce. Here we test whether or not (1) prescription of symptomatic drugs against Alzheimer's disease (AD) may approximate the number of patients affected by dementia in Italy and (2) adherence to this treatment affects the pattern of prescription of drugs (i.e. antipsychotics and antidepressants) for behavioural and psychological symptoms of dementia (BPSD) and the previously reported limited prescription of analgesics. METHODS: This retrospective observational study concerns 84,235 subjects older than 60 years and registered in the provincial prescription database of the health district of Cosenza accounting for a population of 298,000 inhabitants. The prescribing pattern of antipsychotics, antidepressants, and analgesics has been investigated in patients receiving concurrent prescriptions of acetylcholinesterase inhibitors (AChEI) and/or memantine. Data from a single centre for cognitive disturbances and dementia (CDCD) in the same health district were used to explore at which stage dementia was diagnosed. The study was approved by Calabria Region Ethical Committee no. 31/2017 and registered on October 31, 2017. RESULTS: The data show that 859 patients are treated with AChEI and/or memantine; 420 patients (48.89%) receive at least 80% of the recommended medications. CDCD data indicate a delay in dementia diagnosis, which often was made when the patients were moderately to severely demented (Mini Mental State Examination, MMSE ≤ 20). Adherence did not influence prescription of most of the drugs explored, but use of non-steroidal anti-inflammatory drugs was higher in non-adherent patients. Antipsychotics and antidepressants are frequently used (20.61-20.71% and 42.37-51.43%, respectively), and this, at least in part, might stem from the observed under-treatment of chronic pain (opioids are prescribed in the 4.76% and 12.46% of adherent and non-adherent patients and gabapentin and pregabalin are used in the 4.29% and 4.07% of adherent and non-adherent patients respectively), resulting in more frequent BPSD. 16.43% of patients receive antipsychotics for longer than 6-12 weeks. CONCLUSION: This 2-year period study, including a wide cohort of community demented patients, shows that dementia is diagnosed late and that prevalence of BPSD prescriptions is high and not impacted by adherence to anti-dementia drugs. The rate of prescription of potentially harmful antipsychotics and antidepressants appears to be high though whether the concomitantly observed limited prescription of analgesics might be a contributing factor needs to be further investigated. Our data support the development of strategies to improve the management of BPSD.

Bergamot rehabilitation AgaINst agitation in dementia (BRAINAID): Study protocol for a randomized, double-blind, placebo-controlled trial to assess the efficacy of furocoumarin-free bergamot loaded in a nanotechnology-based delivery system of the essential oil in the treatment of agitation in elderly affected by severe dementia.

Pain is underdiagnosed and often not adequately treated, contributing to behavioral and psychological symptoms of dementia (BPSD). BPSD are treated with atypical antipsychotics that are associated with severe cerebrocardiovascular effects. Interestingly, treatment of pain may reduce agitation. Research is focusing on nonpharmacological treatment, such as aromatherapy, for pain and BPSD in dementia. This clinical study will assess the effect on agitation in severely demented elderly of BEO loaded in a nanotechnological odorless cream indistinguishable from placebo. This is a protocol for a randomized, double-blind, placebo-controlled trial (NCT04321889). A total of 134 patients aged ≥65 years with severe dementia (mini-mental state examination <12) will be recruited and randomly allocated 1:1 to either BEO or placebo group. After baseline screening, BEO (80 mg) cream or placebo cream will be trans-dermally applied on both arms twice a day for 4 weeks with a 4-week follow-up period. The effect on agitation will be the primary endpoint. Any adverse events will be reported. A double-blind, clinical trial evaluating efficacy and safety of an essential oil endowed with strong analgesic properties has never been carried out before. This study could form the basis for a safer and more effective treatment of BPSD in severe dementia.

Role of CGRP pathway polymorphisms in migraine: a systematic review and impact on CGRP mAbs migraine therapy.

BACKGROUND: the interest of clinical reaseach in polymorphisms and epigenetics in migraine has been growing over the years. Due to the new era of preventative migraine treatment opened by monoclonal antibodies (mAbs) targeting the signaling of the calcitonin-gene related peptide (CGRP), the present systematic review aims at identifying genetic variants occurring along the CGRP pathway and at verifying whether these can affect the clinical features and the course of disease and the responsiveness of patients to therapy. METHODS: the literature search has been conducted consulting the most relevant scientific databases, i.e. PubMed/MEDLINE, Scopus, Web of Science, the Human Genome Epidemiology (HuGE) Published Literature database (Public Health Genomics Knowledge Base) and Clinicaltrials.gov from database inception until April 1, 2021. The process of identification and selection of the studies included in the analysis has followed the PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) criteria for systematic reviews and meta-analyses and the guidance from the Human Genome Epidemiology Network for reporting gene-disease associations. RESULTS: the search has retrieved 800 results, among which only 7 studies have met the eligibility criteria for inclusion in the analysis. The latter are case-control studies of genetic association and an exploratory analysis and two polymorphisms have been detected as the most recurring: the rs3781719 (T > C) of the CALC A gene encoding CGRP and the rs7590387 of the gene encoding the receptor activity-modifying protein (RAMP) 1 (C > G). Only one study assessing the methylation pattern with regard to CGRP pathway has been found from the search. No genetic association studies investigating the possible effect of genetic variants affecting CGRP signaling on the responsiveness to the most recent pharmacological approaches, i.e. anti-CGRP(R) mAbs, gepants and ditans, have been published. According to the Human Genome Epidemiology (HuGE) systematic reviews and meta-analyses risk-of-bias score for genetic association studies, the heterogeneity between and across studies and the small sample size do not allow to draw conclusions and prompt future studies. CONCLUSIONS: adequately powered, good quality genetic association studies are needed to understand the impact of genetic variants affecting the pathway of CGRP on migraine susceptibility and clinical manifestation and to predict the response to therapy in terms of efficacy and safety.

Effects of the autophagy modulators d-limonene and chloroquine on vimentin levels in SH-SY5Y cells.

The molecular target and mechanism by which d-limonene induces LC3 lipidation and autophagosome formation remain elusive. Here, we report that this monoterpene rapidly enhances Ca2+ levels in SH-SY5Y cells; yet this effect does not lead to calpain- or caspase-mediated proteolysis of α-spectrin, nor calpain activity is required for the established enhancement of LC3-II levels by d-limonene. However, d-limonene rapidly reduced vimentin levels, an unexpected effect also induced by the autophagy inhibitor chloroquine (CQ). The magnitude of vimentin reduction parallels accumulation of LC3-II caused by a brief incubation with d-limonene or CQ. For longer exposure (48 h), d-limonene does not reduce vimentin, nor it increases LC3-II levels; conversely, a clear reduction of vimentin along with a massive accumulation of LC3-II is evident in cells treated with CQ. Vimentin participates in organelle positioning and in other cellular processes that have linked this intermediate filament protein to various diseases, including cancer, inflammatory and autoimmune disorders, and to virus replication and internalization. Our findings suggest an inverse relationship between vimentin reduction and LC3-II accumulation, whose causal link needs to be examined. Further experiments are needed to dissect the role of vimentin reduction in the mechanisms through which CQ impairs fusion of autophagosome with lysosomes as well as in other effects of this drug.

Role of 5-HT1A Receptor in the Anxiolytic-Relaxant Effects of Bergamot Essential Oil in Rodent.

The essential oil obtained by the fresh fruit of Citrus bergamia Risso et Poiteau is used worldwide in aromatherapy to reduce pain, facilitate sleep induction, and/or minimize the effects of stress-induced anxiety. Preclinical pharmacological data demonstrate that bergamot essential oil (BEO) modulates specific neurotransmissions and shows an anxiolytic-relaxant effect not superimposable to that of the benzodiazepine diazepam, suggesting that neurotransmissions, other than GABAergic, could be involved. Several studies on essential oils indicate a role for serotonergic (5-HT) neurotransmission in anxiety. Interestingly, among serotonergic receptors, the 5-HT1A subtype seems to play a key role in the control of anxiety. Here, we report that modulation of the 5-HT1A receptor by selective agonist ((±)8-OH-DPAT) or antagonist (WAY-100635) may influence some of the anxiolytic-relaxant effects of BEO in Open Field and Elevated Plus Maze tests.

Neuropharmacology of the Neuropsychiatric Symptoms of Dementia and Role of Pain: Essential Oil of Bergamot as a Novel Therapeutic Approach.

Aging of the population makes of dementia a challenge for health systems worldwide. The cognitive disturbance is a serious but not the only issue in dementia; behavioral and psychological syndromes known as neuropsychiatric symptoms of dementia remarkably reduce the quality of life. The cluster of symptoms includes anxiety, depression, wandering, delusions, hallucinations, misidentifications, agitation and aggression. The pathophysiology of these symptoms implicates all the neurotransmitter systems, with a pivotal role for the glutamatergic neurotransmission. Imbalanced glutamatergic and GABAergic neurotransmissions, over-activation of the extrasynaptic N-methyl-D-aspartate (NMDA) receptors and alterations of the latter have been linked to the development of neuropsychiatric symptoms experienced by almost the entire demented population. Drugs with efficacy and safety for prevention or long term treatment of these disorders are not available yet. Aromatherapy provides the best evidence for positive outcomes in the control of agitation, the most resistant symptom. Demented patients often cannot verbalize pain, resulting in unrelieved symptoms and contributing to agitation. Bergamot essential oil provides extensive preclinical evidence of analgesic properties. Incidentally, the essential oil of bergamot induces anxyolitic-like effects devoid of sedation, typical of benzodiazepines, with a noteworthy advantage for demented patients. These data, together with the reported safety profile, form the rational basis for bergamot as a neurotherapeutic to be trialed for the control of behavioral and psychological symptoms of dementia.

Post-ischemic treatment with azithromycin protects ganglion cells against retinal ischemia/reperfusion injury in the rat.

Purpose: Retinal ischemic phenomena occur in several ocular diseases that share the degeneration and death of retinal ganglion cells (RGCs) as the final event. We tested the neuroprotective effect of azithromycin, a widely used semisynthetic macrolide antibiotic endowed with anti-inflammatory and immunomodulatory properties, in a model of retinal ischemic injury induced by transient elevation of intraocular pressure in the rat. Methods: Retinal ischemia was induced in adult rats with transient elevation of intraocular pressure. RGCs were retrogradely labeled with Fluoro-Gold, and survival was assessed following a single dose of azithromycin given systemically at the end of the ischemia. The expression of death-associated proteins and extracellular signal-regulated kinase (ERK) activation was studied with western blotting. Expression and activity of matrix metalloproteinase-2 (MMP-2) and -9 were analyzed with gelatin zymography. Results: Acute post-injury administration of azithromycin significantly prevented RGC death. This effect was accompanied by reduced calpain activity and prevention of Bcl-2-associated death promoter (Bad) upregulation. The observed neuroprotection was associated with a significant inhibition of MMP-2/-9 gelatinolytic activity and ERK1/2 phosphorylation. Conclusions: Azithromycin provides neuroprotection by modifying the inflammatory state of the retina following ischemia/reperfusion injury suggesting potential for repurposing as a drug capable of limiting or preventing retinal neuronal damage.

Bergamot Essential Oil Attenuates Anxiety-Like Behaviour in Rats.

Preclinical studies have recently highlighted that bergamot essential oil (BEO) is endowed with remarkable neurobiolological effects. BEO can affect synaptic transmission, modulate electroencephalographic activity and it showed neuroprotective and analgesic properties. The phytocomplex, along with other essential oils, is also widely used in aromatherapy to minimize symptoms of stress-induced anxiety and mild mood disorders. However, only limited preclinical evidences are actually available. This study examined the anxiolytic/sedative-like effects of BEO using an open field task (OFT), an elevated plus-maze task (EPM), and a forced swimming task (FST) in rats. This study further compared behavioural effects of BEO to those of the benzodiazepine diazepam. Analysis of data suggests that BEO induces anxiolytic-like/relaxant effects in animal behavioural tasks not superimposable to those of the DZP. The present observations provide further insight to the pharmacological profile of BEO and support its rational use in aromatherapy.

Spinal autophagy is differently modulated in distinct mouse models of neuropathic pain.

BACKGROUND: Autophagy is a homeostatic degradative process essential for basal turnover of long-lived proteins and organelles as well as for removal of dysfunctional cellular components. Dysregulation of the autophagic machinery has been recently associated to several conditions including neurodegenerative diseases and cancer, but only very few studies have investigated its role in pain processing. RESULTS: We previously described autophagy impairment at the spinal cord in the experimental model of neuropathic pain induced by spinal nerve ligation (SNL). In this study, we characterized the main autophagic markers in two other common experimental models of neuropathic pain, the chronic constriction injury (CCI) and the spared nerve injury (SNI). The different modulation of LC3-I, Beclin 1 and p62 suggested that autophagy is differentially affected in the spinal dorsal horn depending on the type of peripheral injury. Confocal analysis of p62 distribution in the spinal dorsal horn indicated its presence mainly in NeuN-positive cell bodies and occasionally in glial processes, thus suggesting a predominant expression in the neuronal compartment. Finally, we investigated the consequences of autophagy impairment on pain behaviour by using the autophagy blocker cloroquine. Intrathecal chloroquine injection in naïve mice induced spinal accumulation of LC3 and p62 paralleled by significant mechanical hypersensitivity thus confirming the block in autophagosome clearance and suggesting the participation of the autophagic process in spinal mechanisms of pain processing. Altogether, our data indicate that spinal autophagy is differentially altered in different experimental pain models of neuropathic pain and that this process may be relevant for pain control.

Intravitreal injection of forskolin, homotaurine, and L-carnosine affords neuroprotection to retinal ganglion cells following retinal ischemic injury.

PURPOSE: Retinal ganglion cell (RGC) death is the final event leading to visual impairment in glaucoma; therefore, identification of neuroprotective strategies able to slow down or prevent the process is one of the main challenges for glaucoma research. The purpose of this study was to evaluate the neuroprotective potential of RGC death induced by the in vivo transient increase in intraocular pressure (IOP) of a combined treatment with forskolin, homotaurine, and L-carnosine. Forskolin (7beta-acetoxy-8, 13-epoxy-1a, 6ß, 9a-trihydroxy-labd-14-en-11-one) is an activator of adenylate cyclase that decreases IOP by reducing aqueous humor production and functions as a neuroprotector due to its neurotrophin-stimulating activity. Homotaurine is a natural aminosulfonate compound endowed with neuromodulatory effects, while the dipeptide L-carnosine is known for its antioxidant properties. METHODS: Retinal ischemia was induced in the right eye of adult male Wistar rats by acutely increasing the IOP. Forskolin, homotaurine, and L-carnosine were intravitreally injected and RGC survival evaluated following retrograde labeling with FluoroGold. Total and phosphorylated Akt and glycogen synthase kinase-3ß (GSK-3ß) protein levels, as well as calpain activity, were analyzed with western blot. Protein kinase A (PKA) was inhibited by intravitreal injection of H89. RESULTS: A synergic neuroprotective effect on RGC survival was observed following the combined treatment with forskolin, homotaurine, and L-carnosine compared to forskolin alone. The observed neuroprotection was associated with reduced calpain activity, upregulation of phosphoinositide 3-kinase (PI3K)/Akt pathway, and inhibition of GSK-3ß but was independent from PKA activation and distinct from the hypotensive effects of forskolin. CONCLUSIONS: A multidrug/multitarget approach, by interfering with several pathways involved in RGC degeneration, may be promising to achieve glaucoma neuroprotection.

Activation of RXR/PPARγ underlies neuroprotection by bexarotene in ischemic stroke.

The identification of novel drug targets for the treatment of ischemic stroke is currently an urgent challenge. Recent experimental findings have highlighted the neuroprotective potential of immunomodulatory strategies, based on polarization of myeloid cells toward non-inflammatory, beneficial phenotypes. Given the role of retinoid X receptors (RXR) in myeloid cells differentiation and polarization, here we have explored the neuroprotective potential of the RXR agonist bexarotene in mice subjected to focal cerebral ischemia. Acute administration of bexarotene significantly reduced blood brain barrier leakage, brain infarct damage and neurological deficit produced by transient middle cerebral artery occlusion in mice, without affecting cerebral blood flow. The rexinoid exerted neuroprotection with a wide time-window, being effective when administered up to 4.5h after the insult. The amelioration of histological outcome, as well as the ability of bexarotene to revert middle cerebral artery occlusion (MCAo)-induced spleen atrophy, was antagonised by BR1211, a pan-RXR antagonist, or by the selective peroxisome proliferator-activated receptor (PPAR)γ antagonist bisphenol A diglycidyl ether (BADGE), highlighting the involvement of the RXR/PPARγ heterodimer in the beneficial effects exerted by the drug. Immunofluorescence analysis revealed that bexarotene elevates Ym1-immunopositive N2 neutrophils both in the ipsilateral hemisphere and in the spleen of mice subjected to transient middle cerebral artery occlusion, pointing to a major role for peripheral neutrophil polarization in neuroprotection. Thus, our findings suggest that the RXR agonist bexarotene exerts peripheral immunomodulatory effects under ischemic conditions to be effectively repurposed for the acute therapy of ischemic stroke.