Joanna Rzemieniec

Background. Neuroinflammation, driven by chronic microglial activation and blood–brain barrier dysfunction, is increasingly recognised as a key pathogenic mechanism in neurodegenerative disorders. Achillea erba-rotta subsp. moschata, an alpine medicinal plant traditionally employed for inflammatory conditions, has demonstrated antioxidant and anti-inflammatory properties; however, its effects on brain-related cell types and the underlying neuropharmacological mechanisms remain largely unexplored. This study investigated the molecular pathways by which A. erba-rotta subsp. moschata modulates neuroinflammation in key cellular components of the neurovascular unit. Methods. We evaluated the pharmacological activity of A. erba-rotta subsp. moschata aqueous extract (20–200 µg/mL) in LPS-stimulated BV2 microglial cells and human brain microvascular endothelial cells (hBMECs). Molecular mechanisms were characterised using qRT-PCR and Western blot analysis, focusing on inflammatory, antioxidant (Nrf2/HO-1), and AhR signalling pathways. Results. A. erba-rotta subsp. moschata extract significantly attenuates inflammatory responses in both cell types. In BV2 microglia, the extract reduced pro-inflammatory mediators and promoted anti-inflammatory signalling, including dose-dependent upregulation of TGF-β. In parallel, in hBMECs, the extract preserved endothelial integrity and mitigated inflammation-induced alterations without affecting cell viability. At the molecular level, the extract modulated key transcriptional pathways involved in inflammation and redox homeostasis, including NF-κB and the Nrf2/HO-1 axis. Importantly, robust CYP1A1 induction indicated aryl hydrocarbon receptor (AhR) activation, revealing coordinated crosstalk between inflammatory and antioxidant pathways. Conclusions. A. erba-rotta subsp. moschata exerts balanced, tissue-dependent immunomodulatory activity through multi-target neuropharmacological mechanisms. The anti-inflammatory effects in microglia combined with barrier-preserving actions in brain endothelium, support its therapeutic potential as a neuropharmacological agent for neuroinflammatory disorders.

BACKGROUND: Basic science studies have reported remote ischemic conditioning (RIC) as neuroprotective in acute ischemic stroke, although clinical evidence remains conflicting. The TRICS BASIC study (Multicenter Translational Trial of Remote Ischemic Conditioning in Acute Ischemic Stroke) investigated the efficacy and safety of RIC in experimental ischemic stroke using a rigorous clinical trial methodology. METHODS: Multicenter, multispecies, parallel group, randomized, controlled, preclinical trial of transient femoral artery clipping to induce RIC in female and male rats and mice subjected to transient endovascular occlusion of the middle cerebral artery. Animals were randomized to receive RIC, or sham surgery, after reperfusion. The primary end point was a good functional outcome at 48 hours, assessed using a composite functional neuroscore. Secondary end points were infarct volume at 48 hours and safety, assessed using a standardized health report at 24 and 48 hours. Preenrollment harmonization, centralized monitoring, allocation concealment, blinded outcome assessment, and intention-to-treat analysis were applied. RESULTS: The trial enrolled 164 rodents (82 mice and 82 rats) of both sexes (53% females), across 7 laboratories. A greater proportion of RIC-treated rodents achieved a favorable functional outcome compared with controls, at 48 hours postischemia (55% versus 36%; odds ratio, 2.2 [95% CI, 1.23–4.4]; P =0.009). RIC was associated with a small reduction in infarct volume (standardized mean difference, −0.38 [95% CI, −0.70 to −0.05]; P =0.024). Health monitoring indicated no major safety concerns, and postoperative analgesia requirements were lower in RIC-treated mice. CONCLUSIONS: Surgically induced RIC provided a modest but evident neuroprotective effect in experimental ischemic stroke, underscoring the potential of this strategy as an adjunctive treatment in stroke care. The findings of the TRICS BASIC study highlighted the importance of multicenter preclinical trials in addressing variability and enhancing translational validity. REGISTRATION: URL: https://www.preclinicaltrials.eu ; Unique identifier: PCTE0000177.

Phytochemicals gained substantial interest for their protective action in cerebral ischemia. Additionally, there is a growing body of evidence suggesting that miRs play a role in stroke therapy. However, no systematic review to date has summarized the impact of phytochemicals on miRs modulation and their role in protecting the brain against ischemia. This systematic review aims to assess the current literature on the protective action of phytochemicals in cellular and animal models of stroke, with special focus on the miRs modulation. A literature search was conducted across three databases (PubMed, Scopus, Embase), adhering to PRISMA guidelines. Studies published in English between 2014 and 2024 were included based on relevant keywords. A total of 224 articles were excluded based on their title and/or abstract as they were deemed irrelevant. Furthermore, 4 articles were excluded due to the lack of an English or full‐text version. Finally, 14 articles were included in this review. Phytochemicals reverted ischemia‐induced changes in miRs expression; that is, they upregulated miR‐556‐3p, miR‐223‐3p, miR‐1287‐5p, miR‐214, miR‐96, miR‐450b‐5p, miR‐149‐5p, miR‐375, and downregulated miR‐145‐5p, miR‐124, miR‐128‐3p, miR‐122, miR‐181b, miR‐155, miR‐134, and miR‐128‐3p. These, in turn, led to an inhibition of apoptosis, inflammation, oxidative stress, ferroptosis, pyroptosis, and activation of Wnt and AMPK signaling. Our analysis indicates that targeting miRs with phytochemicals may inhibit the ischemic injury; thus, it represents a promising tool for the development of new therapeutic strategies for stroke. Given that all of the reviewed studies utilized cellular or animal models, further human clinical trials are necessary to confirm these findings.

Benzophenone-3 (BP-3), commonly used as a UV filter in personal care products and as a stabilizer, is an alleged endocrine disruptor with potential neurodevelopmental impacts. Despite its abundance in the environment, the studies on its effect on brain development are scarce, especially in terms of multigenerational impact. In this work, for the first time, we examined neurotoxic and pro-apoptotic effects of BP-3 on mouse brain regions (cerebral cortex and hippocampus) in both the first (F₁) and second (F₂) generations after maternal exposure to environmentally relevant BP-3 levels. We found disregulated markers of cell damage (LDH, H₂O₂, caspase-3 and -8) and observed increased expression of pro-apoptotic Fas/FAS or Fasl/FASL. BP-3 exposure disrupted the BAX/BCL2 pathway, showing stronger effects in the F₁ than in the F₂ generation, with a dominance of extrinsic pathway (FAS, FASL, caspase-8) over intrinsic one (BAX, BCL2), suggesting that BP-3-induced apoptosis primarily operates via the extrinsic pathway and could impair brain homeostasis across generations. This study underscores the potential of BP-3 to increase multigenerational risks associated with disrupted neurodevelopment and highlights the importance of understanding its long-term neurotoxic effects.

Triclocarban is a highly effective and broadly used antimicrobial agent. Humans are continually exposed to triclocarban, but the safety of prenatal exposure to triclocarban in the context of neurodevelopment remains unknown. In this study, we demonstrated for the first time that mice that had been prenatally exposed to environmentally relevant doses of triclocarban had impaired estrogen receptor 1 (ESR1) signaling in the brain. These mice displayed decreased mRNA and protein expression levels of ESR1 as well as hypermethylation of the Esr1 gene in the cerebral cortex. Prenatal exposure to triclocarban also diminished the mRNA expression of Esr2, Gper1, Ahr, Arnt, Cyp19a1, Cyp1a1, and Atg7, and the protein levels of CAR, ARNT, and MAP1LC3AB in female brains and decreased the protein levels of BCL2, ARNT, and MAP1LC3AB in male brains. In addition, exposure to triclocarban caused sex-specific alterations in the methylation levels of global DNA and estrogen receptor genes. Microarray and enrichment analyses showed that, in males, triclocarban dysregulated mainly neurogenesis-related genes, whereas, in females, the compound dysregulated mainly neurotransmitter-related genes. In conclusion, our data identified triclocarban as a neurodevelopmental risk factor that particularly targets ESR1, affects apoptosis and autophagy, and in sex-specific ways disrupts the epigenetic status of brain tissue and dysregulates the postnatal expression of neurogenesis- and neurotransmitter-related genes.

Nearly 18 million people died from cardiovascular diseases in 2019, of these 85% were due to heart attack and stroke. The available therapies although efficacious, have narrow therapeutic window and long list of contraindications. Therefore, there is still an urgent need to find novel molecular targets that could protect the brain and heart against ischemia without evoking major side effects. Nuclear receptors are one of the promising targets for anti-ischemic drugs. Modulation of estrogen receptors (ERs) and peroxisome proliferator-activated receptors (PPARs) by their ligands is known to exert neuro-, and cardioprotective effects through anti-apoptotic, anti-inflammatory or anti-oxidant action. Recently, it has been shown that the expression of aryl hydrocarbon receptor (AhR) is strongly increased after brain or heart ischemia and evokes an activation of apoptosis or inflammation in injury site. We hypothesize that activation of ERs and PPARs and inhibition of AhR signaling pathways could be a promising strategy to protect the heart and the brain against ischemia. In this Review, we will discuss currently available knowledge on the mechanisms of action of ERs, PPARs and AhR in experimental models of stroke and myocardial infarction and future perspectives to use them as novel targets in cardiovascular diseases.

Introduction Multicentre preclinical randomised controlled trials (pRCT) are emerging as a necessary step to confirm efficacy and improve translation into the clinic. The aim of this project is to perform two multicentre pRCTs (one in rats and one in mice) to investigate the efficacy of remote ischaemic conditioning (RIC) in an experimental model of severe ischaemic stroke. Methods and analysis Seven research laboratories within the Italian Stroke Organization (ISO) Basic Science network will participate in the study. Transient endovascular occlusion of the proximal right middle cerebral artery will be performed in two species (rats and mice) and in both sexes. Animals will be randomised to receive RIC by transient surgical occlusion of the right femoral artery, or sham surgery, after reperfusion. Blinded outcome assessment will be performed for dichotomised functional neuroscore (primary endpoint) and infarct volume (secondary endpoint) at 48 hours. A sample size of 80 animals per species will yield 82% power to detect a significant difference of 30% in the primary outcome in both pRCTs. Analyses will be performed in a blind status and according to an intention-to-treat paradigm. The results of this study will provide robust, translationally oriented, high-quality evidence on the efficacy of RIC in multiple species of rodents with large ischaemic stroke. Ethics and dissemination This is approved by the Animal Welfare Regulatory Body of the University of Milano Bicocca, under project license from the Italian Ministry of Health. Trial results will be subject to publication according to the definition of the outcome presented in this protocol. Trial registration number PCTE0000177.

Each year, 1 million children die due to perinatal asphyxia; however, there are no effective drugs to protect the neonatal brain against hypoxic/ischemic damage. In this study, we demonstrated for the first time the neuroprotective capacity of 3,3’-diindolylmethane (DIM) in an in vivo model of rat perinatal asphyxia, which has translational value and corresponds to hypoxic/ischemic episodes in human newborns. Posttreatment with DIM restored the weight of the ipsilateral hemisphere and normalized cell number in the brain structures of rats exposed to perinatal asphyxia. DIM also downregulated the mRNA expression of HIF1A-regulated Bnip3 and Hif1a which is a hypoxic marker, and the expression of miR-181b which is an indicator of perinatal asphyxia. In addition, DIM inhibited apoptosis and oxidative stress accompanying perinatal asphyxia through: downregulation of FAS, CASP-3, CAPN1, GPx3 and SOD-1, attenuation of caspase-9 activity, and upregulation of anti-apoptotic Bcl2 mRNA. The protective effects of DIM were accompanied by the inhibition of the AhR and NMDA signaling pathways, as indicated by the reduced expression levels of AhR, ARNT, CYP1A1, GluN1 and GluN2B, which was correlated with enhanced global DNA methylation and the methylation of the Ahr and Grin2b genes. Because our study provided evidence that in rat brain undergoing perinatal asphyxia, DIM predominantly targets AhR and NMDA, we postulate that compounds that possess the ability to inhibit their signaling are promising therapeutic tools to prevent stroke.