Fondazione IRET ONLUS

Prolonged marine heat waves are a major manifestation of climate change, threatening marine biodiversity. Using the Mytilus galloprovincialis mussel as a sentinel organism, we investigated the impact of experimental, prolonged high temperatures as expected in 2050 on byssus-producing organ. We demonstrated that 30 days exposure at 28 °C, alters molecular and functional properties of mussel foot. Results revealed severe downregulation in the synthesis of the proximal, distal and non-gradient pre-collagen byssus components. The stressed mussels produced less than half the number of byssus filaments compared to the control. Byssus threads showed reduced mechanical resistance to traction, with a 55% decrease in maximum force. At the organism’s health status level, a long-term collapse in heat shock protein 70 subspecies expression suggests energy depletion. This is confirmed by a reduction of the hepatosomatic index during exposure and by the physiological impairment with clearance rate reduction and loss of air exposure tolerance. These effects compromise the mussel’s substrate adhesion, increasing the risk of detachment but also threaten bivalve survival. The potential consequences at the ecological level could be severe, including a reduction in coastal biodiversity, in the ecosystem services provided by these animals, as well as risks to the sustainability of Mediterranean mussel farming.

The international guidelines outlining the mandatory developmental toxicology studies of new molecules on pre-implantation, post-implantation and organogenesis phases, require a minimum of 60 pregnant female rats for each molecule to be tested. To date, available in vitro methods still have many limitations, resulting in poor translational power. In the present study, an innovative in vitro platform is proposed, based on rat embryonic stem cells (RESCs), which is easy to use and suitable for wide-scale screening, mimicking two different developmental stages: i) pre-implant model (undifferentiated pluripotent cells), ii) post-implant model (neuroectodermal lineage differentiation). The in vitro platform was validated by testing the toxicity on the pre-implant model of RA itself, as a known teratogen, a member of the environmental pollutant family per- and polyfluoroalkyl substances (PFAS), the perfluorooctanic acid (PFOA), and the endocrine disruptor chemical 2,2',6,6'-tetrabromobisphenol A (TBBPA) as test compound, targeting the thyroid hormone (TH) signal. The post-implant model showed inactivation of the pluripotent markers and activation of the neuroectodermal markers. The preimplant model resulted high responsive and sensitive to the embryotoxic effect of the tested compounds. The TBBPA was selected to test the potential effects of on viability and neuroectodermal differentiation, assessed through colorimetric and cell-based high-content screening methods establishing sub-toxic (20 μM) and toxic (40 μM) doses. A high-throughput gene expression array-based analysis showed a prompt response of the in vitro testing platform to TBBPA treatment. A rescue experiment exploiting a pan-thyroid receptor (pan-TR) inhibitor (1-850) showed that the effects of TBBPA on RESCs was blocked, demonstrating its activity through TRs. The RESCs-based platform allowed reproducible, robust and highly predictable results, thanks to the coupling of RESCs with high-throughput technologies. These results support the possible use of RESCs-based models as a screening platform for developmental toxicity testing to reduce the number of animals currently used for this aim.

The complexity of the central nervous system (CNS) requires researchers to consider all the variables linked to the interaction between the different cell inhabitants. On this basis, any in vitro study of the physiological and pathological processes regarding the CNS should consider the balance between the standardization of the assay and the complexity of the cellular system which mimics the in vivo microenvironment. One of the main structural and functional components of the CNS is the oligodendrocyte precursor cell (OPC), responsible for developmental myelination and myelin turnover and repair during adulthood following differentiation into mature oligodendrocytes. In the present brief research report, we describe a 3D culture tool (VITVO) based on an inert and biocompatible synthetic polymer material scaffold, functionalized with laminin coating, and tested as a new culture microenvironment for neural stem/precursor cell (NSPC) differentiation compared to standard 2D cultures. NSPCs spontaneously differentiate in the three neural lineages (neurons, astrocytes and OPCs), identified by specific markers, along the fibers in the 3D structure. Analysis of the mRNA levels for lineage differentiation markers reveals a higher expression compared to those seeded on a 2D surface, suggesting an acceleration of the differentiation process. We then focused on the oligodendroglial lineage, showing that in VITVO, mature oligodendrocytes exhibit a myelinating morphology, proven by 3D image elaboration, linked to a higher expression of mature oligodendrocyte markers. This preliminary study on an innovative 3D culture system is the first robust step in producing new microenvironment-based strategies to investigate in vitro OPC and oligodendrocyte biology.

Nerve growth factor (NGF) was the first-discovered member of the neurotrophin family, a class of bioactive molecules which exerts powerful biological effects on the CNS and other peripheral tissues, not only during development, but also during adulthood. While these molecules have long been regarded as potential drugs to combat acute and chronic neurodegenerative processes, as evidenced by the extensive data on their neuroprotective properties, their clinical application has been hindered by their unexpected side effects, as well as by difficulties in defining appropriate dosing and administration strategies. This paper reviews aspects related to the endogenous production of NGF in healthy and pathological conditions, along with conventional and biomaterial-assisted delivery strategies, in an attempt to clarify the impediments to the clinical application of this powerful molecule.

Neurotrophins (NTs) are molecules regulating differentiation, maintenance, and functional plasticity of vertebrate nervous systems. Nerve growth factor (NGF) was the first to be identified in the neurotrophin family. The long scientific history of NTs provided not only advancement in the neuroscience field but opened new scenarios involving different body districts in physiological and pathological conditions, which include the immune, endocrine, and skeletal system, vascular districts, inflammation, etc. To date, many biological aspects of NTs have been clarified, but the new discoveries are still opening new insights on molecular and cellular mechanisms and systemic effects, also affecting the possible therapeutic application of NTs. This short review summarizes the main aspects of NGF biology and biochemistry, including the role of the NGF precursor molecule, high- and low-affinity receptors and related intracellular pathways, and target cells.

Spinal cord injury (SCI) is an incurable condition, in which a cascade of cellular and molecular events triggered by inflammation and excitotoxicity impairs endogenous regeneration, namely remyelination and axonal outgrowth. We designed a treatment solution based on an implantable biomaterial (electrospun poly (l-lactic acid) [PLLA]) loaded with ibuprofen and triiodothyronine (T3) to counteract inflammation, thus improving endogenous regeneration. In vivo efficacy was tested by implanting the drug-loaded PLLA in the rat model of T8 contusion SCI. We observed the expected recovery of locomotion beginning on day 7. In PLLA-implanted rats (i.e., controls), the recovery stabilized at 21 days post-lesion (DPL), after which no further improvement was observed. On the contrary, in PLLA + ibuprofen (Ibu) + T3 (PLLA-Ibu-T3) rats a further recovery and a significant treatment effect were observed, also confirmed by the gait analysis on 49 DPL. Glutamate release at 24 h and 8 DPL was reduced in PLLA-Ibu-T3- compared to PLLA-implanted rats, such as the estimated lesion volume at 60 DPL. The myelin- and 200-neurofilament-positive area fraction was higher in PLLA-Ibu-T3-implanted rats, where the percentage of astrocytes was significantly reduced. The implant of a PLLA electrospun scaffold loaded with Ibu and T3 significantly improves the endogenous regeneration, leading to an improvement of functional locomotion outcome in the SCI.

The lack of reliable diagnostic and prognostic markers for spinal cord injured (SCI) patients is a severe obstacle in development and testing of new therapies, and it also impairs appropriate rehabilitation care. The sparse available data on the biochemical composition of cerebrospinal fluid (CSF) during the acute and/or chronic phase of the lesion provide, up until now, inconsistent results. In this pilot study, we then explored the possibility of combining a multi-parametric and bioinformatic analysis of CSF for its biological properties tested on different cells types, suitable for investigating inflammation and re-myelination. The patient enrollment was based on stringent inclusion criteria; that is, cervical and thoracic SCI trauma, CSF collection within 24 h of trauma, type of surgical approach for spine stabilization, and absence of steroid therapy before CSF collection. Eleven SCI patients and four healthy controls were included, and in three patients, CSF was also collected at 3 months after lesion. We identified 19 proteins among the 60 investigated cytokines, chemokines, growth factors, and structural biomarkers, which are transiently regulated 24 h after SCI. A bioinformatic analysis indicated that interleukin (IL)-6 and IL-10 are in the core of the interconnected net of activated proteins. Cell-based experiments indicate that CSF from SCI patients stimulates astroglia derivation from neural precursor cells, and an inverse correlation between IL-8 CSF level and oligodendrocyte precursor cells generated from neural stem cells was also observed. Results from this pilot study suggest that using a combined bioanalytic and biological approach to analyze SCI CSF at different times after injury could be a useful approach for identifying reliable diagnostic and prognostic markers in SCI.

Background: Contrary to the most credited theories on laser therapy that see power/energy as the major factors to its effectiveness, a technique using an extremely low power/energy laser stimulation to treat musculoskeletal pain and dysfunction is proposed. The stimulus consists of a 20 s train of modulated pulses with an average power below 0.02 mW and is applied on sequences of acupuncture points selected according to the impaired segment of the patient’s body. Methods: Modifications on the extracellular soft tissue matrix and on the “fascia” were sonographically demonstrated. Laboratory and clinical tests confirmed the effectiveness. Results: Responses similar to those experienced in acupuncture were observed. The device—a CE Class IIa certified medical device named Libralux—affords a clinically proven effectiveness exceeding 80% in the treatment of musculoskeletal conditions and associated motor dysfunctions. An average of just three application sessions was generally sufficient to overcome the dysfunction. Conclusions: The development of the method is supported by over 20 years of R&D activities, with a range of experiments discussed in several papers published in indexed peer-reviewed journals. A few considerations regarding the possible physiological action mechanisms involved are proposed in this paper.

We describe age-related histological structure and molecular changes of the neurovascular unit (NVU) in the cerebral cortex of Tg2576 and age-matched wild-type (WT) mice. Major results can be summarized as follows: (i) β-amyloid (6E10)-immunoreactivity progressively increases in neurons and astrocytes of Tg2576 mice, reaching the highest concentration at 5 months and then decreasing as soon as extracellular plaque deposition begins; (ii) the synaptic puncta density of glutamatergic and GABAergic neurons in Tg2576 mice is unbalanced versus WT at all investigated ages, with a decrease in synaptophysin and VGLUT1; density of VGAT contacts is higher in 27-month-old Tg2576 versus WT mice; (iii) capillary density is higher in 5-month-old Tg2576 versus WT mice, then decreases to a lower density at 27 months, when the capillary-astrocyte interface is lower; and (iv) mRNA expression of genes involved in microvessel dynamics indicates age- and genotype-dependent changes in the expression levels of hypoxia-related genes, i.e. the highest level is in 5-month-old animals and there is impaired regulation in Tg2576. We conclude that at 5 months, when learning and memory impairment is already present in the absence of extracellular amyloid plaque deposition, Tg2576 mice display alterations in the structure and molecular regulation of the NVU.