Development and Characterization of scaffold based three-dimensional neuronal cultures

Tissue engineering, through different approaches, strives to create functional components of organs and cell types in vitro. A major bottle neck in recreating in vivo-like phenotypes in these in vitro systems is to find biocompatible materials to scaffold both as an interface with living tissues, and also to support the survival and function of the cells during in vitro culture. It is clear nowadays that 2D in vitro models poorly reflect the complexity of the body organs. This is particularly true for the brain, and many research group are working, using different approaches, to have a more appropriate system to investigate developmental processes, neuronal network dynamics and molecular pathways, as well as for drug screening. In my Ph.D., I aimed to circumvent these major bottlenecks by developing and carefully characterizing 3D neural cultures. To do so, I used optical imaging methods, particularly calcium imaging, to investigate the neuronal network dynamics of reconstructed 3D primary cultures from rats. In particular, I developed and tested new scaffolds for these culture systems. In the brain both conductivity and stiffness are important parameters that help shape neural networks. To mimick these properties of the brain, I tested the scaffolding properties of elastic organic polymers like Polydimethylsiloxane (PDMS) and gelatin. In addition, I used graphene-based materials as electrically conductive scaffolds. Through these studies, I developed a number of functional 3D-neural networks using different scaffolds with distinct structure, topology and compositions. In particular I determined that: 1) Patch substrate with a monolayer of gelatin nanofibers electrospun and crosslinked on a honeycomb microframe of poly (ethylene glycol) diacrylate (PEGDA) are suitable substrates for neuronal studies in a 3D environment. This method allows us to minimize exogenous material contact of cells and largely increase the exposure area of cells to the culture medium. Even though there are no connections in vertical dimension, I found that neurons, and especially astrocytes, have a more in vivo like morphology comparing to that on culture dish or on glass slide. We also found that neurons were preferentially located in the suspended areas of the monolayer nanofibers. Finally, calcium imaging revealed that primary neurons have a higher degree of neural activity on the patch than on glass. These results suggest that crosslinked and monolayer gelatin nanofibers closely mimic the extracellular matrix structure and allow more effective culture of primary neurons than conventional methods, thus facilitating advanced studies of neural functions as well as cell-based assays. 2) PDMS microlattices scaffolds, produced by conventional photolithography techniques, can be used as a soft scaffold for in vitro cell culture for both cell lines and primary neuronal cultures. The photomask with micro-scale dots array spin coated with photoresist is downward mounted on a rotating stage with a 45\ub0angle to UV irradiated direction. After three irradiation times the UV exposed area could be developed to form a three dimensional (3D) porous photoresist template. PDMS is poured in and cured and a 3D PDMS lattice is obtained after etching. I determined that our 3D PDMS lattices are suitable for: a. Culturing NIH-3T3 cell line into the microlattices. We observed homogenously cell adhesion and extension to form a 3D in vitro culture. Cell nuclear shape could also be controlled by adjusting the unit-cell architecture of the lattice. b. Easy cell observation. This 3D scaffold is biocompatible and also transparent and it can be applied to study the differences between 2D and 3D cell cultures in vitro and enable cells maintain their in vivo morphology of tissue-like structures within an in vitro platform. c. Culturing primary hippocampal cells from rats. We observed a great cell-material interaction. Neurons and astrocytes grow over and among the pillars creating a 3D in vitro neuronal network. These results show that adjusting the size of 3D structure it is not only possible to control the cell growth and shape, but also to allow primary hippocampal cells to be cultured on a softer substrates and have suspended connection among the pillars reaching an unexpected three-dimensionality. 3) Graphene foam scaffold is particularly suitable for 3D-neural cultures as it permitted the formation of a modular network between cultured neurons, which is characterized by a higher firing frequency and synchronization. I was able to culture through the whole scaffolds (1-1.5 mm in heights) a viable neuronal network which, because of its third dimension, has many connections among distant neurons leading to small-world networks and their characteristic dynamics. This new in vitro model reflects some of the observation done on the nervous system dynamics in vivo. More in details, I defined: a. The presence of a Moderately Synchronized (MS) regime and a Highly Synchronized (HS) regime. The HS regime was never observed in 2D networks and was observed in the cases of highly connected portion of the network. During the MS regime, neuronal assemblies in synchrony changed with time as observed in mammalian brains. After two weeks, the degree of synchrony in 3D networks decreased, as observed in vivo. b. The absence of a disequilibrium between excitatory and inhibitory neurons by staining the culture for the GABA neurotransmitter. c. The presence of astrocytes with a ramified shape that resemble the in vivo shape that allows the engulfment of many neuronal terminals. d. The maturation of the network follow the pattern of maturation observed in vivo with a decrease synchronization and frequency. These results show that dimensionality determines properties of neuronal networks and that several features of brain dynamics are a consequence of its 3D topology. Moreover this scaffold allows to have a 3D neuronal network in vitro that has similar in vivo dynamics and could represent a better in vitro model to study some of the aspects of the nervous system. All together, these results show that to achieve complex connectivity among neurons, 3D-scaffolds with distinct electrical and mechanical properties can be applied. What makes the difference is the aim that every research group wants to achieve. In the future it could be useful to compare a 3D scaffold based in vitro model to a positive control, such as organotipic slices or acute slices, in order to understand how close we are to the network dynamics of an ex-vivo preparation. Moreover, it could be interesting to investigate the reasons behind the more elevated frequency and synchronization of the 3D neuronal networks. It could be possible that the different morphology of astrocytes affects the on-going activity of neurons or that there is a different speed of maturation of the network since we have another growing dimension (the z-plane). Beyond the basic research, it would be interesting to apply this new in vitro model to study the progression of some diseases like tumours, and in particular brain tumours, in a 3D environment and in a 3D brain culture

Genetic Polymorphisms and Ischemic Heart Disease

Although the progression in diagnostic tools, prevention strategies, and therapies, ischemic heart disease still represents the major cause of mortality and morbidity worldwide that globally represents an important problem for individuals and healthcare resources. By convention, ischemic heart disease is associated with the presence of an atherosclerotic plaque that is able to limit the flow in large-medium-sized coronary arteries. Nevertheless, several findings suggest a more complex pathophysiology of ischemic heart disease. At this time, there is no well-defined assessment of myocardial ischemia pathophysiology. Moreover, several data have identified genetic variations at different loci that are linked with ischemic heart disease susceptibility. This chapter aims to examine this complicated disease and to review the evidence on the genetic heritability acting with other factors in determining the presence of ischemic heart disease, due to either an obstruction in epicardial vessels or a dysfunction of coronary microcirculation

Effect of non-forage roughage replacement on feeding behaviour and milk production in dairy cows

The objective of this study was to determine whether the partial replacement of roughage from forage with non-forage fiber sources, in a total mixed ration (TMR), could reduce feed sorting by dairy cows without modifying behaviour and milk production. Twelve Holstein cows were fed two TMR maize silage based diets in a cross-over experiment. Compared to the control diet (C-diet), experimental diet (E-diet) was formulated by replacing 8% neutral detergent fibre (NDF) from straw and alfalfa hay with soybean hulls and wheat bran. E-diet had a lower physical effectiveness factor (pef) (0.37 vs 0.34; P<0.001) and physical effective NDF (peNDF) (15.5 vs 14.6%; P<0.01). Feeding and sorting behaviour, ruminal activity, milk yield and composition and blood metabolites were measured. The results indicated that dry matter intake was not affected by diet. Cows fed the experimental TMR spent less time eating (192 vs. 178 min/d; P<0.05) but showed the same number of meals per day. C-diet fed cows sorted against peNDF in a greater extent (98.3 100.9%; P<0.05). Treatments did not affect cows time budget of general behaviors, with particular regard to ruminating activity. Despite different forage sources in TMR, no significant differences in milk yield and quality were detected

Bi-Ventricular Myocardial Performance in Heart Failure: A New Approach to Evaluate Interventricular Dyssynchrony

Aims: Patients with heart failure (HF) exhibit ventricular dyssynchrony with negative effects on ventricular systolic and diastolic performance and poor prognosis. There is no consensus about the best approach for estimating the dyssynchrony and for selecting candidates for resynchronization therapy (CRT). We sought to evaluate whether Myocardial Performance Index (MPI), calculated as differences between left and right ventricle (LV, RV), ΔMPI, represents a marker of interventricular dyssynchrony. Methods: The study included 40 patients (22 males, 18 females, mean age 71±13) with NYHA functional class II-III, chronic heart failure (77% ischaemic), in optimal drug therapy for at least three months. All patients underwent a complete two-dimensional and Tissue Doppler Echocardiography (TDE), including an assessment of MPI in both ventricles. Results: Significant correlations were found between ΔMPI and QRS (r = 0.41, p &lt; 0.001), with NYHA (r = 0.66, p &lt; 0.001), with SPWMD (r = 0.32, p &lt; 0.05), with LV ejection fraction (r = -0.32, p &lt; 0.05), with Spv wave at the septal site of LV (r = -0.32, p &lt; 0.05), and with IVMD (r = 0.44, p &lt; 0.001). Ten patients have been re-evaluated six months after CRT implantation, and ΔMPI significantly correlated with the difference between basal LVEF and six months after CRT implantation (r = 0.43, p &lt; 0.04). Conclusion: The ΔMPI could represent an integrative marker of interventricular dyssynchrony and could be considered as a new parameter in the patient selection process to be undergone CRT

Diabetes mellitus and ischemic heart disease. the role of ion channels

Diabetes mellitus is one the strongest risk factors for cardiovascular disease and, in particular, for ischemic heart disease (IHD). The pathophysiology of myocardial ischemia in diabetic patients is complex and not fully understood: some diabetic patients have mainly coronary stenosis obstructing blood flow to the myocardium; others present with coronary microvascular disease with an absence of plaques in the epicardial vessels. Ion channels acting in the cross-talk between the myocardial energy state and coronary blood flow may play a role in the pathophysiology of IHD in diabetic patients. In particular, some genetic variants for ATP-dependent potassium channels seem to be involved in the determinism of IH

Triggers for atrial fibrillation. the role of anxiety

Atrial fibrillation (AF) is the most widely recognized arrhythmia. Systemic arterial hypertension, diabetes, obesity, heart failure, and valvular heart diseases are major risk factors for the onset and progression of AF. Various studies have emphasized the augmented anxiety rate among AF patients due to the poor quality of life; however, little information is known about the possibility of triggering atrial fibrillation by anxiety. +e present review sought to underline the possible pathophysiological association between AF and anxiety disorders and suggests that anxiety can be an independent risk factor for AF, acting as atrigger, creating an arrhythmogenic substrate, and modulating the autonomic nervous system.+e awareness of the role of anxietydisorders as a risk factor for AF may lead to the development of new clinical strategies for the management of AF

Silver as antibacterial toward Listeria monocytogenes

Listeria monocytogenes is a serious foodborne pathogen that can contaminate food during processing and can grow during food shelf-life. New types of safe and effective food contact materials embedding antimicrobial agents, like silver, can play an important role in the food industry. The present work aimed at evaluating the in vitro growth kinetics of different strains of L. monocytogenes in the presence of silver, both in its ionic and nano form. The antimicrobial effect was determined by assaying the number of culturable bacterial cells, which formed colonies after incubation in the presence of silver nanoparticles (AgNPs) or silver nitrate (AgNO(3)). Ionic release experiments were performed in parallel. A different reduction of bacterial viability between silver ionic and nano forms was observed, with a time delayed effect exerted by AgNPs. An association between antimicrobial activity and ions concentration was shown by both silver chemical forms, suggesting the major role of ions in the antimicrobial mode of action

ANALISI SPAZIALE DELLE AREE HNVF NEL TERRITORIO LUCANO Spatial Analysis of HNVF Areas in the Region of Basilicata

In this paper it was proposed a spatial multicriteria analysis model for the identification of the High Nature Value Farmland (HNVF) areas. Six criteria was identified and analyzed. The application was conducted in the region of Basilicata, Italy. The result highlight four HNVF classes. Large part of agricultural land of Basilicata has a high nature value. The obtained results are useful in the agricultural programming, such as, for example, in the next Rural Development Plan (2014-2020)