Safety of Innovative Nanotechnology Oral Formulations Loaded with Bioactive Menopause Molecules: Influence of Genotoxicity and Biochemical Parameters on a Menopausal Rat Model.

In recent years, nanoparticles have gained significant importance due to their unique properties, such as pharmacological, electrical, optical, and magnetic abilities, contributing to the growth of the science and technology sector. Particular naturally derived biomolecules with beneficial effects on menopause disorder have been the subject of studies of pharmaceutical formulation to obtain alternative pharmaceutical forms with increased bioavailability and without side effects, as in nanostructured lipid carriers (NLCs) loaded with such active ingredients. In the present study, one stage of a broader project, we have performed pharmacotoxicology studies for six combinatory innovative nanocapsule pharmaceutical forms containing active natural biomolecules before considering them as oral formulas for (1) in vitro toxicity studies on culture cells and (2) in vivo preclinical studies on a surgically induced menopause model of Wistar female rats, and the influence of the NLCs on key biochemical parameters: lipid profile (TG, Chol, HDL), glycemic markers (Gli), bone markers (Pac, Palc, Ca, phosphorus), renal markers (Crea, urea, URAC), inflammation (TNF), oxidative stress (GSH, MDA), and estrogen-progesterone hormonal profile. The micronucleus test did not reveal the genotoxicity of the tested compounds; the menopause model showed no significant safety concerns for the six tested formulas evaluated using the blood biochemical parameters; and the results showed the potential hypoglycemic, hypolipidemic, hypouricemic, and antioxidant potential of one of the tested formulas containing nano diosgenin and glycyrrhizic acid.

Modifications in cellular viability, DNA damage and stress responses inflicted in cancer cells by copper-64 ions.

Due to combined therapeutical emissions, a high linear energy transfer Auger-electrons with the longer ranged ß- particles, 64Cu-based radiopharmaceuticals raise particular theragnostic interest in cancer, by joined therapeutic and real-time PET imaging properties. The in vitro study aimed to investigate the biological and molecular background of 64CuCl2 therapy by analyzing the damages and stress responses inflicted in various human normal and tumor cell lines. Colon (HT29 and HCT116) and prostate carcinoma (DU145) cell lines, as well as human normal BJ fibroblasts, were treated up to 72 h with 2-40 MBq/mL 64CuCl2. Radioisotope uptake and retention were assessed, and cell viability/death, DNA damage, oxidative stress, and the expression of 84 stress genes were investigated at various time points after [64Cu]CuCl2 addition. All the investigated cells incorporated 64Cu ions similarly, independent of their tumoral or normal status, but their fate after exposure to [64Cu]CuCl2 was cell-dependent. The most striking cytotoxic effects of the radioisotope were registered in colon carcinoma HCT116 cells, for which a substantial decrease in the number of metabolically active cells, and an increased DNA damage and oxidative stress were registered. The stress gene expression study highlighted the activation of both death and repair mechanisms in these cells, related to extrinsic apoptosis, necrosis/necroptosis or autophagy, and cell cycle arrest, nucleotide excision repair, antioxidant, and hypoxic responses, respectively. The in vitro study indicated that 40 MBq/mL [64Cu]CuCl2 delivers a therapeutic effect in human colon carcinoma, but its use is limited by harmful, yet lower effects on normal fibroblasts. The exposure of tumor cells to 20 MBq/mL [64Cu]CuCl2, might be used for a softer approach aiming for a lower radiotoxicity in normal fibroblasts as compared to tumor cells. This radioactive concentration was able to induce a persistent decrease in the number of metabolically active cells, accompanied by DNA damage and oxidative stress, associated with significant changes in stress gene expression in HCT116 colon cancer cells.

Chemistry-Induced Effects on Cell Behavior upon Plasma Treatment of pNIPAAM.

In the field of bioengineering, depending on the required application, the attachment of various biological entities to the biomaterial is either favored or needs to be prevented. Therefore, different surfaces modification strategies were developed in combination with the characteristics of the materials. The present contribution reports on the use of the specific surface property of a thermoresponsive polymer poly(N-isopropylacrylamide) pNIPAAM obtained by spin coating in combination with plasma treatment for tuning cell behavior on treated polymeric surfaces. Topographical information for the plasma-treated pNIPAAM coatings obtained by Atomic Force Microscopy (AFM) measurements evidenced a more compact surface for Ar treatment due to combined etching and redeposition, while for oxygen, a clear increase of pores diameter is noticed. The chemical surface composition as determined by X-ray Photoelectron Spectroscopy showed the specific modifications induced by plasma treatment, namely strong oxidation for oxygen plasma treatment illustrated by eight times increase of O-C=O contribution and respectively an increase of C-N/O=C-N bonds in the case of ammonia plasma treatment. Structural information provided by FTIR spectroscopy reveals a significant increase of the carboxylic group upon argon and mostly oxygen plasma treatment and the increase in width and intensity of the amide-related groups for the ammonia plasma treatment. The biological investigations evidenced that L929 fibroblast cells viability is increased by 25% upon plasma treatment, while the cell attachment is up to 2.8 times higher for the oxygen plasma-treated surface compared to the initial spin-coated pNIPAAM. Moreover, the cell detachment process proved to be up to 2-3 times faster for the oxygen and argon plasma-treated surfaces and up to 1.5 times faster for the ammonia-treated surface. These results show the versatility of plasma treatment for inducing beneficial chemical modifications of pNIPAAM surfaces that allows the tuning of cellular response for improving the attachment-detachment process in view of tissue engineering.

Laser Direct Writing via Two-Photon Polymerization of 3D Hierarchical Structures with Cells-Antiadhesive Properties.

We report the design and fabrication by laser direct writing via two photons polymerization of innovative hierarchical structures with cell-repellency capability. The structures were designed in the shape of "mushrooms", consisting of an underside (mushroom's leg) acting as a support structure and a top side (mushroom's hat) decorated with micro- and nanostructures. A ripple-like pattern was created on top of the mushrooms, over length scales ranging from several µm (microstructured mushroom-like pillars, MMP) to tens of nm (nanostructured mushroom-like pillars, NMP). The MMP and NMP structures were hydrophobic, with contact angles of (127 ± 2)° and (128 ± 4)°, respectively, whereas flat polymer surfaces were hydrophilic, with a contact angle of (43 ± 1)°. The cell attachment on NMP structures was reduced by 55% as compared to the controls, whereas for the MMP, a reduction of only 21% was observed. Moreover, the MMP structures preserved the native spindle-like with phyllopodia cellular shape, whereas the cells from NMP structures showed a round shape and absence of phyllopodia. Overall, the NMP structures were more effective in impeding the cellular attachment and affected the cell shape to a greater extent than the MMP structures. The influence of the wettability on cell adhesion and shape was less important, the cellular behavior being mainly governed by structures' topography.

Preparation and Preliminary Evaluation of Neurotensin Radiolabelled with 68Ga and 177Lu as Potential Theranostic Agent for Colon Cancer.

The neurotensin is a tridecapeptide involved in the proliferation of colon cancer, the overexpression of neurotensin receptors occurring at an early stage development of many tumours. Targeting neurotensin receptors by using the same biological active molecule is an effective approach for both imaging quantification and treatment. The present work aimed to demonstrate the ability of radiolabelled neurotensin to specifically target colon cancer cells, and substantiate its usefulness in targeted imaging and radiotherapy, depending on the emission of the coupled radioisotope. Syntheses of 68Ga-DOTA-NT and 177Lu-DOTA-NT were developed to obtain a level of quality suitable for preclinical use with consistent high synthesis yields. Radiochemical purity meets the pharmaceutical requirements, and it is maintained 4 h for 68Ga-DOTA-NT and 48 h for 177Lu-DOTA-NT. Extensive in vitro studies were conducted to assess the uptake and retention of 68Ga-DOTA-NT, the amount of non-specific binding of neurotensin and the effect of 177Lu-DOTA-NT on HT-29 cells. In vivo biodistribution of 68Ga-DOTA-NT revealed significant uptake at the tumour site, along with fast clearance evidenced by decreasing activity in kidneys and blood after 60 min p.i. 177Lu-DOTA-NT exhibited similar uptake in the tumour, but also a significant uptake at 14 days p.i. in the bone marrow was reported. These results successfully demonstrated the potential of neurotensin to deliver imaging/therapeutic 68Ga/177Lu radioisotopes pair, but also the need for further evaluation of the possible radiotoxicity effects on the liver, kidneys or bone marrow.

Enhanced Internalization of Nanoparticles Following Ionizing Radiation Leads to Mitotic Catastrophe in MG-63 Human Osteosarcoma Cells.

This study aims to investigate whether ionizing radiation combined with doxorubicin-conjugated iron oxide nanoparticles (NP-DOX) improves the internalization and cytotoxic effects of the nano-carrier-mediated drug delivery in MG-63 human osteosarcoma cells. NP-DOX was designed and synthesized using the co-precipitation method. Highly stable and crystalline nanoparticles conjugated with DOX were internalized in MG-63 cells through macropinocytosis and located in the perinuclear area. Higher nanoparticles internalization in MG-63 cells previously exposed to 1 Gy X-rays was correlated with an early accumulation of cells in G2/M, starting at 12 h after treatment. After 48 h, the application of the combined treatment led to higher cytotoxic effects compared to the individual treatment, with a reduction in the metabolic capacity and unrepaired DNA breaks, whilst a low percent of arrested cells, contributing to the commitment of mitotic catastrophe. NP-DOX showed hemocompatibility and no systemic cytotoxicity, nor histopathological alteration of the main organs.

Collagen/Chitosan Functionalization of Complex 3D Structures Fabricated by Laser Direct Writing via Two-Photon Polymerization for Enhanced Osteogenesis.

The fabrication of 3D microstructures is under continuous development for engineering bone substitutes. Collagen/chitosan (Col/CT) blends emerge as biomaterials that meet the mechanical and biological requirements associated with bone tissue. In this work, we optimize the osteogenic effect of 3D microstructures by their functionalization with Col/CT blends with different blending ratios. The structures were fabricated by laser direct writing via two-photons polymerization of IP-L780 photopolymer. They comprised of hexagonal and ellipsoidal units 80 µm in length, 40 µm in width and 14 µm height, separated by 20 µm pillars. Structures' functionalization was achieved via dip coating in Col/CT blends with specific blending ratios. The osteogenic role of Col/CT functionalization of the 3D structures was confirmed by biological assays concerning the expression of alkaline phosphatase (ALP) and osteocalcin secretion as osteogenic markers and Alizarin Red (AR) as dye for mineral deposits in osteoblast-like cells seeded on the structures. The structures having ellipsoidal units showed the best results, but the trends were similar for both ellipsoidal and hexagonal units. The strongest osteogenic effect was obtained for Col/CT blending ratio of 20/80, as demonstrated by the highest ALP activity, osteocalcin secretion and AR staining intensity in the seeded cells compared to all the other samples.

Preclinical Evaluation of NHS-Activated Gold Nanoparticles Functionalized with Bombesin or Neurotensin-Like Peptides for Targeting Colon and Prostate Tumours.

Recent advances and large-scale use of hybrid imaging modalities like PET-CT have led to the necessity of improving nano-drug carriers that can facilitate both functional and metabolic screening in nuclear medicine applications. In this study, we focused on the evaluation of four potential imaging nanoparticle structures labelled with the 68Ga positron emitter. For this purpose, we functionalized NHS-activated PEG-gold nanoparticles with 68Ga-DOTA-Neuromedin B, 68Ga-DOTA-PEG(4)-BBN(7-14), 68Ga-DOTA-NT and 68Ga-DOTA-Neuromedin N. In vitro binding kinetics and specific binding to human HT-29 colon carcinoma cells and DU-145 prostate carcinoma cells respectively were assessed, over 75% retention being obtained in the case of 68Ga-DOTA-PEG(4)-BBN(7-14)-AuNP in prostate tumour cells and over 50% in colon carcinoma cells. Biodistribution in NU/J mice highlighted a three-fold uptake increase in tumours at 30 min post-injection of 68Ga-DOTA-NT-AuNP and 68Ga-DOTA-PEG(4)-BBN(7-14)-AuNP compared to 68Ga-DOTA-NT and 68Ga-DOTA-PEG(4)-BBN(7-14) respectively, therewith fast distribution in prostate and colon tumours and minimum accumulation in non-targeted tissues.

3D Superparamagnetic Scaffolds for Bone Mineralization under Static Magnetic Field Stimulation.

We reported on three-dimensional (3D) superparamagnetic scaffolds that enhanced the mineralization of magnetic nanoparticle-free osteoblast cells. The scaffolds were fabricated with submicronic resolution by laser direct writing via two photons polymerization of Ormocore/magnetic nanoparticles (MNPs) composites and possessed complex and reproducible architectures. MNPs with a diameter of 4.9 ± 1.5 nm and saturation magnetization of 30 emu/g were added to Ormocore, in concentrations of 0, 2 and 4 mg/mL. The homogenous distribution and the concentration of the MNPs from the unpolymerized Ormocore/MNPs composite were preserved after the photopolymerization process. The MNPs in the scaffolds retained their superparamagnetic behavior. The specific magnetizations of the scaffolds with 2 and 4 mg/mL MNPs concentrations were of 14 emu/g and 17 emu/g, respectively. The MNPs reduced the shrinkage of the structures from 80.2 ± 5.3% for scaffolds without MNPs to 20.7 ± 4.7% for scaffolds with 4 mg/mL MNPs. Osteoblast cells seeded on scaffolds exposed to static magnetic field of 1.3 T deformed the regular architecture of the scaffolds and evoked faster mineralization in comparison to unstimulated samples. Scaffolds deformation and extracellular matrix mineralization under static magnetic field (SMF) exposure increased with increasing MNPs concentration. The results are discussed in the frame of gradient magnetic fields of ~3 × 10-4 T/m generated by MNPs over the cells bodies.

RNA-Binding Proteins HuB, HuC, and HuD are Distinctly Regulated in Dorsal Root Ganglia Neurons from STZ-Sensitive Compared to STZ-Resistant Diabetic Mice.

The neuron-specific Elav-like Hu RNA-binding proteins were described to play an important role in neuronal differentiation and plasticity by ensuring the post-transcriptional control of RNAs encoding for various proteins. Although Elav-like Hu proteins alterations were reported in diabetes or neuropathy, little is known about the regulation of neuron-specific Elav-like Hu RNA-binding proteins in sensory neurons of dorsal root ganglia (DRG) due to the diabetic condition. The goal of our study was to analyze the gene and protein expression of HuB, HuC, and HuD in DRG sensory neurons in diabetes. The diabetic condition was induced in CD-1 adult male mice with single-intraperitoneal injection of streptozotocin (STZ, 150 mg/kg), and 8-weeks (advanced diabetes) after induction was quantified the Elav-like proteins expression. Based on the glycemia values, we identified two types of responses to STZ, and mice were classified in STZ-resistant (diabetic resistant, glycemia < 260 mg/dL) and STZ-sensitive (diabetic, glycemia > 260 mg/dL). Body weight measurements indicated that 8-weeks after STZ-induction of diabetes, control mice have a higher increase in body weight compared to the diabetic and diabetic resistant mice. Moreover, after 8-weeks, diabetic mice (19.52 ± 3.52 s) have longer paw withdrawal latencies in the hot-plate test than diabetic resistant (11.36 ± 1.92 s) and control (11.03 ± 1.97 s) mice, that correlates with the installation of warm hypoalgesia due to the diabetic condition. Further on, we evidenced the decrease of Elav-like gene expression in DRG neurons of diabetic mice (Elavl2, 0.68 ± 0.05 fold; Elavl3, 0.65 ± 0.01 fold; Elavl4, 0.53 ± 0.07 fold) and diabetic resistant mice (Ealvl2, 0.56 ± 0.07 fold; Elavl3, 0.32 ± 0.09 fold) compared to control mice. Interestingly, Elav-like genes have a more accentuated downregulation in diabetic resistant than in diabetic mice, although hypoalgesia was evidenced only in diabetic mice. The Elav-like gene expression changes do not always correlate with the Hu protein expression changes. To detail, HuB is upregulated and HuD is downregulated in diabetic mice, while HuB, HuC, and HuD are downregulated in diabetic resistant mice compared to control mice. To resume, we demonstrated HuD downregulation and HuB upregulation in DRG sensory neurons induced by diabetes, which might be correlated with altered post-transcriptional control of RNAs involved in the regulation of thermal hypoalgesia condition caused by the advanced diabetic neuropathy.

Beta-Estradiol Regulates Voltage-Gated Calcium Channels and Estrogen Receptors in Telocytes from Human Myometrium.

Voltage-gated calcium channels and estrogen receptors are essential players in uterine physiology, and their association with different calcium signaling pathways contributes to healthy and pathological conditions of the uterine myometrium. Among the properties of the various cell subtypes present in human uterine myometrium, there is increasing evidence that calcium oscillations in telocytes (TCs) contribute to contractile activity and pregnancy. Our study aimed to evaluate the effects of beta-estradiol on voltage-gated calcium channels and estrogen receptors in TCs from human uterine myometrium and to understand their role in pregnancy. For this purpose, we employed patch-clamp recordings, ratiometric Fura-2-based calcium imaging analysis, and qRT-PCR techniques for the analysis of cultured human myometrial TCs derived from pregnant and non-pregnant uterine samples. In human myometrial TCs from both non-pregnant and pregnant uterus, we evidenced by qRT-PCR the presence of genes encoding for voltage-gated calcium channels (Cav3.1, Ca3.2, Cav3.3, Cav2.1), estrogen receptors (ESR1, ESR2, GPR30), and nuclear receptor coactivator 3 (NCOA3). Pregnancy significantly upregulated Cav3.1 and downregulated Cav3.2, Cav3.3, ESR1, ESR2, and NCOA3, compared to the non-pregnant condition. Beta-estradiol treatment (24 h, 10, 100, 1000 nM) downregulated Cav3.2, Cav3.3, Cav1.2, ESR1, ESR2, GRP30, and NCOA3 in TCs from human pregnant uterine myometrium. We also confirmed the functional expression of voltage-gated calcium channels by patch-clamp recordings and calcium imaging analysis of TCs from pregnant human myometrium by perfusing with BAY K8644, which induced calcium influx through these channels. Additionally, we demonstrated that beta-estradiol (1000 nM) antagonized the effect of BAY K8644 (2.5 or 5 µM) in the same preparations. In conclusion, we evidenced the presence of voltage-gated calcium channels and estrogen receptors in TCs from non-pregnant and pregnant human uterine myometrium and their gene expression regulation by beta-estradiol in pregnant conditions. Further exploration of the calcium signaling in TCs and its modulation by estrogen hormones will contribute to the understanding of labor and pregnancy mechanisms and to the development of effective strategies to reduce the risk of premature birth.

3D Biomimetic Magnetic Structures for Static Magnetic Field Stimulation of Osteogenesis.

We designed, fabricated and optimized 3D biomimetic magnetic structures that stimulate the osteogenesis in static magnetic fields. The structures were fabricated by direct laser writing via two-photon polymerization of IP-L780 photopolymer and were based on ellipsoidal, hexagonal units organized in a multilayered architecture. The magnetic activity of the structures was assured by coating with a thin layer of collagen-chitosan-hydroxyapatite-magnetic nanoparticles composite. In vitro experiments using MG-63 osteoblast-like cells for 3D structures with gradients of pore size helped us to find an optimum pore size between 20-40 µm. Starting from optimized 3D structures, we evaluated both qualitatively and quantitatively the effects of static magnetic fields of up to 250 mT on cell proliferation and differentiation, by ALP (alkaline phosphatase) production, Alizarin Red and osteocalcin secretion measurements. We demonstrated that the synergic effect of 3D structure optimization and static magnetic stimulation enhances the bone regeneration by a factor greater than 2 as compared with the same structure in the absence of a magnetic field.

Laser-direct writing by two-photon polymerization of 3D honeycomb-like structures for bone regeneration.

A major limitation of existing 3D implantable structures for bone tissue engineering is that most of the cells rapidly attach on the outer edges of the structure, restricting the cells penetration into the inner parts and causing the formation of a necrotic core. Furthermore, these structures generally possess a random spatial arrangement and do not preserve the isotropy on the whole volume. Here, we report on the fabrication and testing of an innovative 3D hierarchical, honeycomb-like structure (HS), with reproducible and isotropic arhitecture, that allows in 'volume' migration of osteoblasts. In particular, we demonstrate the possibility to control the 3D spatial cells growth inside these complex architectures by adjusting the free spaces inside the structures. The structures were made of vertical microtubes arranged in a mulitlayered configuration, fabricated via laser direct writing by two photons polymerization of the IP-L780 photopolymer. In vitro tests performed in MG-63 osteoblast-like cells demonstrated that the cells migration inside the 3D structures is conducted by the separation space between the microtubes layers. Specifically, for layers separation between 2 and 10 µm, the cells gradually penetrated between the microtubes. Furthermore, these structures induced the strongest cells osteogenic differentiation and mineralization, with ALP activity 1.5 times stronger, amount of calcified minerals 1.3 times higher and osteocalcin secretion increased by 2.3 times compared to the other structures. On the opposite, for layers separation less than 2 µm and above 10 µm, the cells were not able to make interconnections and exhibited poor mineralization ability.

Bystander effects and compartmental stress response to X-ray irradiation in L929 cells.

Bystander effects are indirect consequences of radiation and many other stress factors. They occur in cells that are not directly exposed to these factors, but receive signals from affected cells either by gap junctions or by molecules released in the medium. Characterizing these effects and deciphering the underlying mechanisms involved in radiation-induced bystander effects are relevant for cancer radiotherapy and radioprotection. At doses of X-ray radiation 0.5 and 1 Gy, we detected bystander effects as increased numbers of micronuclei shortly after the treatment, through medium transfer and by co-cultures. Interestingly, bystander cells did not exhibit long-term adverse changes in viability. Evaluation of several compartmental stress markers (CHOP, BiP, mtHsp60, cytHsp70) by qRT-PCR did not reveal expression changes at transcriptional level. We investigated the involvement of ROS and NO in this process by addition of specific scavengers of these molecules, DMSO or c-PTIO in the transferred medium. This approach proved that ROS but not NO is involved in the induction of lesions in the acceptor cells. These results indicate that L929 cells are susceptible to stress effects of radiation-induced bystander signaling.

Electrically stimulated osteogenesis on Ti-PPy/PLGA constructs prepared by laser-assisted processes.

This work describes a versatile laser-based protocol for fabricating micro-patterned, electrically conductive titanium-polypyrrole/poly(lactic-co-glycolic)acid (Ti-PPy/PLGA) constructs for electrically stimulated (ES) osteogenesis. Ti supports were patterned using fs laser ablation in order to create high spatial resolution microstructures meant to provide mechanical resistance and physical cues for cell growth. Matrix Assisted Pulsed Laser Evaporation (MAPLE) was used to coat the patterned Ti supports with PPy/PLGA layers acting as biocompatible surfaces having chemical and electrical properties suitable for cell differentiation and mineralization. In vitro biological assays on osteoblast-like MG63 cells showed that the constructs maintained cell viability without cytotoxicity. At 24 h after cell seeding, electrical stimulation with currents of 200 µA was applied for 4 h. This treatment was shown to promote earlier onset of osteogenesis. More specifically, the alkaline phosphatase activity of the stimulated cultures reached the maximum before that of the non-stimulated ones, i.e. controls, indicating faster cell differentiation. Moreover, mineralization was found to occur at an earlier stage in the stimulated cultures, as compared to the controls, starting with Day 6 of cell culture. At later stages, calcium levels in the stimulated cultures were higher than those in control samples by about 70%, with Ca/P ratios similar to those of natural bone. In all, the laser-based protocol emerges as an efficient alternative to existing fabrication technologies.

Compartmental stress responses correlate with cell survival in bystander effects induced by the DNA damage agent, bleomycin.

Physical or chemical stress applied to a cell system trigger a signal cascade that is transmitted to the neighboring cell population in a process known as bystander effect. Despite its wide occurrence in biological systems this phenomenon is mainly documented in cancer treatments. Thus understanding whether the bystander effect acts as an adaptive priming element for the neighboring cells or a sensitization factor is critical in designing treatment strategies. Here we characterize the bystander effects induced by bleomycin, a DNA-damaging agent, and compartmental stress responses associated with this phenomenon. Mouse fibroblasts were treated with increasing concentrations of bleomycin and assessed for DNA damage, cell death and induction of compartmental stress response (endoplasmic reticulum, mitochondrial and cytoplasmic stress). Preconditioned media were used to analyze bystander damage using the same end-points. Bleomycin induced bystander response was reflected primarily in increased DNA damage. This was dependent on the concentration of bleomycin and time of media conditioning. Interestingly, we found that ROS but not NO are involved in the transmission of the bystander effect. Consistent transcriptional down-regulation of the stress response factors tested (i.e. BiP, mtHsp60, Hsp70) occurred in the direct effect indicating that bleomycin might induce an arrest of transcription correlated with decreased survival. We observed the opposite trend in the bystander effect, with specific stress markers appearing increased and correlated with increased survival. These data shed new light on the potential role of stress pathways activation in bystander effects and their putative impact on the pro-survival pro-death balance.

Dual effect of methylglyoxal on the intracellular Ca2+ signaling and neurite outgrowth in mouse sensory neurons.

The formation of advanced glycation end products is one of the major factors involved in diabetic neuropathy, aging, and neurodegenerative diseases. Reactive carbonyl compounds, such as methylglyoxal (MG), play a key role in cross-linking to various proteins in the extracellular matrix, especially in neurons, which have a high rate of oxidative metabolism. The MG effect was tested on dorsal root ganglia primary neurons in cultures from adult male Balb/c mice. Lower MG doses contribute to an increased adherence of neurons on their support and an increased glia proliferation, as proved by MTS assay and bright-field microscopy. Time-lapse fluorescence microscopy by Fura-2 was performed for monitoring the relative fluorescence ratio changes (ΔR/R(0)) upon depolarization and immunofluorescence staining for quantifying the degree of neurites extension. The relative change in fluorescence ratio modifies the amplitude and dispersion depending on the subtype of sensory neurons, the medium-sized neurons are more sensitive to MG treatment when compared to small ones. Low MG concentrations (0-150 µM) increase neuronal viability, excitability, and the capacity of neurite extension, while higher concentrations (250-750 µM) are cytotoxic in a dose-dependent manner. In our opinion, MG could be metabolized by the glyoxalase system inside sensory neurons up to a threshold concentration, afterwards disturbing the cell equilibrium. Our study points out that MG has a dual effect concentration dependent on the neuronal viability, excitability, and neurite outgrowth, but only the excitability changes are soma-sized dependent. In conclusion, our data may partially explain the distinct neuronal modifications in various neurodegenerative pathologies.