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Spectral imaging is a fluorescence microscopy technique with several applications, including imaging of environment-sensitive probes, spectral unmixing and identification of fluorescent species. In confocal microscopes not equipped with a spectral detection unit, spectral images can be obtained using the lambda scan mode of the microscope, namely the sequential acquisition of images using a tunable emission filter or other dispersive optical elements. Unfortunately, the lambda scan mode has poor temporal resolution, is a photon-wasting technique, and is not ideal for the spectral imaging of live samples. Here, we describe a spectral imaging method that can be implemented on commercial confocal microscopes not equipped with a spectral detector. The method is based on simultaneous image acquisition in 4 contiguous spectral channels and spectral phasor analysis. We demonstrate that this method can be easily implemented on a Leica confocal laser scanning microscope, with better photon efficiency and temporal resolution than the lambda scan mode. We perform a 4-channel (4 C) spectral phasor analysis of live cells stained with the environment-sensitive ACDAN and Nile Red dyes. We can distinguish changes in spectral emission in the order of 5 nm between different subcellular compartments. We show that 4 C-spectral phasor can be used to decompose the Nile Red signal into 2 components and perform 3-color imaging in combination with a DNA dye in live organoids. Finally, we show that the 4 C-spectral phasor can be also used to unmix the signal of fluorescent proteins with overlapping emission spectra such as mEmerald and EYFP.

Different drug classes such as antineoplastic drugs (anthracyclines, cyclophosphamide, 5-fluorouracil, taxanes, tyrosine kinase inhibitors), antiretroviral drugs, antipsychotic, and immunosuppressant drugs are known to induce cardiotoxic and neurotoxic effects. Recent studies have demonstrated that the impairment of the nuclear factor erythroid 2–related factor 2 (Nrf2) pathway is a primary event in the pathophysiology of drug-induced cardiotoxicity and neurotoxicity. The Nrf2 pathway regulates the expression of different genes whose products are involved in antioxidant and inflammatory responses and the detoxification of toxic species. Cardiotoxic drugs, such as the anthracycline doxorubicin, or neurotoxic drugs, such as paclitaxel, suppress or impair the Nrf2 pathway, whereas the rescue of this pathway counteracts both the oxidative stress and inflammation that are related to drug-induced cardiotoxicity and neurotoxicity. Therefore Nrf2 represents a novel pharmacological target to develop new antidotes in the field of clinical toxicology. Interestingly, carnosine (β-alanyl-l-histidine), an endogenous dipeptide that is characterized by strong antioxidant, anti-inflammatory, and neuroprotective properties is able to rescue/activate the Nrf2 pathway, as demonstrated by different preclinical studies and preliminary clinical evidence. Starting from these new data, in the present review, we examined the evidence on the therapeutic potential of carnosine as an endogenous antidote that is able to rescue the Nrf2 pathway and then counteract drug-induced cardiotoxicity and neurotoxicity.

The activity of microglia is fundamental for the regulation of numerous physiological processes including brain development, synaptic plasticity, and neurogenesis, and its deviation from homeostasis can lead to pathological conditions, including numerous neurodegenerative disorders. Carnosine is a naturally occurring molecule with well-characterized antioxidant and anti-inflammatory activities, able to modulate the response and polarization of immune cells and ameliorate their cellular energy metabolism. The better understanding of microglia characteristics under basal physiological conditions, as well as the possible modulation of the mechanisms related to its response to environmental challenges and/or pro-inflammatory/pro-oxidant stimuli, are of utmost importance for the development of therapeutic strategies. In the present study, we assessed the activity of carnosine on human HMC3 microglial cells, first investigating the effects of increasing concentrations of carnosine on cell viability. When used at a concentration of 20 mM, carnosine led to a decrease of cell viability, paralleled by gene expression increase and decrease, respectively, of interleukin 6 and heme oxygenase 1. When using the maximal non-toxic concentration (10 mM), carnosine decreased nitric oxide bioavailability, with no changes in the intracellular levels of superoxide ion. The characterization of energy metabolism of HMC3 microglial cells under basal conditions, never reported before, demonstrated that it is mainly based on mitochondrial oxidative metabolism, paralleled by a high rate of biosynthetic reactions. The exposure of HMC3 cells to carnosine seems to ameliorate microglia energy state, as indicated by the increase in the adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio and energy charge potential. The improvement of cell energy metabolism mediated by 10 mM carnosine could represent a useful protective weapon in the case of human microglia undergoing stressing conditions.

In point-scanning microscopy, optical sectioning is achieved using a small aperture placed in front of the detector, i.e. the detection pinhole, which rejects the out-of-focus background. The maximum level of optical sectioning is theoretically obtained for the minimum size of the pinhole aperture, but this is normally prevented by the dramatic reduction of the detected signal when the pinhole is closed, leading to a compromise between axial resolution and signal-to-noise ratio. We have recently demonstrated that, instead of closing the pinhole, one can reach a similar level of optical sectioning by tuning the pinhole size in a confocal microscope and by analyzing the resulting image series. The method, consisting in the application of the separation of photons by lifetime tuning (SPLIT) algorithm to series of images acquired with tunable pinhole size, is called SPLIT-pinhole (SPLIT-PIN). Here, we share and describe a SPLIT-PIN software for the processing of series of images acquired at tunable pinhole size, which generates images with reduced out-of-focus background. The software can be used on series of at least two images acquired on available commercial microscopes equipped with a tunable pinhole, including confocal and stimulated emission depletion (STED) microscopes. We demonstrate applicability on different types of imaging modalities: (1) confocal imaging of DNA in a non-adherent cell line; (2) removal of out-of-focus background in super-resolved STED microscopy; (3) imaging of live intestinal organoids stained with a membrane dye.

Depression is a risk factor for the development of Alzheimer’s disease (AD). A neurobiological and clinical continuum exists between AD and depression, with neuroinflammation and oxidative stress being involved in both diseases. Second-generation antidepressants, in particular selective serotonin reuptake inhibitors (SSRIs), are currently investigated as neuroprotective drugs in AD. By employing a non-transgenic AD model, obtained by intracerebroventricular (i.c.v.) injection of amyloid-β (Aβ) oligomers in 2-month-old C57BL/6 mice, we recently demonstrated that the SSRI fluoxetine (FLX) and the multimodal antidepressant vortioxetine (VTX) reversed the depressive-like phenotype and memory deficits induced by Aβ oligomers rescuing the levels of transforming growth factor-β1 (TGF-β1). Aim of our study was to test FLX and VTX for their ability to prevent oxidative stress in the hippocampus of Aβ-injected mice, a brain area strongly affected in both depression and AD. The long-term intraperitoneal (i.p.) administration of FLX (10 mg/kg) or VTX (5 and 10 mg/kg) for 24 days, starting 7 days before Aβ injection, was able to prevent the over-expression of inducible nitric oxide synthase (iNOS) and NADPH oxidase 2 (Nox2) induced by Aβ oligomers. Antidepressant pre-treatment was also able to rescue the mRNA expression of glutathione peroxidase 1 (Gpx1) antioxidant enzyme. FLX and VTX also prevented Aβ-induced neurodegeneration in mixed neuronal cultures treated with Aβ oligomers. Our data represent the first evidence that the long-term treatment with the antidepressants FLX or VTX can prevent the oxidative stress phenomena related to the cognitive deficits and depressive-like phenotype observed in a non-transgenic animal model of AD.

Carnosine (beta-alanyl-L-histidine) is an endogenous dipeptide synthesized via the activity of the ATP-dependent enzyme carnosine synthetase 1 and can be found at a very high concentration in tissues with a high metabolic rate, including muscles (up to 20 mM) and brain (up to 5 mM). Because of its well-demonstrated multimodal pharmacodynamic profile, which includes anti-aggregant, antioxidant, and anti-inflammatory activities, as well as its ability to modulate the energy metabolism status in immune cells, this dipeptide has been investigated in numerous experimental models of diseases, including Alzheimer’s disease, and at a clinical level. The main limit for the therapeutic use of carnosine is related to its rapid hydrolysis exerted by carnosinases, especially at the plasma level, reason why the development of new strategies, including the chemical modification of carnosine or its vehiculation into innovative drug delivery systems (DDS), aiming at increasing its bioavailability and/or at facilitating the site-specific transport to different tissues, is of utmost importance. In the present review, after a description of carnosine structure, biological activities, administration routes, and metabolism, we focused on different DDS, including vesicular systems and metallic nanoparticles, as well as on possible chemical derivatization strategies related to carnosine. In particular, a basic description of the DDS employed or the derivatization/conjugation applied to obtain carnosine formulations, followed by the possible mechanism of action, is given. To the best of our knowledge, this is the first review that includes all the new formulations of carnosine (DDS and derivatives), allowing a decrease or complete prevention of the hydrolysis of this dipeptide exerted by carnosinases, the simultaneous blood–brain barrier crossing, the maintenance or enhancement of carnosine biological activity, and the site-specific transport to different tissues, which then offers perspectives for the development of new drugs.

Vinpocetine (VNP), a semisynthetic active pharmaceutical ingredient, is used for oral management of cerebrovascular diseases because of its ability to enhance the blood flow to the brain. However, despite that, the therapeutic application of VNP is restricted due to its reduced bioavailability and diminished brain levels that could be attributed to its low aqueous solubility, short half-life, and presystemic metabolism exposure. Accordingly, the goal of this work was to explore the ability of surface-tailored intranasal emulsomes to boost brain delivery of the drug. A 3 2 2 1 factorial design was implemented to explore the impact of phospholipid (PL) to solid lipid weight ratio, PL to cholesterol molar ratio, and type of solid lipid on vesicle size, zeta potential, drug entrapment, and release efficiency of the new developed VNP emulsomes. Tailoring of the optimized emulsomal surface formulation was performed using either cationization or PEGylation approaches to boost blood-brain barrier penetration. The pharmacokinetic assessment in rats showed significantly improved bioavailability of VNP emulsomal formulations compared to the oral market product. Additionally, surface-tailored emulsomes exhibited significantly higher brain levels compared to the optimized emulsomes. Based on these findings, the proposed surface-tailored emulsomes could be considered as a promising platform for achieving high brain levels of VNP following intranasal administration.

Stressful experiences early in life, especially in the prenatal period, can increase the risk to develop depression during adolescence. However, there may be important qualitative and quantitative differences in outcome of prenatal stress (PNS), where some individuals exposed to PNS are vulnerable and develop a depressive-like phenotype, while others appear to be resilient. PNS exposure, a well-established rat model of early life stress, is known to increase vulnerability to depression and a recent study demonstrated a strong interaction between transforming growth factor-β1 (TGF-β1) gene and PNS in the pathogenesis of depression. Moreover, it is well-known that the exposure to early life stress experiences induces brain oxidative damage by increasing nitric oxide levels and decreasing antioxidant factors. In the present work, we examined the role of TGF-β1 pathway in an animal model of adolescent depression induced by PNS obtained by exposing pregnant females to a stressful condition during the last week of gestation. We performed behavioral tests to identify vulnerable or resilient subjects in the obtained litters (postnatal day, PND > 35) and we carried out molecular analyses on hippocampus, a brain area with a key role in the pathogenesis of depression. We found that female, but not male, PNS adolescent rats exhibited a depressive-like behavior in forced swim test (FST), whereas both male and female PNS rats showed a deficit of recognition memory as assessed by novel object recognition test (NOR). Interestingly, we found an increased expression of type 2 TGF-β1 receptor (TGFβ-R2) in the hippocampus of both male and female resilient PNS rats, with higher plasma TGF-β1 levels in male, but not in female, PNS rats. Furthermore, PNS induced the activation of oxidative stress pathways by increasing inducible nitric oxide synthase (iNOS), NADPH oxidase 1 (NOX1) and NOX2 levels in the hippocampus of both male and female PNS adolescent rats. Our data suggest that high levels of TGF-β1 and its receptor TGFβ-R2 can significantly increase the resiliency of adolescent rats to PNS, suggesting that TGF-β1 pathway might represent a novel pharmacological target to prevent adolescent depression in rats.

Microglia are a key driver of neurodegenerative disease, orchestrating inflammatory signaling, metabolic stress responses, synaptic remodeling, and neuronal fate within the central nervous system (CNS). Among experimental models, the human microglial cell line, HMC3, is one of the most widely used models for mechanistic investigation and pharmacological screening of microglial dysfunction, particularly in neurodegenerative contexts. Nevertheless, a key question remains: how faithfully does HMC3 reflect human microglial biology? This review integrates current evidence on HMC3 cells, including their molecular and metabolic features, functional plasticity, and disease-oriented applications. HMC3 cells reproduce hallmark neurodegeneration-associated programs, such as stimulus-dependent polarization, oxidative and endoplasmic reticulum stress signaling, inflammasome activation, autophagy dysregulation, lipid remodeling, angiogenic cross-talk, and phagocytic clearance of amyloid and apoptotic debris, modeling processes relevant to Alzheimer's disease, Parkinson's disease, ischemic injury, and metabolic neurodegeneration. Neuron-microglia co-culture systems further demonstrate the direct impacts of HMC3 activation states on neuronal vulnerability and survival. We also summarize the expanding repertoire of pharmacological and genetic interventions applied to HMC3, highlighting their compatibility with high-throughput and multi-omics discovery platforms. Despite inherent limitations of immortalized models, HMC3 represents a powerful front-line tool for dissecting neurodegenerative microglial mechanisms and steering early therapeutic discovery.

Carnosine (β-alanyl-L-histidine) is a naturally occurring endogenous peptide widely distributed in excitable tissues such as the brain. This dipeptide possesses well-demonstrated antioxidant, anti-inflammatory, and anti-aggregation properties, and it may be useful for treatment of pathologies characterized by oxidative stress and energy unbalance such as depression and Alzheimer’s disease (AD). Microglia, the brain-resident macrophages, are involved in different physiological brain activities such synaptic plasticity and neurogenesis, but their dysregulation has been linked to the pathogenesis of numerous diseases. In AD brain, the activation of microglia towards a pro-oxidant and pro-inflammatory phenotype has found in an early phase of cognitive decline, reason why new pharmacological targets related to microglia activation are of great importance to develop innovative therapeutic strategies. In particular, microglia represent a common model of lipopolysaccharides (LPS)-induced activation to identify novel pharmacological targets for depression and AD and numerous studies have linked the impairment of energy metabolism, including ATP dyshomeostasis, to the onset of depressive episodes. In the present study, we first investigated the toxic potential of LPS + ATP in the absence or presence of carnosine. Our studies were carried out on human microglia (HMC3 cell line) in which LPS + ATP combination has shown the ability to promote cell death, oxidative stress, and inflammation. Additionally, to shed more light on the molecular mechanisms underlying the protective effect of carnosine, its ability to modulate reactive oxygen species production and the variation of parameters representative of cellular energy metabolism was evaluated by microchip electrophoresis coupled to laser-induced fluorescence and high performance liquid chromatography, respectively. In our experimental conditions, carnosine prevented LPS + ATP-induced cell death and oxidative stress, also completely restoring basal energy metabolism in human HMC3 microglia. Our results suggest a therapeutic potential of carnosine as a new pharmacological tool in the context of multifactorial disorders characterize by neuroinflammatory phenomena including depression and AD.