Modeling the three-dimensional connectivity of in vitro cortical ensembles coupled to Micro-Electrode Arrays

: Nowadays, in vitro three-dimensional (3D) neuronal networks are becoming a consolidated experimental model to overcome most of the intrinsic limitations of bi-dimensional (2D) assemblies. In the 3D environment, experimental evidence revealed a wider repertoire of activity patterns, characterized by a modulation of the bursting features, than the one observed in 2D cultures. However, it is not totally clear and understood what pushes the neuronal networks towards different dynamical regimes. One possible explanation could be the underlying connectivity, which could involve a larger number of neurons in a 3D rather than a 2D space and could organize following well-defined topological schemes. Driven by experimental findings, achieved by recording 3D cortical networks organized in multi-layered structures coupled to Micro-Electrode Arrays (MEAs), in the present work we developed a large-scale computational network model made up of leaky integrate-and-fire (LIF) neurons to investigate possible structural configurations able to sustain the emerging patterns of electrophysiological activity. In particular, we investigated the role of the number of layers defining a 3D assembly and the spatial distribution of the connections within and among the layers. These configurations give rise to different patterns of activity that could be compared to the ones emerging from real in vitro 3D neuronal populations. Our results suggest that the introduction of three-dimensionality induced a global reduction in both firing and bursting rates with respect to 2D models. In addition, we found that there is a minimum number of layers necessary to obtain a change in the dynamics of the network. However, the effects produced by a 3D organization of the cells is somewhat mitigated if a scale-free connectivity is implemented in either one or all the layers of the network. Finally, the best matching of the experimental data is achieved supposing a 3D connectivity organized in structured bundles of links located in different areas of the 2D network

Stimulus-Evoked Activity Modulation of In Vitro Engineered Cortical and Hippocampal Networks

The delivery of electrical stimuli is crucial to shape the electrophysiological activity of neuronal populations and to appreciate the response of the different brain circuits involved. In the present work, we used dissociated cortical and hippocampal networks coupled to Micro-Electrode Arrays (MEAs) to investigate the features of their evoked response when a low-frequency (0.2 Hz) electrical stimulation protocol is delivered. In particular, cortical and hippocampal neurons were topologically organized to recreate interconnected sub-populations with a polydimethylsiloxane (PDMS) mask, which guaranteed the segregation of the cell bodies and the connections among the sub-regions through microchannels. We found that cortical assemblies were more reactive than hippocampal ones. Despite both configurations exhibiting a fast (<35 ms) response, this did not uniformly distribute over the MEA in the hippocampal networks. Moreover, the propagation of the stimuli-evoked activity within the networks showed a late (35-500 ms) response only in the cortical assemblies. The achieved results suggest the importance of the neuronal target when electrical stimulation experiments are performed. Not all neuronal types display the same response, and in light of transferring stimulation protocols to in vivo applications, it becomes fundamental to design realistic in vitro brain-on-a-chip devices to investigate the dynamical properties of complex neuronal circuits

Modularity and neuronal heterogeneity: Two properties that influence in vitro neuropharmacological experiments

IntroductionThe goal of this work is to prove the relevance of the experimental model (in vitro neuronal networks in this study) when drug-delivery testing is performed. MethodsWe used dissociated cortical and hippocampal neurons coupled to Micro-Electrode Arrays (MEAs) arranged in different configurations characterized by modularity (i.e., the presence of interconnected sub-networks) and heterogeneity (i.e., the co-existence of neurons coming from brain districts). We delivered increasing concentrations of bicuculline (BIC), a neuromodulator acting on the GABAergic system, and we extracted the IC50 values (i.e., the effective concentration yielding a reduction in the response by 50%) of the mean firing rate for each configuration. ResultsWe found significant lower values of the IC50 computed for modular cortical-hippocampal ensembles than isolated cortical or hippocampal ones. DiscussionAlthough tested with a specific neuromodulator, this work aims at proving the relevance of ad hoc experimental models to perform neuropharmacological experiments to avoid errors of overestimation/underestimation leading to biased information in the characterization of the effects of a drug on neuronal networks

3F(eature)s model: modularity, heterogeneity and three-dimensionality to design in vitro neuronal model

In this work, we present a novel experimental platform to build in vitro interconnected (i.e., modular) heterogeneous (e.g., cortical-hippocampal) and three-dimensional (3D) neuronal cultures plated on Micro-Electrode Arrays (MEAs) to extracellularly record the electrophysiological activity continuously

Modularity and neuronal heterogeneity: Two properties that influence in vitro neuropharmacological experiments

IntroductionThe goal of this work is to prove the relevance of the experimental model (in vitro neuronal networks in this study) when drug-delivery testing is performed.MethodsWe used dissociated cortical and hippocampal neurons coupled to Micro-Electrode Arrays (MEAs) arranged in different configurations characterized by modularity (i.e., the presence of interconnected sub-networks) and heterogeneity (i.e., the co-existence of neurons coming from brain districts). We delivered increasing concentrations of bicuculline (BIC), a neuromodulator acting on the GABAergic system, and we extracted the IC50 values (i.e., the effective concentration yielding a reduction in the response by 50%) of the mean firing rate for each configuration.ResultsWe found significant lower values of the IC50 computed for modular cortical-hippocampal ensembles than isolated cortical or hippocampal ones.DiscussionAlthough tested with a specific neuromodulator, this work aims at proving the relevance of ad hoc experimental models to perform neuropharmacological experiments to avoid errors of overestimation/underestimation leading to biased information in the characterization of the effects of a drug on neuronal networks

Frailty and rehabilitation outcome in older patients with cardiorespiratory disease: Preliminary multidimensional data

Chronic heart failure (CHF) and chronic obstructive pulmonary disease (COPD) are two clinical conditions often associated with cognitive dysfunctions, psychological distress, poor quality of life (QoL), and functional worsening. In addition, since patients suffering from these conditions are often older adults, frailty syndrome represented a further and important issue to be investigated. The present preliminary study aimed to perform a multidimensional assessment of CHF COPD older patients (age ≥65) undergoing cardiac or pulmonary rehabilitation. The characteristics of the included patients (30 CHF and 30 COPD) resulted almost similar, except for the COPD patients' longer duration of illness and better performances in Addenbrooke's cognitive examination III subtests and short physical performance battery (SPPB). No significant differences were found in the frailty evaluation, but a consistent number of patients resulted to be frail (CHF=36.7% vs COPD=26.6%). After the rehabilitation program, a significant improvement was found in the whole sample concerning the executive functions (14.34±2.49 vs 15.62±2.22, p=0.001), quality of life (58.77±18.87 vs 65.82±18.45, p=0.003), depressive and anxious symptoms (6.27±4.21 vs 3.77±3.39, p=0.001 and 5.17±3.40 vs 3.38±3.21, p=0.001), frailty status [4.00 (3.00,5.00) vs 3.00 (3.00,5.00) p=0.035] and functional exercise abilities [SPPB, 7.40±3.10 vs 9.51±3.67, p=0.0002; timed up and go test, 14.62±4.90 vs 11.97±4.51, p=<0.0001; 6-minute walking test, 353.85±127.62 vs 392.59±123.14, p=0.0002]. Preliminary results showed a substantial homogeneity of CHF and COPD older patients' cognitive, psychosocial, frailty, and functional characteristics. Nevertheless, the specific rehabilitation intervention appears promising in both clinical populations. This trial has been registered with the ClinicalTrials.gov, NCT05230927 registration number (clinicaltrials.gov/ct2/show/NCT05230927)

Designing an effective dissolution test for bilayer tablets tailored for optimal melatonin release in sleep disorder management

This project aims to investigate the release performance of bilayer tablet (BL-Tablet) designed with both fast and slow-release technology, targeting sleep disorders. The tablet incorporates Melatonin, extracts of Eschscholzia californica and Melissa officinalis. In order to validate the effectiveness of the extended-release profile, an advanced dissolution test was herein proposed. This new method utilizes biorelevant intestinal fluid media and incorporates a stomach-to-intestine fluid changing (SIFC) system. To demonstrate the advantages of employing this method for assessing the controlled release profile of active ingredients, the dissolution results were compared with those obtained using the conventional EU Pharmacopoeia approach. Furthermore, the comparative analysis was extended to include a monolayer tablet version (ML-Tablet) lacking the slow-release technology. Technological characterization and bioaccessibility studies, including intestinal permeability test, were conducted as well to assess the pharmacological performance and bioavailability of active ingredients. The dissolution data recovered revealed that the two dissolution methods did not exhibit any significant differences in the release of ML-Tablet’s. However, the dissolution profile of the BL-Tablet exhibited notable differences between the two methods particularly when assessing the behavior of the slow-release layer. In this scenario, both methods initially exhibited a similar release pattern within the first approximately 0.5 h, driven by the fast-release layer of the tablet. Following this, distinct gradual and sustained releases were observed, spanning 2.5 h for the EU Pharmacopoeia method and 8 h for the new SIFC-biorelevant dissolution method, respectively. Overall, the novel method demonstrated a substantial improvement compared to conventional EU Pharmacopoeia test in evaluating the performance of a controlled slow-release technology. Remarkably, the prolonged release technology did not have an adverse impact on melatonin intestinal absorption, and, consequently, maintaining its potential bioavailability of around 78%. Concluding, this research provides valuable insights into how the innovative dissolution test can assist formulators in developing controlled release formulations

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5–11 December, to 17.5% (25/143 samples) in the week 12–18, to 65.9% (89/135 samples) in the week 19–25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased fromone in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool