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Modeling human neuronal properties in physiological and pathological conditions is essential to identify novel potential drugs and to explore pathological mechanisms of neurological diseases. For this purpose, we generated a three-dimensional (3D) neuronal culture, by employing the readily available human neuroblastoma SH-SY5Y cell line, and a new differentiation protocol. The entire differentiation process occurred in a matrix and lasted 47 days, with 7 days of pre-differentiation phase and 40 days of differentiation, and allowed the development of a 3D culture in conditions consistent with the physiological environment. Neurons in the culture were electrically active, were able to establish functional networks, and showed features of cholinergic neurons. Hence here we provide an easily accessible, reproducible, and suitable culture method that might empower studies on synaptic function, vesicle trafficking, and metabolism, which sustain neuronal activity and cerebral circuits. Moreover, this novel differentiation protocol could represent a promising cellular tool to study physiological cellular processes, such as migration, differentiation, maturation, and to develop novel therapeutic approaches.

Palmitoylethanolamide (PEA) is an endogenous lipid produced on demand by neurons and glial cells that displays neuroprotective properties. It is well known that inflammation and neuronal damage are strictly related processes and that microglia play a pivotal role in their regulation. The aim of the present work was to assess whether PEA could exert its neuroprotective and antiinflammatory effects through the modulation of microglia reactive phenotypes. In N9 microglial cells, the pre-incubation with PEA blunted the increase of M1 pro-inflammatory markers induced by lipopolysaccharide (LPS), concomitantly increasing those M2 anti-inflammatory markers. Images of microglial cells were processed to obtain a set of morphological parameters that highlighted the ability of PEA to inhibit the LPS-induced M1 polarization and suggested that PEA might induce the anti-inflammatory M2a phenotype. Functionally, PEA prevented Ca2+ transients in both N9 cells and primary microglia and antagonized the neuronal hyperexcitability induced by LPS, as revealed by multi-electrode array (MEA) measurements on primary cortical cultures of neurons, microglia, and astrocyte. Finally, the investigation of the molecular pathway indicated that PEA effects are not mediated by toll-like receptor 4 (TLR4); on the contrary, a partial involvement of cannabinoid type 2 receptor (CB2R) was shown by using a selective receptor inverse agonist.

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Heating has been recently used as an alternative application to electrical stimulation to modulate excitability and to induce neuritogenesis and the expression of neuronal markers, but a long-term functional differentiation has not been described so far. Here we present the results obtained by a new approach for scalable thermal stimulation on the behavior of a model of dorsal root ganglion neurons, the F-11 cell line. Initially, we performed experiments of bulk stimulation in incubator for different time intervals and temperatures, and significant differences in neurite elongation and in electrophysiological properties were observed in cultures exposed at 41,5°C for 30 minutes. Thus, we exposed the cultures to the same temperature increase by irradiating, with a near infrared laser, a disc of Prussian Blue nanoparticles and poly-vinyl alcohol, that we stuck on the outer surface of the petri dish. In irradiated cells neurites were significantly longer and the electrophysiological properties (action potential firing frequency and spontaneous activity) were significantly increased compared to the control. These results suggest that a targeted thermal stimulation could be a promising technique to induce differentiation and support the future application of this method as a strategy to modify neuronal behavior in vivo.

We induced differentiation in F-11 cells to verify if they could show similarities with sensory neurons, in order to develop an alternative to animal models for research studies in the biomedical field.

Purpose: The aim is discussing the origins of worsening of external eye condition (EEC) and of tear film (TF) instability after wear of silicone-hydrogel contact lenses (CLs) with hydrogen-peroxide (H2O2) care system. Methods: EEC and TF stability were evaluated before and after 15 days of wear combined with different care systems: (1) H2O2, (2) detergent solution and H2O2, (3) multipurpose solution (MPS), (4) H2O2 and artificial tears. In-vitro cell mortality tests were performed after 24 h cell incubation with CLs treated with H2O2. Photon correlation spectroscopy (PCS) was carried out on tears of non-wearers and CL wearers who used MPS or H2O2 solution. Results: Worsening of EEC was observed only for the group using H2O2 (group 1). In-vitro, cell mortality was found higher for worn CL than for unworn CLs. Worsening of TF stability was observed regardless of care system and also PCS results on tears of CL wearers were found different compared to non-wearers regardless of care system. The only observed remedy for tear instability of CL wearers was found to be the administration of artificial tears. Conclusions: Worsening of EEC of CL wearers using H2O2 is attributed to H2O2 scarce cleaning efficacy, which can be solved by adding a CL detergent solution. The origin of TF instability is found to be different. A remedy was found to be the administration of artificial tears, whose effect could be attributed either to the role of specific components or to rinsing and replacement of TF during wear.

Sodium channel myotonia is a form of muscle channelopathy due to mutations that affect the Nav1.4 channel. We describe seven families with a series of symptoms ranging from asymptomatic to clearly myotonic signs that have in common two novel mutations, p.Ile215Thr and p.Gly241Val, in the first domain of the Nav1.4 channel. The families described have been clinically and genetically evaluated. p.Ile215Thr and p.Gly241Val lie, respectively, on extracellular and intracellular loops of the first domain of the Nav1.4 channel. We assessed that the p.Ile215Thr mutation can be related to a founder effect in people from Southern Italy. Electrophysiological evaluation of the channel function showed that the voltage dependence of the activation for both the mutant channels was significantly shifted toward hyperpolarized potentials (Ile215Thr: -28.6 ± 1.5 mV and Gly241Val: -30.2 ± 1.3 mV vs. WT: -18.5 ± 1.3 mV). The slow inactivation was also significantly affected, whereas fast inactivation showed a different behavior in the two mutants. We characterized two novel mutations of the SCN4A gene expanding the knowledge about genetics of mild forms of myotonia, and we present, to our knowledge, the first homozygous patient with sodium channel myotonia.

The isolation and culture of dorsal root ganglion (DRG) neurons cause adaptive changes in the expression and regulation of ion channels, with consequences on neuronal excitability. Considering that not all neurons survive the isolation and that DRG neurons are heterogeneous, it is difficult to find the cellular subtype of interest. For this reason, researchers opt for DRG-derived immortal cell lines to investigate endogenous properties. The F-11 cell line is a hybridoma of embryonic rat DRG neurons fused with the mouse neuroblastoma line N18TG2. In the proliferative condition, F-11 cells do not display a gene expression profile correspondent with specific subclasses of sensory neurons, but the most significant differences when compared with DRGs are the reduction of voltage-gated sodium, potassium and calcium channels, and the small amounts of TRPV1 transcripts. To investigate if functional properties of mature F-11 cells showed more similarities with those of isolated DRG neurons, we differentiated them by serum deprivation. Potassium and sodium currents significantly increased with differentiation, and biophysical properties of tetrodotoxin (TTX)-sensitive currents were similar to those characterized in small DRG neurons. The analysis of the voltage-dependence of calcium currents demonstrated the lack of low threshold activated components. The exclusive expression of high threshold activated Ca2+ currents and of TTX-sensitive Na+ currents correlated with the generation of a regular tonic electrical activity, which was recorded in the majority of the cells (80%) and was closely related to the activity of afferent TTX-sensitive A fibers of the proximal urethra and the bladder. Responses to capsaicin and substance P were also recorded in ~20% and ~80% of cells, respectively. The percentage of cells responsive to acetylcholine was consistent with the percentage referred for rat DRG primary neurons and cell electrical activity was modified by activation of non-NMDA rec

Three-dimensional cell cultures are leading the way to the fabrication of tissue-like constructs useful to developmental biology and pharmaceutical screenings. However, their reproducibility and translational potential have been limited by biomaterial and culture media compositions, as well as cellular sources. We developed a construct comprising synthetic multifunctionalized hydrogels, serum-free media, and densely seeded good manufacturing practice protocol-grade human neural stem cells (hNSC). We tracked hNSC proliferation, differentiation, and maturation into GABAergic, glutamatergic, and cholinergic neurons, showing entangled electrically active neural networks. The neuroregenerative potential of the "engineered tissue" was assessed in spinal cord injuries, where hNSC-derived progenitors and predifferentiated hNSC progeny, embedded in multifunctionalized hydrogels, were implanted. All implants decreased astrogliosis and lowered the immune response, but scaffolds with predifferentiated hNSCs showed higher percentages of neuronal markers, better hNSC engraftment, and improved behavioral recovery. Our hNSC-construct enables the formation of 3D functional neuronal networks in vitro, allowing novel strategies for hNSC therapies in vivo

The emerging trends in regenerative medicine rely among others on biomaterial-based therapies, with the use of biomaterials as a central delivery system for biochemical and physical cues to manipulate transplanted or ingrowth cells and to orchestrate tissue regeneration. Cell adhesion properties of a biomaterial strongly depend on its surface characteristics. Among others poly(ε-caprolactone) (PCL) is a biocompatible and biodegradable material with low cytotoxicity that is widely adopted as synthetic polymer in several applications. However, it is hydrophobic, which limits its use in tissue engineering. In order to improve its hydrophilicity and cellular compatibility, PCL surface was grafted with maltose through a two-step procedure in which controlled aminolysis of PCL ester bonds by hexanediamine was followed by reductive amination with the carbohydrate reducing end. The modified PCL surface was then characterized in detail by x-ray Photoelectron Spectroscopy (XPS) and Near Edge x-ray Absorption Fine Structure (NEXAFS) spectroscopies. In addition, the biocompatibility of the proposed biomaterial was investigated in preliminary biological assays

Background: This investigation used image data generated by a physical phantom over a wide range of count statistics to evaluate the effectiveness of several of the newer commercially available SPECT reconstruction iterative algorithms (IRR) in improving perfusion defect contrast and spatial resolution, while controlling image noise. Methods: A cardiac phantom was imaged using four different gamma cameras over a wide range of counts statistics (from 6 to 0.8 Mcounts). Images were reconstructed with FBP, OSEM, and the IRR available on site. IRR were applied without corrections (IRR NC), with attenuation correction (IRR AC), scatter correction (IRR SC), and attenuation + scatter corrections (IRR SCAC). Four image performance indices related to spatial resolution, contrast, and image noise were analyzed. Results: IRR NC always determined significant improvements in all indices in comparison to FBP or OSEM. Improvements were emphasized with IRR SC and IRR SCAC. Count reduction from 6 to 1.5 Mcounts did not impair the performances of any of the considered indices. Conclusions: This is the first study comparing the relative performance of different, commercially available, IRR software, over a wide range of count statistics; the additional effect of scatter and attenuation corrections, alone or in combination, was also evaluated. Our results confirm that IRR algorithms produce substantial benefits with respect to conventional FBP or OSEM reconstruction methods, as assessed through different figures of merit, in particular when SC and/or SCAC are also included. © 2013 American Society of Nuclear Cardiology.

Context: Children’s cough is a daily concern for most pediatricians. The management of both acute and chronic cough requires a systematic and comprehensive approach. Despite the approved protocols for management, the pediatric assessment of cough and the corresponding prescribing attitude frequently do not fit these protocols, which can be affected by parental suggestions - sometimes substantially. Objective: The objective of this study was to investigate both the perception and the behavior of a representative sample of Italian pediatricians toward cough in real life. Methods: A specific questionnaire consisting of 18 questions was prepared. The questionnaire was completed by 300 pediatricians (all members of PAIDOSS: Italian National Observatory on Health of Childhood and Adolescence) who represented ~300,000 children. Results: A vast majority of children have cough throughout the year (99.3% of respondents have cough during autumn/winter and 64.7% in spring/summer). Allergic disease is the most frequent suspected cause of chronic cough in children (53%), and this is supported by the high demand for consultations: 73% seek the opinion of allergologists, 62% of otorhinolaryngologists and only 33% of pulmonologists. The majority of pediatricians (92%) reported that they prescribe therapy in acute cough regardless of cough guidelines. Moreover, the survey pointed out the abuse of aerosol therapy (26% in acute cough and 38% in chronic cough) and of antibiotics prescription (22% in acute cough and 42% in chronic cough). Conclusion: Our survey suggests that some Italian pediatricians’ therapeutic attitudes should be substantially improved in order to achieve better management of cough in children and to minimize the burden of cough

The previous exploration of the structure-affinity relationships concerning 4-phenyl-2-quinolinecarboxamide peripheral benzodiazepine receptor (PBR) ligands 6 showed as an interesting result the importance of the presence of a chlorine atom in the methylene carbon at position 3 of the quinoline nucleus. The subnanomolar PBR affinity shown by N-benzyl-3-chloromethyl-N-methyl-4-phenyl-2-quinolinecarboxamide (6b) suggested its chlorine atom to be replaced with other halogens in order to optimize the interaction of the quinolinecarboxamide derivatives with PBR and to develop suitable candidates for positron emission tomography (PET) or single photon emission computed tomography (SPECT) studies. The binding studies led to the discovery of fluoromethyl derivative 6a, which showed an IC(50) value of 0.11nM and is, therefore, one of the most potent PBR ligands so far described. Fluoromethyl derivative 6a has been labeled with (11)C (t(1/2)=20.4min, beta(+)=99.8%) starting from the corresponding des-methyl precursor (14) using [(11)C]CH(3)I in the presence of tetrabutylammonium hydroxide in DMF with a 35-40% radiochemical yield (corrected for decay) and 1.5Ci/mumol of specific radioactivity. Ex vivo rat biodistribution and inhibition (following intravenous pre-administration of PK11195) studies showed that [(11)C]6a rapidly and specifically accumulated in PBR-rich tissues such as heart, lung, kidney, spleen, and adrenal, and at a lower level in other peripheral organs and in the brain. The images obtained in mouse with small animal YAP-(S)PET essentially confirmed the result of the ex vivo biodistribution experiments. The biological data suggest that [(11)C]6a is a promising radioligand for peripheral benzodiazepine receptor PET imaging in vivo.