Alcian blue staining has been used to visualise nanoparticles at transmission electron microscop

F. Carton

G. Lollo

M. Malatesta

REPELLIN, MATHIEU

English

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rerio fish1, optimal model organism for ecotoxicological analysisand for the study of neurodegenerative and neurobehavioral dis-eases2. In order to assess the toxic effects of this metal, Daniorerio embryos at shield stage were exposed to AlCl3 at the con-centrations of 50, 100 and 200 μM respectively for 72 h. Wecompared the swimming performances of treated larvae withthose of the control larvae, assessing different parameters likeDistance moved, Velocity mean, Cumulative movement, Meanderand Heading using the DanioVision instrument. Collected datashowed that AlCl3 significantly affected the behavioural parame-ters with a trend inversely proportional to the concentrations, infact the performances worsen at low concentrations compared tohigher doses3. In this light, we analysed mRNA expression levelby qPCR of different marker genes of neural development andfunction, including c-fos, appa and appb. C-fos is an immediate-early gene often used as indirect marker of neuronal activity4,while appa and appb are the homolog genes of the mammalianamyloid precursor protein (APP), an essential gene fornormal brain development and a key player for the Alzheimer’sdisease pathogenesis5. We observed that the expression of thesegenes was affected by AlCl3. The results confirmed toxic effect ofAlCl3 on D. rerio larvae, suggesting the need for further experi-ments to uncover the mechanisms by which the aluminium expo-sure affects the normal developmental processes and might be atbasis of neurological and behavioural disorders.References1. Monaco A, et al. J Appl Toxicol 2017; 36:1629-38.2. Senger MB, et al. Cell Biol Toxicol 2011 ;27:199-205.3. Capriello T, et al. Chemosphere 2019 ;222:243-249.4. Chung L. Dev Reprod 2015;19(2):61-7.5. Abramsson A, et al. Dev Biol 2013;381(2):377-88.NOVEL APPLICATIONS OF LONG-ESTABLISHED HISTO-CHEMICAL TECHNIQUES TO STUDY NANOPARTICLE-CELL INTERACTIONS AT TRANSMISSION ELECTRONMICROSCOPYF. Carton1,2, M. Repellin1,2*, G. Lollo2, M. Malatesta11Dept. of Neurosciences, Biomedicine and Movement Sciences,University of Verona, Italy; 2University of Lyon, Université ClaudeBernard Lyon 1, CNRS, LAGEPP UMR 5007, FranceE-mail: flavia.carton@univr.itTransmission electron microscopy (TEM) is the technique ofchoice to explore the effects of nanocomposites on biological sys-tems. The high resolution of TEM allows the location and dynam-ic tracking of nanoparticles (NPs) inside cells and tissues, pro-viding crucial information on their actual interactions. It is thusmandatory that NPs are unequivocally detected in the inter- andintracellular space. This is easily obtained for NPs containingelectron dense components such as metal ions, but it may be dif-ficult when they are made of organic components (e.g., polymersor lipids) whose moderate electron density makes them hardlydiscernible in the cytosolic milieu. We faced this situation withvarious types of NPs and solved the problem by setting up novelapplications for long-established histochemical techniques.Chitosan-based and phospholipidic NPs were made clearly visi-ble at TEM by labelling them with fluorochromes during theirsynthesis, and subsequently applying diaminobenzidine (DAB)photo-oxidation, that gives rise to a finely granular electrondense product thanks to the reactive oxygen species originatingupon fluorochrome irradiation1,2. By this method, not only theuptake mechanisms and intracellular distribution of these NPswere revealed, but also their degradation pathways, thanks to thepresence of DAB precipitates on the NPs remnants inside sec-ondary lysosomes and residual bodies. However, in the absence offluorochrome labelling DAB photo-oxidation cannot be applied:as an alternative approach, specific histochemical methods givingrise to electron dense products may be used, such as Alcian bluestaining3. Hyaluronic acid-based NPs were thus visualized atTEM, and we were able to describe the very early step of theiruptake as well as their degradation, which were impossible to getby conventional morphology. *INVITE project received funding from EU Horizon 2020 GANo 754345.References1. Malatesta M, et al. Eur J Histochem 2012 ;56(2):e20.2. Costanzo M, et al. Int J Pharm 2016;508(1-2):83-91.3. Schofield BH, et al. Histochem J 1975;7(2):139-49.EFFECTS OF MOLECULES WITH NEUROTROPHICACTIVITY IN AN IN VITRO MODEL OF PARKINSON’SDISEASEM. Catanesi1, M. d’Angelo1, R. Benot1, A. Antonosante1, E. Benedetti1, A. Cimini1,21Department of Life, Health e Environmental Sciences; Universityof l’Aquila, Italy; 2Sbarro Institute Dept of Biology, TempleUniversity, Philadelphia, USA E-mail: mariano.catanesi86@gmail.comOxidative stress results from an in balance between oxidativespecies and scavenging antioxidant systems. Reactive oxygenspecies (ROS) and reactive nitrogen species (RNS) may be eitherharmful or beneficial to the cells, depending on their concentra-tions. Methionine acts as amino acid precursor for glutathionethat protects the cells from oxidative damage and plays pivotalrole in cell detoxification from oxidative stress1. The aim of thisresearch was to study the neurotrophic and antioxidant activitiesof methionine and taurine on an in vitro model of PD. Themethionine and taurine effects were first evaluated on an oxida-tive model based on H2O2 treatment, then followed by the set-upof a PD in vitro model by treating dopaminergic neurons with 6-OHDA2. The effects of methionine and taurine were evaluated byMTS assay, Western Blotting analysis and Immunofluorescence.Methionine and taurine were both able to counteract the pro-oxidative death effects of H2O2 and 6-OHDA by decreasing theapoptotic markers (caspase 9, Bcl-2), by modulating oxidativestress markers (Mn-sod, catalase) and the oxidative stress index(ratio Mn-sod/catalase); by increasing the PI3K/AKT survivalpathway and antioxidant markers like a Nrf2. The results so farobtained confirmed the potential neuroprotective activities ofmethionine and taurine in the PD in vitro model. The study willcontinue by evaluating the possible protective activities of thesemolecules on mitochondrial dysfunctions using an oxidativephosphorylation uncoupling like a TMRM in live cell, JC-1 andthe activation of pro-survival pathways depending on neu-rotrophine modulation in the PD in vitro model.References1. Martínez Y, et al Amino Acids 2017;49:2091-98. 2. Lopes FM, et al.  Brain Res 2010;1337:85-94.A NEW INSIGHT IN THE AXON AND DENDRITIC DEVEL-OPMENT: THE FMRP-RACK1 PARTYM. Ceci1, B. Di Giacomo1, V. Nobile2, S. Proietti1, M. Agrawal3, K. Welshhans3, N. Romano11Department of Ecological and Biological Sciences (DEB),University of Tuscia, Largo dell’Università,Viterbo, Italy;2Institute of Genomic Medicine, Catholic University, andFondazione Policlinico Universitario “A. Gemelli” IRCCS, Rome,Italy; 3School of Biomedical Sciences, Kent State University, E Summit St., Kent Ohio, USA,E-mail: m.ceci@unitus.it8Proceedings of the 65th Congress of the Italian Embryological Group (GEI - SIBSC) - 38th Congress of the Italian Society of Histochemistry (SII)

Novel applications of long-established histochemical techniques to study nanoparticle-cell interactions at transmission electron microscopy

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Novel applications of long-established histochemical techniques to study nanoparticle-cell interactions at transmission electron microscopy

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