Chemogenetic inhibition of central amygdala CRF-expressing neurons decreases alcohol intake but not trauma-related behaviors in a rat model of post-traumatic stress and alcohol use disorder.

Post-traumatic stress disorder (PTSD) and alcohol use disorder (AUD) are often comorbid. Few treatments exist to reduce comorbid PTSD/AUD. Elucidating the mechanisms underlying their comorbidity could reveal new avenues for therapy. Here, we employed a model of comorbid PTSD/AUD, in which rats were subjected to a stressful shock in a familiar context followed by alcohol drinking. We then examined fear overgeneralization and irritability in these rats. Familiar context stress elevated drinking, increased fear overgeneralization, increased alcohol-related aggressive signs, and elevated peripheral stress hormones. We then examined transcripts of stress- and fear-relevant genes in the central amygdala (CeA), a locus that regulates stress-mediated alcohol drinking. Compared with unstressed rats, stressed rats exhibited increases in CeA transcripts for Crh and Fkbp5 and decreases in transcripts for Bdnf and Il18. Levels of Nr3c1 mRNA, which encodes the glucocorticoid receptor, increased in stressed males but decreased in stressed females. Transcripts of Il18 binding protein (Il18bp), Glp-1r, and genes associated with calcitonin gene-related peptide signaling (Calca, Ramp1, Crlr-1, and Iapp) were unaltered. Crh, but not Crhr1, mRNA was increased by stress; thus, we tested whether inhibiting CeA neurons that express corticotropin-releasing factor (CRF) suppress PTSD/AUD-like behaviors. We used Crh-Cre rats that had received a Cre-dependent vector encoding hM4D(Gi), an inhibitory Designer Receptors Exclusively Activated by Designer Drugs. Chemogenetic inhibition of CeA CRF neurons reduced alcohol intake but not fear overgeneralization or irritability-like behaviors. Our findings suggest that CeA CRF modulates PTSD/AUD comorbidity, and inhibiting CRF neural activity is primarily associated with reducing alcohol drinking but not trauma-related behaviors that are associated with PTSD/AUD.

Novel Dual-Acting Hybrids Targeting Type-2 Cannabinoid Receptors and Cholinesterase Activity Show Neuroprotective Effects In Vitro and Amelioration of Cognitive Impairment In Vivo.

Alzheimer's disease (AD) is a neurodegenerative form of dementia characterized by the loss of synapses and a progressive decline in cognitive abilities. Among current treatments for AD, acetylcholinesterase (AChE) inhibitors have efficacy limited to symptom relief, with significant side effects and poor compliance. Pharmacological agents that modulate the activity of type-2 cannabinoid receptors (CB2R) of the endocannabinoid system by activating or blocking them have also been shown to be effective against neuroinflammation. Herein, we describe the design, synthesis, and pharmacological effects in vitro and in vivo of dual-acting compounds that inhibit AChE and butyrylcholinesterase (BChE) and target CB2R. Within the investigated series, compound 4g proved to be the most promising. It achieved IC50 values in the low micromolar to submicromolar range against both human cholinesterase isoforms while antagonizing CB2R with Ki of 31 nM. Interestingly, 4g showed neuroprotective effects on the SH-SY5Y cell line thanks to its ability to prevent oxidative stress-induced cell toxicity and reverse scopolamine-induced amnesia in the Y-maze forced alternation test in vivo.

Novel Combination of Choline with Withania somnifera (L.) Dunal, and Bacopa monnieri (L.) Wetts Reduced Oxidative Stress in Microglia Cells, Promoting Neuroprotection.

Memory deficit is one of the major negative outcomes of chronic stress. Cholinergic system modulates memory not only through the neuronal cells, but also via interactions with non-neuronal cells, suggesting that microglia can influence synaptic function and plasticity, contributing to cognition and memory function. Withania somnifera (L.) Dunal (WS) and Bacopa monnieri (L.) Wettst (BM), are traditional herbal medicinal products used for the temporary relief of symptoms of stress. The aim of this study was to investigate whether choline (CLN) activity could be enhanced via an association with adaptogens: WS and BM extracts. First, we optimized an in vitro model of corticotropin-releasing hormone (CRH)-induced oxidative stress on microglial BV2 cells. CRH 100 nM reduced BV2 cell viability and induced morphological changes and neurotoxicity after 24 h of microglia stimulation. Moreover, it induced an increase in the production of reactive oxygen species (ROS) and dysregulated antioxidant protein (i.e., SIRT-1 and NRF-2). The association between choline and adaptogens (CBW) 10 µg/mL counteracted the effect of CRH on BV2 cells and reduced the neurotoxicity produced by BV2 CRH-conditioned medium in the SH-SY5Y cell lines. CBW 200 mg/kg produced an ameliorative effect on recognition memory in the novel object recognition test (NORT) test in mice. In conclusion, combining choline with adaptogen plant extracts might represent a promising intervention in chronic stress associated with memory disturbances through the attenuation of microglia-induced oxidative stress.

Honokiol-Rich Magnolia officinalis Bark Extract Attenuates Trauma-Induced Neuropathic Pain.

Neuropathic pain (NP) affects about 8% of the general population. Current analgesic therapies have limited efficacy, making NP one of the most difficult to treat pain conditions. Evidence indicates that excessive oxidative stress can contribute to the onset of chronic NP and several natural antioxidant compounds have shown promising efficacy in NP models. Thus, this study aimed to investigate the pain-relieving activity of honokiol (HNK)-rich standardized extract of Magnolia officinalis Rehder & E. Wilson bark (MOE), well known for its antioxidant and anti-inflammatory properties, in the spared nerve injury (SNI) model. The molecular mechanisms and efficacy toward neuroinflammation were investigated in spinal cord samples from SNI mice and LPS-stimulated BV2 microglia cells. MOE and HNK showed antioxidant activity. MOE (30 mg/kg p.o.) produced an antiallodynic effect in SNI mice in the absence of locomotor impairment, reduced spinal p-p38, p-JNK1, iNOS, p-p65, IL-1ß, and Nrf2 overexpression, increased IL-10 and MBP levels and attenuated the Notch signaling pathway by reducing Jagged1 and NEXT. These effects were prevented by the CB1 antagonist AM251. HNK reduced the proinflammatory response of LPS-stimulated BV2 and reduced Jagged1 overexpression. MOE and HNK, by modulating oxidative and proinflammatory responses, might represent interesting candidates for NP management.

Sex-dependent factors of alcohol and neuroimmune mechanisms.

Excessive alcohol use disrupts neuroimmune signaling across various cell types, including neurons, microglia, and astrocytes. The present review focuses on recent, albeit limited, evidence of sex differences in biological factors that mediate neuroimmune responses to alcohol and underlying neuroimmune systems that may influence alcohol drinking behaviors. Females are more vulnerable than males to the neurotoxic and negative consequences of chronic alcohol drinking, reflected by elevations of pro-inflammatory cytokines and inflammatory mediators. Differences in cytokine, microglial, astrocytic, genomic, and transcriptomic evidence suggest females are more reactive than males to neuroinflammatory changes after chronic alcohol exposure. The growing body of evidence supports that innate immune factors modulate synaptic transmission, providing a mechanistic framework to examine sex differences across neurocircuitry. Targeting neuroimmune signaling may be a viable strategy for treating AUD, but more research is needed to understand sex-specific differences in alcohol drinking and neuroimmune mechanisms.

Glucocorticoid receptors regulate central amygdala GABAergic synapses in Marchigian-Sardinian alcohol-preferring rats.

Impairments in the function of the hypothalamic-pituitary-adrenal (HPA) axis and enhanced glucocorticoid receptor (GR) activity in the central amygdala (CeA) are critical mechanisms in the pathogenesis of alcohol use disorder (AUD). The GR antagonist mifepristone attenuates craving in AUD patients, alcohol consumption in AUD models, and decreases CeA γ-aminobutyric acid (GABA) transmission in alcohol-dependent rats. Previous studies suggest elevated GR activity in the CeA of male alcohol-preferring Marchigian-Sardinian (msP) rats, but its contribution to heightened CeA GABA transmission driving their characteristic post-dependent phenotype is largely unknown. We determined Nr3c1 (the gene encoding GR) gene transcription in the CeA in male and female msP and Wistar rats using in situ hybridization and studied acute effects of mifepristone (10 µM) and its interaction with ethanol (44 mM) on pharmacologically isolated spontaneous inhibitory postsynaptic currents (sIPSCs) and electrically evoked inhibitory postsynaptic potentials (eIPSPs) in the CeA using ex vivo slice electrophysiology. Female rats of both genotypes expressed more CeA GRs than males, suggesting a sexually dimorphic GR regulation of CeA activity. Mifepristone reduced sIPSC frequencies (GABA release) and eIPSP amplitudes in msP rats of both sexes, but not in their Wistar counterparts; however, it did not prevent acute ethanol-induced increase in CeA GABA transmission in male rats. In msP rats, GR regulates CeA GABAergic signaling under basal conditions, indicative of intrinsically active GR. Thus, enhanced GR function in the CeA represents a key mechanism contributing to maladaptive behaviors associated with AUD.

IL-18 Signaling in the Rat Central Amygdala Is Disrupted in a Comorbid Model of Post-Traumatic Stress and Alcohol Use Disorder.

Alcohol use disorder (AUD) and anxiety disorders are frequently comorbid and share dysregulated neuroimmune-related pathways. Here, we used our established rat model of comorbid post-traumatic stress disorder (PTSD)/AUD to characterize the interleukin 18 (IL-18) system in the central amygdala (CeA). Male and female rats underwent novel (NOV) and familiar (FAM) shock stress, or no stress (unstressed controls; CTL) followed by voluntary alcohol drinking and PTSD-related behaviors, then all received renewed alcohol access prior to the experiments. In situ hybridization revealed that the number of CeA positive cells for Il18 mRNA increased, while for Il18bp decreased in both male and female FAM stressed rats versus CTL. No changes were observed in Il18r1 expression across groups. Ex vivo electrophysiology showed that IL-18 reduced GABAA-mediated miniature inhibitory postsynaptic currents (mIPSCs) frequencies in CTL, suggesting reduced CeA GABA release, regardless of sex. Notably, this presynaptic effect of IL-18 was lost in both NOV and FAM males, while it persisted in NOV and FAM females. IL-18 decreased mIPSC amplitude in CTL female rats, suggesting postsynaptic effects. Overall, our results suggest that stress in rats with alcohol access impacts CeA IL-18-system expression and, in sex-related fashion, IL-18's modulatory function at GABA synapses.

Helichrysum stoechas (L.) Moench reduces body weight gain and modulates mood disorders via inhibition of silent information regulator 1 (SIRT1) by arzanol.

The prevalence of obesity is steadily rising, making safe and more efficient anti-obesity treatments an urgent medical need. Growing evidence correlates obesity and comorbidities, including anxiety and depression, with the development of a low-grade inflammation in peripheral and central tissues. We hypothesized that attenuating neuroinflammation might reduce weight gain and improve mood. We investigated the efficacy of a methanolic extract from Helichrysum stoechas (L.) Moench (HSE), well-known for its anti-inflammatory properties, and its main constituent arzanol (AZL). HPLC-ESI-MS2 and HPLC-UV were used to characterize the extract. HSE effects on mood and feeding behavior was assessed in mice. The mechanism of action of HSE and AZL was investigated in hippocampus samples and SH-SY5Y cells by western blotting and immunofluorescence. Oral administration of HSE for 3 weeks limited weight gain with no significant decrease in food intake. HSE produced an anxiolytic-like and antidepressant-like phenotype comparable to diazepam and amitriptyline, respectively, in the absence of locomotor and cognitive impairments and induced neuroprotective effects in glutamate-exposed SH-SY5Y cells. A dose-dependent reduction of SIRT1 expression was detected in SH-SY5Y cells and in hippocampal samples from HSE-treated mice. The inhibition of the SIRT1-FoxO1 pathway was induced in the hypothalamus. Molecular docking studies proposed a mechanism of SIRT1 inhibition by AZL, confirmed by the evaluation of inhibitory effects on SIRT1 enzymatic activity. HSE limited weight gain and comorbidities through an AZL-mediated SIRT1 inhibition. These activities indicate HSE an innovative therapeutic perspective for obesity and associated mood disorders.

The Selective CB2 Agonist COR167 Reduced Symptoms in a Mice Model of Trauma-Induced Peripheral Neuropathy through HDAC-1 Inhibition.

Neuropathic pain is a chronic disabling condition with a 7-10% of prevalence in the general population that is largely undertreated. Available analgesic therapies are poorly effective and are often accompanied by numerous side effects. Growing evidence indicates cannabinoids are a valuable treatment opportunity for neuropathic pain. The endocannabinoid system is an important regulator of pain perception through the CB1 receptors, but CB1 agonists, while largely effective, are not always satisfactory pain-relieving agents in clinics because of their serious adverse effects. Recently, several CB2 agonists have shown analgesic, anti-hyperalgesic, and anti-allodynic activity in the absence of CB1-induced psychostimulant effects, offering promise in neuropathic pain management. The aim of this study was to evaluate the anti-neuropathic activity of a novel selective CB2 agonist, COR167, in a preclinical model of peripheral neuropathy, the spared nerve injury (SNI). Oral COR167, in a dose-dependent manner, attenuated mechanical allodynia and thermal hyperalgesia after acute and repeated administration, showing the absence of tolerance induction. At anti-neuropathic doses, COR167 did not show any alteration in the locomotor behavior. SNI mice showed increased microglial levels of HDAC1 protein in the ipsilateral side of the spinal cord, along with NF-kB activation. COR167 treatment prevented the HDAC1 overexpression and the NF-kB activation and increased the levels of the anti-inflammatory cytokine IL-10 through a CB2-mediated mechanism. Oral administration of COR167 shows promising therapeutic potential in the management of neuropathic pain conditions.

Chronic alcohol induced mechanical allodynia by promoting neuroinflammation: A mouse model of alcohol-evoked neuropathic pain.

BACKGROUND AND PURPOSE: Chronic pain is considered a key factor contributing to alcohol use disorder (AUD). The mechanisms responsible for chronic pain associated with chronic alcohol consumption are unknown. We evaluated the development of chronic pain in a mouse model of alcohol dependence and investigate the role of neuroinflammation. EXPERIMENTAL APPROACH: The chronic-intermittent ethanol two-bottle choice CIE-2BC paradigm generates three groups: alcohol-dependent with escalating alcohol intake, nondependent (moderate drinking) and alcohol-naïve control male and female mice. We measured mechanical allodynia during withdrawal and after the last voluntary drinking. Immunoblotting was used to evaluate the protein levels of IBA-1, CSFR, IL-6, p38 and ERK2/1 in spinal cord tissue of dependent and non-dependent animals. KEY RESULTS: We found significant escalation of drinking in the dependent group in male and female compared with the non-dependent group. The dependent group developed mechanical allodynia during 72 h of withdrawal, which was completely reversed after voluntary drinking. We observed an increased pain hypersensitivity compared with the naïve in 50% of non-dependent group. Increased IBA-1 and CSFR expression was observed in spinal cord tissue of both hypersensitivity-abstinence related and neuropathy-alcohol mice, and increased IL-6 expression and ERK1/2 activation in mice with hypersensitivity-related to abstinence, but not in mice with alcohol-evoked neuropathic pain. CONCLUSIONS AND IMPLICATIONS: The CIE-2BC model induces two distinct pain conditions specific to the type of ethanol exposure: abstinence-related hypersensitivity in dependent mice and alcohol-evoked neuropathic pain in about a half of the non-dependent mice.

Zingiber officinale Roscoe Rhizome Extract Exerts Senomorphic and Anti-Inflammatory Activities on Human Endothelial Cells.

Aging is related to a low-grade and sterile inflammation called inflammaging, recognized as the main risk factor for age-related disease (ARD) development. Inflammaging is fostered by the repeated activation of immune cells, as well as by the accumulation of senescent cells. Recently, a number of natural compounds have gained attention to be tested as anti-aging therapies, based on their anti-inflammatory activity and/or ability to reduce the pro-inflammatory secretome of senescent cells (senomorphyc activity). Here, we investigated the anti-inflammatory and senomorphic properties of an Asian-native Zingiber officinale Roscoe extract (ZOE), commonly consumed as a food spice and herbal medicine. We employed two models of primary endothelial cells (HUVECs), such as the replicative-senescence and LPS-induced response, to investigate the anti-inflammatory/senomorphic effect of ZOE, and one cellular model of neuroinflammation, i.e., immortalized murine microglial cells (BV2). First, we found that the ZOE treatment induced the inhibition of NF-kB activation in BV2 cells. Among the constituents of ZOE, we showed that the terpenoid-enriched fraction (ZTE) was the component able to counteract the phosphorylation of NF-kB(p65), while 6-gingerol (GIN) and 6-shogaol (SHO) did not produce any significant effect. Further, we observed that the treatment with 10 µg/mL of ZOE exerted anti-inflammatory activity on LPS-stimulated young (y)HUVEC and senomorphyc activity on replicative senescent (s)HUVEC, significantly reducing the expression levels of IL-1ß, TNF -α, IL-8, MCP-1, and ICAM-1. Moreover, the ZTE treatment was able to significantly reduce the IL-8 levels secreted in the medium of both LPS-stimulated yHUVEC and sHUVEC. Overall, our data suggest a potential protective role of ZOE on neuroinflammation and endothelial inflammation/activation, thus suggesting its potential relevance in delaying/postponing ARD development and progression, characterized by endothelial dysfunction.

A Novel Perilla frutescens (L.) Britton Cell-Derived Phytocomplex Regulates Keratinocytes Inflammatory Cascade and Barrier Function and Preserves Vaginal Mucosal Integrity In Vivo.

In the last years, the medicinal plant Perilla frutescens (L.) Britton has gained scientific interest because leaf extracts, due to the presence of rosmarinic acid and other polyphenols, have shown anti-allergic and skin protective potential in pre-clinical studies. Nevertheless, the lack of standardized extracts has limited clinical applications to date. In this work, for the first time, a standardized phytocomplex of P. frutescens, enriched in rosmarinic acid and total polyphenols, was produced through innovative in vitro cell culture biotechnology and tested. The activity of perilla was evaluated in an in vitro inflammatory model of human keratinocytes (HaCaT) by monitoring tight junctions, filaggrin, and loricrin protein levels, the release of pro-inflammatory cytokines and JNK MAPK signaling. In a practical health care application, the perilla biotechnological phytocomplex was tested in a multilayer model of vaginal mucosa, and then, in a preliminary clinical observation to explore its capacity to preserve vaginal mucosal integrity in women in peri-menopause. In keratinocytes cells, perilla phytocomplex demonstrated to exert a marked activity in epidermis barrier maintenance and anti-inflammatory effects, preserving tight junction expression and downregulating cytokines release through targeting JNK activation. Furthermore, perilla showed positive effects in retaining vaginal mucosal integrity in the reconstructed vaginal mucosa model and in vivo tests. Overall, our data suggest that the biotechnological P. frutescens phytocomplex could represent an innovative ingredient for dermatological applications.

Posttranscriptional Regulation of Gene Expression Participates in the Myelin Restoration in Mouse Models of Multiple Sclerosis: Antisense Modulation of HuR and HuD ELAV RNA Binding Protein.

Neuropathic pain is the most difficult-to-treat pain syndrome in multiple sclerosis. Evidence relates neuropathic pain to demyelination, which often originates from unresolved neuroinflammation or altered immune response. Posttranscriptional regulation of gene expression might play a fundamental role in the regulation of these processes. The ELAV RNA-binding proteins HuR and HuD are involved in the promotion of inflammatory phenomena and in neuronal development and maintenance, respectively. Thus, the aim of this study was to investigate the role of HuR and HuD in demyelination-associated neuropathic pain in the mouse experimental autoimmune encephalomyelitis (EAE) model. HuR resulted overexpressed in the spinal cord of MOG35-55-EAE and PLP139-151-EAE mice and was detected in CD11b + cells. Conversely, HuD was largely downregulated in the MOG-EAE spinal cord, along with GAP43 and neurofilament H, while in PLP-EAE mice, HuD and neuronal markers remained unaltered. Intranasal antisense oligonucleotide (ASO) delivery to knockdown HuR, increased myelin basic protein expression, and Luxol Fast Blue staining in both EAE models, an indication of increased myelin content. These effects temporally coincided with attenuation of pain hypersensitivity. Anti-HuR ASO increased the expression of HuD in GAP43-expressing cells and promoted a HuD-mediated neuroprotective activity in MOG-EAE mice, while in PLP-EAE mice, HuR silencing dampened pro-inflammatory responses mediated by spinal microglia activation. In conclusion, anti-HuR ASO showed myelin protection at analgesic doses with multitarget mechanisms, and it deserves further consideration as an innovative agent to counteract demyelination in neuropathic pain states.

Zingiberene, a non-zinc-binding class I HDAC inhibitor: A novel strategy for the management of neuropathic pain.

BACKGROUND: Even though numerous Histone deacetylase inhibitors (HDACi) have been approved for the treatment of different types of cancer, and others are in clinical trials for the treatment of neurodegenerative diseases, the main problem related to the clinical use of available HDACi is their low isoform selectivity which causes undesirable effects and inevitably limits their therapeutic application. Previously, we demonstrated that a standardized Zingiber officinalis Roscoe rhizome extract (ZOE) reduced neuroinflammation through HDAC1 inhibition in a mice model of neuropathy, and this activity was related to terpenes fraction. HYPOTHESIS/PURPOSE: The aim of this work was to identify the ZOE constituent responsible for the activity on HDAC1 and to study its possible application in trauma-induced neuropathic pain. METHODS: The ability of ZOE and its terpenes fraction (ZTE) to inhibit HDAC and SIRT isoforms activity and protein expression was assessed in vitro. Then, a structure-based virtual screening approach was applied to predict which constituent could be responsible for the activity. In the next step, the activity of selected compound was tested in an in vitro model of neuroinflammation and in an in vivo model of peripheral neuropathy (SNI). RESULTS: ZTE resulted to be more potent than ZOE on HDAC1, 2, and 6 isoforms, while ZOE was more active on HDAC8. Zingiberene (ZNG) was found to be the most promising HDAC1 inhibitor, with an IC50 of 2.3 ± 0.1 µM. A non-zinc-binding mechanism of inhibition was proposed based on molecular docking. Moreover, the oral administration of ZNG reduced thermal hyperalgesia and mechanical allodynia in animals with neuropathy after 60 min from administration, and decreased HDAC-1 levels in the spinal cord microglia. CONCLUSION: We found a new non-zinc-dependent inhibitor of HDAC class I, with a therapeutic application in trauma-related neuropathic pain forms in which microglia-spinal overexpression of HDAC1 occurs. The non-zinc-binding mechanism has the potential to reduce off target effects, leading to a higher selectivity and better safety profile, compared to other HDAC inhibitors.

Cannabidiol-rich non-psychotropic Cannabis sativa L. oils attenuate peripheral neuropathy symptoms by regulation of CB2-mediated microglial neuroinflammation.

Neuropathic pain (NP) is a chronic disease that affects the normal quality of life of patients. To date, the therapies available are only symptomatic and they are unable to reduce the progression of the disease. Many studies reported the efficacy of Cannabis sativa L. (C. sativa) on NP, but no Δ9 -tetrahydrocannabinol (Δ9 -THC)-free extracts have been investigated in detail for this activity so far. The principal aim of this work is to investigate the potential pain-relieving effect of innovative cannabidiol-rich non-psychotropic C. sativa oils, with a high content of terpenes (K2), compared to the same extract devoid of terpenes (K1). Oral administration of K2 (25 mg kg-1 ) induced a rapid and long-lasting relief of pain hypersensitivity in a mice model of peripheral neuropathy. In spinal cord samples, K2 reduced mitogen-activated protein kinase (MAPKs) levels and neuroinflammatory factors. These effects were reverted by the administration of a CB2 antagonist (AM630), but not by a CB1 antagonist (AM251). Conversely, K1 showed a lower efficacy in the absence of CB1/CB2-mediated mechanisms. In LPS-stimulated murine microglial cells (BV2), K2 reduced microglia pro-inflammatory phenotype through the downregulation of histone deacetylase 1 (HDAC-1) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IKBα) and increased interleukin-10 (IL-10) expression, an important antiinflammatory cytokine. In conclusion, these results suggested that K2 oral administration attenuated NP symptoms by reducing spinal neuroinflammation and underline the important role of the synergism between cannabinoids and terpenes.

Microglia senescence is related to neuropathic pain-associated comorbidities in the spared nerve injury model.

ABSTRACT: The increased presence of senescent cells in different neurological diseases suggests the contribution of senescence in the pathophysiology of neurodegenerative disorders. Microglia can adapt to any type of disturbance of the homeostasis of the central nervous system, and its altered activity can lead to permanent and unresolvable damage. The aim of this work was to characterize the behavioural phenotype of spared nerve injury mice and then associate it with senescence-related mechanisms. In this work, we investigated the timing of the onset of anxiety, depression, or memory decline associated with peripheral neuropathic pain and their correlation with the presence of microglial cellular senescence. Spared nerve injury mice showed a persistent pain hypersensitivity from 3 days after surgery. Twenty-eight days after nerve injury, they also developed anxiety, depression, and cognitive impairment. The appearance of these symptoms was coincident to a significant increase of senescence markers, such as ß-galactosidase and senescent-associated secretory phenotype, at the microglial level in the spinal cord and hippocampus of spared nerve injury animals. These markers were unaltered at previous time points. In murine immortalized microglial cells (BV2) stimulated with LPS 500 ng/mL for 10 days (4 hours/day) every other day, we observed an increase of ß-galactosidase and senescent-associated secretory phenotype appearance, a reduction of cell viability, and an increase of senescence-associated heterochromatin foci. Therefore, present findings could represent an important step to a better understanding of the pathophysiological cellular mechanisms in comorbidities related to neuropathic pain states.

The Histamine H4 Receptor Participates in the Neuropathic Pain-Relieving Activity of the Histamine H3 Receptor Antagonist GSK189254.

Growing evidence points to the histamine system as a promising target for the management of neuropathic pain. Preclinical studies reported the efficacy of H3R antagonists in reducing pain hypersensitivity in models of neuropathic pain through an increase of histamine release within the CNS. Recently, a promising efficacy of H4R agonists as anti-neuropathic agents has been postulated. Since H3R and H4R are both localized in neuronal areas devoted to pain processing, the aim of the study is to investigate the role of H4R in the mechanism of anti-hyperalgesic action of the H3R antagonist GSK189254 in the spared nerve injury (SNI) model in mice. Oral (6 mg/kg), intrathecal (6 µg/mouse), or intra locus coeruleus (LC) (10 µg/µL) administration of GSK189254 reversed mechanical and thermal allodynia in the ipsilateral side of SNI mice. This effect was completely prevented by pretreatment with the H4R antagonist JNJ 10191584 (6 µg/mouse i.t.; (10 µg/µL intraLC). Furthermore, GSK189254 was devoid of any anti-hyperalgesic effect in H4R deficient mice, compared with wild type mice. Conversely, pretreatment with JNJ 10191584 was not able to prevent the hypophagic activity of GSK189254. In conclusion, we demonstrated the selective contribution of H4R to the H3R antagonist-induced attenuation of hypernociceptive behavior in SNI mice. These results might help identify innovative therapeutic interventions for neuropathic pain.

Investigation on the neuroprotective effect of a cannabidiol-enriched non-psychotropic Cannabis sativa L. extract in an in vitro model of excitotoxicity.

The purpose of this study was to evaluate the neuroprotective effect of a cannabidiol-enriched non-psychotropic Cannabis sativa L. extract (CSE) and its main constituents, cannabidiol and ß-caryophyllene. An in vitro model of glutamate-induced neuronal excitotoxicity using SH-SY5Y cells was optimized. The impact of CSE on glutamate-impaired cell viability, brain-derived neurotrophic factor release, CB1 protein expression, and ERK levels was evaluated. The involvement of CB1 modulation was verified by the cotreatment with the CB1 antagonist AM4113. CSE was able to significantly protect SH-SY5Y from glutamate-impaired cell viability, and to counteract the changes in brain-derived neurotrophic factor levels, with a mechanism of action involving ERK modulation. Moreover, CSE completely reversed the reduction of CB1 receptor expression induced by glutamate, and the presence of the CB1 antagonist AM4113 reduced CSE effectiveness, suggesting that CBr play a role in the modulation of neuronal excitotoxicity. This work demonstrated the in vitro effectiveness of CSE as a neuroprotective agent, proposing the whole cannabis phytocomplex as a more effective strategy, compared to its main constituents alone, and suggested further investigations by using more complex cell models before moving to in vivo studies.

Attenuation of neuroinflammation in microglia cells by extracts with high content of rosmarinic acid from in vitro cultured Melissa officinalis L. cells.

Plant cell culture is a biotechnology cultivation method that permit to cultivate plants in a short period of time and to obtain extracts with a high degree of standardization and high safety profile. The aim of our study was to evaluate the anti-inflammatory and neuroprotective activity of a standardized Melissa officinalis L. phytocomplex extract (MD) obtained with an in vitro plant cell culture. The MD has been chemically characterized and the content of total polyphenols was 5.17 ± 0.1 % w/w, with a content of rosmarinic acid (RA), its main constituent, of 4.02 ± 0.1 % w/w. MD was tested in an in vitro model of neuroinflammation, in which microglia cells (BV2) were stimulated with Lipopolysaccharides (LPS; 250 ng/mL) for 24 h and its pharmacological activity was compared with that of RA. MD (10 µg/mL) and RA (0.4 µg/mL) reduced pro-inflammatory factors (NF-kB, HDAC, IL-1ß) in LPS-stimulated BV2 cells and counteracted the toxic effect produced by activated microglia medium on neuronal cells. This work shows the efficacy of MD on reducing microglia-mediated neuroinflammation and promoting neuroprotection, highlighting the innovative use of in vitro plant cell cultures to obtain contaminant-free extracts endowed with marked activity and improved quali-quantitative ratio in the constituents' content.

Rosmarinic Acid Reduces Microglia Senescence: A Novel Therapeutic Approach for the Management of Neuropathic Pain Symptoms.

The worldwide incidence of neuropathic pain is around 7-8% and is associated with significant and disabling comorbidities (sleep disturbances, depression, anxiety). It is now known that cellular ageing of microglia contributes to neurodegenerative diseases, mood disorders, and, even if with less evidence, chronic pain. The aim of this work was to investigate in vitro and in vivo the senolytic activity of rosmarinic acid (RA) to be exploited for the management of NP symptoms. BV2 cells were stimulated with LPS 500 ng/mL for 24 h. Treatment with RA 1 µM improved cell viability and reduced IL-1ß release leading to an attenuation of neuroinflammation. We then moved on to test the efficacy of RA in reducing microglial senescence. In our model, BV2 cells were stimulated with LPS 500 ng/mL every 72 h for 4 h/day, over a period of 10 days. RA 1 µM reduced the expression of the ß-galactosidase enzyme, reduced the release of senescence-associated secretory phenotype (SASP) factors, increased cell viability, and reduced the presence of nuclear foci of senescence (SAHF), well-known cellular senescence markers. In the Spared Nerve Injury (SNI) model, 28 days from surgery, repeated oral administration of RA 5 mg/kg reduced hyperalgesia and NP-associated symptoms, such as anxiety and depression. A reduction of senescence markers was detected on both hippocampal and spinal samples of SNI-treated mice. This study represents a starting point for investigating the role of microglial senescence as a possible pharmacological target in controlling symptoms related to the more advanced stages of peripheral neuropathy.