Search

Gene silencing therapies have successfully suppressed the translation of target proteins, a strategy that holds great promise for the treatment of central nervous system (CNS) disorders. Advances in the current knowledge on multimolecular delivery vehicles are concentrated on overcoming the difficulties in delivery of small interfering (si)RNA to target tissues, which include anatomical accessibility, slow diffusion, safety concerns, and the requirement for specific cell uptake within the unique environment of the CNS. The present work addressed these challenges through the implementation of polyornithine derivatives in the construction of polyplexes used as non-viral siRNA delivery vectors. Physicochemical and biological characterization revealed biodegradability and biocompatibility of our polyornithine-based system and the ability to silence gene expression in primary oligodendrocyte progenitor cells (OPCs) effectively. In summary, the well-defined properties and neurological compatibility of this polypeptide-based platform highlight its potential utility in the treatment of CNS disorders.

The conjugation/de-conjugation of Small Ubiquitin-like Modifier (SUMO) has been shown to be associated with a diverse set of physiologic/pathologic conditions. The clinical significance and ostensible therapeutic utility offered via the selective control of the global SUMOylation process has become readily apparent in ischemic pathophysiology. Herein, we describe the development of a novel quantitative high-throughput screening (qHTS) system designed to identify small molecules capable of increasing SUMOylation via the regulation/inhibition of members of the microRNA (miRNA)-182 family. This assay employs a SHSY5Y human neuroblastoma cell line stably transfected with a dual firefly-Renilla luciferase reporter system for identification of specific inhibitors of either miR-182 or miR-183. In this study, we have identified small molecules capable of inducing increased global conjugation of SUMO in both SHSY5Y cells and rat E18-derived primary cortical neurons. The protective effects of a number of the identified compounds were confirmed via an in vitro ischemic model (oxygen/glucose deprivation). Of note, this assay can be easily repurposed to allow high-throughput analyses of the potential drugability of other relevant miRNA(s) in ischemic pathobiology.

This paper describes two small aperture array demonstrators called Medicina and Sardinia Array Demonstrators (MAD and SAD, respectively). The objectives of these instruments are to acquire experience and test new technologies for a possible application to the low-frequency aperture array of the low-frequency telescope of the Square Kilometer Array phase 1 (SKA1-LOW). The MAD experience was concluded in 2014, and it turned out to be an important test bench for implementing calibration techniques based on an artificial source mounted in an aerial vehicle. SAD is based on 128 dual-polarized Vivaldi antennas and is 1 order of magnitude larger than MAD. The architecture and the station size of SAD, which is along the construction phase, are more similar to those under evaluation for SKA1-LOW, and therefore, SAD is expected to provide useful hints for SKA1-LOW.

Peripheral nervous system injury is a frequent traumatic event, affecting a growing field of population, according to the change in lifestyle. Nerve autograph is still the golden standard, however it is invasive and limited in terms of amount and dimension of the available source. Various alternative therapeutic approaches have been proposed and results have been, so far, inconsistent, in terms of both quality and extent of nerve regeneration and re-innervation. Here, a novel technique is presented able to produce tubular collagen-based scaffolds, characterized by a radially/axially patterned microporosity (MPCS). The process has been modelled by means of the Lamm differential equation, according to appropriate scaling laws. In the present investigation, we tested in vivo the clinico-pathological impact of this MPCS over a 10-mm critical size defects in the adult rat sciatic nerve. Rats with transection of the sciatic nerve and implanted with either commercial collagen or silicon conduits were used as controls. MPCS-implanted rats showed significantly improved nerve regeneration at both neurophysiological and neuropathological levels, as compared to control rats. Our data demonstrate that this specific tubular scaffold micro-patterning orchestrates physiological regeneration in the adult rat sciatic nerve over a 10-mm critical size defect. Indeed, whole genome gene expression analyses confirm that the MPCS induces selective gene expression patterns and enhanced cells proliferation, motility and myelination, with a remarkable cell-instructing behavior. The development of morphogenesis phenomena during nerve regeneration process opens new perspectives towards the clinical application of MPCS in this field, owing to its ease of production, cost-effectiveness, favorable degradation rate and remarkable cell-instructing behavior.

Oligodendrocyte progenitor cell (OPC) differentiation is an important therapeutic target to promote remyelination in multiple sclerosis (MS). We previously reported hyperphosphorylated and aggregated microtubule-associated protein tau in MS lesions, suggesting its involvement in axonal degeneration. However, the influence of pathological tau-induced axonal damage on the potential for remyelination is unknown. Therefore, we investigated OPC differentiation in human P301S tau (P301S-htau) transgenic mice, both in vitro and in vivo following focal demyelination. In 2-month-old P301S-htau mice, which show hyperphosphorylated tau in neurons, we found atrophic axons in the spinal cord in the absence of prominent axonal degeneration. These signs of early axonal damage were associated with microgliosis and an upregulation of IL-1β and TNFα. Following in vivo focal white matter demyelination we found that OPCs differentiated more efficiently in P301S-htau mice than wild type (Wt) mice. We also found an increased level of myelin basic protein within the lesions, which however did not translate into increased remyelination due to higher susceptibility of P301S-htau axons to demyelination-induced degeneration compared to Wt axons. In vitro experiments confirmed higher differentiation capacity of OPCs from P301S-htau mice compared with Wt mice-derived OPCs. Because the OPCs from P301S-htau mice do not ectopically express the transgene, and when isolated from newborn mice behave like Wt mice-derived OPCs, we infer that their enhanced differentiation capacity must have been acquired through microenvironmental priming. Our data suggest the intriguing concept that damaged axons may signal to OPCs and promote their differentiation in the attempt at rescue by remyelination. GLIA 2016;64:457-471.

Introduction Nerve injury is a frequent event especially after traumatic lesion, affecting mainly young people. Various therapeutic approaches have been proposed for patients suffering from peripheral nerve injuries. Despite the significant increase in the understanding of the pathophysiology of nerve, results have been, so far, inconsistent, in terms of both quality as well as extent of nerve regeneration and re-innervation. The use of a conduit to reconnect the proximal and distal stumps of a transected nerve and induce peripheral nervous system (PNS) regeneration has been the subject of a large number of investigations. While it is quite established that the presence of a conduit between the transected stumps allows PNS regeneration [1], the micro-structural, mechanical and compositional features of the conduit, as any material inserted within, might significantly affect the quality of regeneration [2, 3]. We have previously developed a micro-patterned collagen scaffold (MPCS), by a spinning technique [4], with a peculiar radially aligned porosity of the tube wall, and predicted that its microstructure might play a significant role in the regulation of cellular and molecular mechanisms sustaining cell behaviour inside the scaffold and, in turn, improving distal induced regeneration. In this work the clinico-pathological impact of this MPCS was evaluated in the adult rat sciatic nerve. Neurophysiological, morphological and whole genome expression studies were conducted to assess the major pathways regulating nerve development and morphogenesis. Materials & Methods A complete and detailed description of the process adopted to produce MPCS has been reported [4]. Scaffolds are prepared using a collagen-based slurry; spinning of the slurry in a proper apparatus and rapid freezing of the sedimented suspension in liquid nitrogen; freeze-drying of the solidified suspension to obtain the final MPCS. Nerve regeneration in MPCS-implanted transected rats was evaluated in vivo over a 10-mm critical size defect in the adult rat sciatic nerve. Rats with transection of the sciatic nerve and implanted with either commercial collagen (NeuraGen®) or silicon conduits were used as controls. To evaluate the degree of regeneration and re-myelination after nerve transection, neurophysiological and morphological studies were performed. Together with sciatic nerves, the tibial plantar nerves at the paw were also investigated, to establish the process of re-innervation at the distal part. Whole gene expression (GE) profiling studies on injured sciatic nerve rats were conducted to investigate the molecular changes and the regulation of biological processes provided by our MPCS. Results & Discussion A damage, which determines a detachment of two nerve stumps, like a traumatic lesion, needs a device that creates an ideal micro-environment in order to facilitate axonal regeneration and re-myelination. Thus, we have developed a MPCS under the hypothesis that the micro-structure of the wall conduit could create a favourable micro-environment for full nerve regeneration after experimental transection. Neurophysiological and morphological studies suggested a faster functional nerve recovery of MPCS-implanted rats compared to rats treated with other conduits, as well as a prompt restoration of a functional vasculature with a physiological blood-nerve barrier. Moreover, in the MPCS, the long-term analysis showed a normal development of nerve, consisting of normal axon diameters and myelin thickness. Up-regulation of the myelin specific-gene observed at early time only on MPCS suggested the ability of our scaffold in providing clues for Schwann cell differentiation. Whole genome gene expression analyses further confirmed that the MPCS induces selective gene expression patterns and enhanced cells proliferation, motility and myelination. Conclusion Our findings provide remarkable molecular and ultra-structural evidence that the optimal micro-patterning of the proposed collagen scaffold plays a key role in turning the inner micro-environment into hospitable for development, rather than induced regeneration, of the proximal nerve stump. Within these optimal conditions, the transected nerve recapitulates in a coordinated fashion its innate programs of differentiation, which ultimately lead to progressive substitution of non-neuronal with neuronal tissues. We hypothesize that the radially aligned porosity of the MPCS promotes nerve regeneration by inducing morphogenetic stimuli and orchestrating cell behaviour towards a physiological regeneration mode.

Experimental Autoimmune Encephalomyelitis (EAE) is a good model of Multiple Sclerosis in rodents. We have applied fMRI to invesigate the alteration in functional response in rats induced with EAE. Cortical activation is altered in EAE rat brain, compared to controls. fMRI showed an increase in the activated volume involving also the cortex ipsilateral and some extra-cortical areas at the observed time points. These results demonstrate brain cortex remodeling in EAE, a remakable feature of MS. The present model seems to be a relevant tool in preclinical MS studies.

Introduction The recent advances in regenerative medicine have led to the development of novel experimental therapies, at both the cellular and molecular levels, which aim at reversing the devastating consequences of acute spinal cord injury [1-2]. Several bioactive molecules able to promote regeneration of the injured nervous system show therapeutic potential only when delivered in a controlled and sustained fashion, via appropriate microcarriers. The aim of this work was to evaluate the suitability of poly(lactic-co-glycolic) acid (PLGA)-based microspheres (MS) as a means to deliver therapeutic amounts of either neurotrophic factors (for axonal protection), or inhibitors of bone morphogenetic proteins (for inhibition of the glial scar), following acute spinal cord injury in mice. Several MS formulations were prepared, in order to achieve proper release kinetics of the selected molecule(s), and the effect of the encapsulation technique on the structure and stability of the loaded molecule was assessed. The encapsulation of environmental regulators (ERs) in bioresorbable PLGA-based MS might be a promising approach to promote the regeneration of the injured nervous system. Materials and Methods Microsphere synthesis: PLGA-based MS were synthesized by means of a double emulsion/solvent evaporation technique (water/oil/water), as previously described [3]. The ER (e.g., recombinant mouse chordin) was loaded in the presence of bovine serum albumin (BSA) as stabilizing molecule, and MS were synthesized starting from different PLGA concentrations. Microsphere characterization: The size distribution of MS was evaluated through dynamic laser light scattering, whereas MS morphology was assessed through scanning electron microscopy (SEM). The encapsulation efficiency was evaluated by UV spectrophotometry. Release study: In vitro release of the ER was monitored in PBS at 37°C with gentle shaking, over a period ranging from 72 hours to 30 days, depending on the given MS formulation tested. In order to assess any effects of the presence of BSA on the ER release kinetics, both the total amount of protein (BSA+ER) released at selected time points and the released amount of ER were measured, by UV spectrophotometry (with a BCA-assay protein kit) and ER-specific ELISA assays respectively. Protein/ER stability and bioactivity: The effect of the encapsulation technique on the structure of the loaded molecule(s) was assessed through sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectrofocalization (IEF), performed at selected time points along the sustained release. Specific bioassays, to assess the biological activity of the released ER, are currently in progress. Results and Discussion The average MS size was found to be significantly affected by the PLGA amount initially dissolved in the organic solvent used to prepare the double emulsion, with lower diameters attained for lower PLGA concentrations. As expected, release kinetics was greatly dependent on the mean MS size. Indeed, for the MS formulations tested, it was possible to modulate the time length of the sustained release of the ER from 3-4 days (for an average MS size of 15 μm, attained for a 10% w/v PLGA concentration) to 35 days (average MS size of 30 μm, attained for a 20% w/v PLGA concentration). The encapsulation efficiency was about 80-90% for all the MS formulations tested. The structural integrity of both BSA and ER was not affected by the encapsulation technique used, as demonstrated by the results of SDS-PAGE and IEF. However, the released fraction of ER as measured by the ELISA assays was found to be significantly lower than the amount expected from the total release study (BSA+ER). Along with in vitro bioactivity assays, specific release studies to understand this discrepancy between the total release of BSA and ER and the release of the only ER are currently being performed. Conclusions ER-loaded PLGA-based MS, possessing different mean diameters, were synthesized by a double emulsion/solvent evaporation technique. This technique yielded MS batches with a small size distribution and a high encapsulation efficiency, and preserved the structural integrity of the loaded molecules. The release kinetics could be tuned by varying the mean MS diameters. Based on these results, PLGA-based MS show potential for the local delivery of ERs to the injured nervous system. However, the suitability of the proposed MS to this purpose will be proved only following in vitro and in vivo bioactivity assays, scheduled in the next future.