SARS-CoV-2-related peptides induce endothelial-to-mesenchymal transition in endothelial capillary cells derived from different body districts: focus on membrane (M) protein.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19, may lead to multiple organ dysfunctions and long-term complications. The induction of microvascular dysfunction is regarded as a main player in these pathological processes. To investigate the possible impact of SARS-CoV-2-induced endothelial-to-mesenchymal transition (EndMT) on fibrosis in "long-COVID" syndrome, we used primary cultures of human microvascular cells derived from the lungs, as the main infection target, compared to cells derived from different organs (dermis, heart, kidney, liver, brain) and to the HUVEC cell line. To mimic the virus action, we used mixed SARS-CoV-2 peptide fragments (PepTivator®) of spike (S), nucleocapsid (N), and membrane (M) proteins. TGFß2 and cytokine mix (IL-1ß, IL-6, TNFα) were used as positive controls. The percentage of cells positive to mesenchymal and endothelial markers was quantified by high content screening. We demonstrated that S+N+M mix induces irreversible EndMT in all analyzed endothelial cells via the TGFß pathway, as demonstrated by ApoA1 treatment. We then tested the contribution of single peptides in lung and brain cells, demonstrating that EndMT is triggered by M peptide. This was confirmed by transfection experiment, inducing the endogenous expression of the glycoprotein M in lung-derived cells. In conclusion, we demonstrated that SARS-CoV-2 peptides induce EndMT in microvascular endothelial cells from multiple body districts. The different peptides play different roles in the induction and maintenance of the virus-mediated effects, which are organ-specific. These results corroborate the hypothesis of the SARS-CoV-2-mediated microvascular damage underlying the multiple organ dysfunctions and the long-COVID syndrome.

Prolonged heat waves reduce the condition index and alter the molecular parameters in the pacific oyster Crassostrea gigas.

The entire shellfish farming sector is negatively affected by heat waves. Predictive models show that while heat waves are not predicted to exceed 28 °C in the northern Adriatic Sea over the coming decades, their duration will increase to periods of up to 30 days. Knowledge regarding the effects of heat waves on bivalves at physiological and molecular level is still limited. This study attempted to simulate what will happen in the future in Pacific oysters exposed to prolonged heat waves, assessing morphometric and physiological indices, and investigating the expression level of a number of genes, including the chaperone heat shock proteins HSP70, HSP72 and HSP90, and the factor P53. A state of stress in the heat wave-exposed animals was found, with loss of body weight and energy resources: despite showing a higher clearance rate, these animals were unable to absorb the nutrients required to maintain homeostasis, as well as demonstrating an alteration in hemolymphatic AST activity, total calcium and magnesium concentration. mRNA levels of all examined genes increased in response to thermal stress, with long-term overexpression, activating cell stress defense mechanisms and modulating the cycle cell. The results of this study indicate that heat waves affect oyster welfare, with consequences for the productivity of the sector due to the lack of salable products.

Stem cell-derived small extracellular vesicles embedded into methacrylated hyaluronic acid wound dressings accelerate wound repair in a pressure model of diabetic ulcer.

Over the past years, the development of innovative smart wound dressings is revolutionizing wound care management and research. Specifically, in the treatment of diabetic foot wounds, three-dimensional (3D) bioprinted patches may enable personalized medicine therapies. In the present work, a methacrylated hyaluronic acid (MeHA) bioink is employed to manufacture 3D printed patches to deliver small extracellular vesicles (sEVs) obtained from human mesenchymal stem cells (MSC-sEVs). The production of sEVs is maximized culturing MSCs in bioreactor. A series of in vitro analyses are carried out to demonstrate the influence of MSC-sEVs on functions of dermal fibroblasts and endothelial cells, which are the primary functional cells in skin repair process. Results demonstrate that both cell populations are able to internalize MSC-sEVs and that the exposure to sEVs stimulates proliferation and migration. In vivo experiments in a well-established diabetic mouse model of pressure ulcer confirm the regenerative properties of MSC-sEVs. The MeHA patch enhances the effectiveness of sEVs by enabling controlled release of MSC-sEVs over 7 days, which improve wound epithelialization, angiogenesis and innervation. The overall findings highlight that MSC-sEVs loading in 3D printed biomaterials represents a powerful technique, which can improve the translational potential of parental stem cell in terms of regulatory and economic impact.

Molecular mechanisms of skin wound healing in non-diabetic and diabetic mice in excision and pressure experimental wounds.

Experimental models for chronic skin lesions are excision and pressure ulcer, defined as "open" and "closed" lesions, respectively, only the latter characterized by tissue hypoxia. Moreover, systemic diseases, such as diabetes mellitus, affect wound repair. Thus, models for testing new therapies should be carefully selected according to the expected targets. In this study, we present an extensive and comparative histological, immunohistochemical, and molecular characterization of these two lesions in diabetic (db/db) and non-diabetic (C57BL/6 J) mice. In db/db mice, we found significant reduction in PGP9.5-IR innervation, reduction of capillary network, and reduced expression of NGF receptors. We found an increase in VEGF receptor Kdr expression, and the PI3K-Akt signaling pathway at the core of the altered molecular network. Db/db mice with pressure ulcers showed an impairment in the molecular regulation of hypoxia-related genes (Hif1a, Flt1, and Kdr), while extracellular matrix encoding genes (Itgb3, Timp1, Fn1, Col4a1) were upregulated by hyperglycemia and lesions. Overall, the molecular analysis suggests that db/db mice have a longer inflammatory phase of the wound repair process, delaying the progression toward the proliferation and remodeling phases.

Clinical and Pathological Features of Severe Gut Dysmotility.

Severe gut motility disorders are characterized by ineffective propulsion of intestinal contents. As a result, patients often develop extremely uncomfortable symptoms, ranging from nausea and vomiting along with alterations of bowel habits, up to radiologically confirmed subobstructive episodes. Chronic intestinal pseudo-obstruction (CIPO) is a typical clinical phenotype of severe gut dysmotility due to morphological and functional alterations of the intrinsic (enteric) innervation and extrinsic nerve supply (hence neuropathy), interstitial cells of Cajal (ICCs) (mesenchymopathy), and smooth muscle cells (myopathy). In this chapter, we highlight some molecular mechanisms of CIPO and review the clinical phenotypes and the genetics of the different types of CIPO. Specifically, we will detail the role of some of the most representative genetic mutations involving RAD21, LIG3, and ACTG2 to provide a better understanding of CIPO and related underlying neuropathic or myopathic histopathological abnormalities. This knowledge may unveil targeted strategies to better manage patients with such severe disease.

NGF and Retinitis Pigmentosa: Structural and Molecular Studies.

Nerve growth factor (NGF) is a neuroprotective molecule performing not only on central and peripheral neurons but also on cells of the visual system. Human retinitis pigmentosa (RP) is a major cause of blindness worldwide, and a resolute therapy is still lacking. Recent studies have shown that ocular NGF administration exerts a protective action on damaged retinal cells of mammalians, including human beings, although whether NGF also protects photoreceptors is not clear.We used the Royal College of Surgeons (RCS) strain in this study. The RCS is a rodent affected by inherited retinitis pigmentosa (RP) during postnatal life. For this study, we investigated whether ocular NGF treatment reduces/stops the progression of photoreceptor degeneration of rats with RP.This study was carried out in vitro on isolated photoreceptors to further investigate the action on these cells and whether the action is direct or mediated.The results indicate that ocular NGF administration can protect photoreceptors from degeneration into a model developing inherited RP and that the NGF action is direct. In this regard, we observed that binding of NGF to its receptor modulates expression of rhodopsin, a specific biological marker for photoreceptor survival and functionality.Part of the data reported in this chapter has been published in a previous study.

NGF and Endogenous Regeneration: From Embryology Toward Therapies.

The self-repair ability of tissues and organs in case of injury and disease is a fundamental biological mechanism and an important therapeutic target. The tissue plasticity and the presence of adult stem cell niches open a new path in the development of pharmacological and non-pharmacological treatments finalized to improve the intrinsic regeneration.In this context, nerve growth factor (NGF) is widely studied for its capability of driving endogenous regeneration of ectoderm-derived tissues, directly acting on the cell targets and through the regulation of the stem cell niches. In fact, this growth factor is very promising for its key role in the development and multiplicity of the cellular targets.In this chapter, we have traveled across the recent history of NGF pleiotropic role in ectodermal tissue generation and repair, from embryonic development to skin wound healing, axonal regrowth, and remyelination.The better understanding of both the biological mechanisms underlying regeneration and the physiological role of NGF in development and injury response will open new therapeutic strategies, driven by the potential applications of this growth factor as an agent for improving endogenous regeneration processes.

Co-Density Distribution Maps for Advanced Molecule Colocalization and Co-Distribution Analysis.

Cellular and subcellular spatial colocalization of structures and molecules in biological specimens is an important indicator of their co-compartmentalization and interaction. Presently, colocalization in biomedical images is addressed with visual inspection and quantified by co-occurrence and correlation coefficients. However, such measures alone cannot capture the complexity of the interactions, which does not limit itself to signal intensity. On top of the previously developed density distribution maps (DDMs), here, we present a method for advancing current colocalization analysis by introducing co-density distribution maps (cDDMs), which, uniquely, provide information about molecules absolute and relative position and local abundance. We exemplify the benefits of our method by developing cDDMs-integrated pipelines for the analysis of molecules pairs co-distribution in three different real-case image datasets. First, cDDMs are shown to be indicators of colocalization and degree, able to increase the reliability of correlation coefficients currently used to detect the presence of colocalization. In addition, they provide a simultaneously visual and quantitative support, which opens for new investigation paths and biomedical considerations. Finally, thanks to the coDDMaker software we developed, cDDMs become an enabling tool for the quasi real time monitoring of experiments and a potential improvement for a large number of biomedical studies.

A Time-Course Study of the Expression Level of Synaptic Plasticity-Associated Genes in Un-Lesioned Spinal Cord and Brain Areas in a Rat Model of Spinal Cord Injury: A Bioinformatic Approach.

"Neuroplasticity" is often evoked to explain adaptation and compensation after acute lesions of the Central Nervous System (CNS). In this study, we investigated the modification of 80 genes involved in synaptic plasticity at different times (24 h, 8 and 45 days) from the traumatic spinal cord injury (SCI), adopting a bioinformatic analysis. mRNA expression levels were analyzed in the motor cortex, basal ganglia, cerebellum and in the spinal segments rostral and caudal to the lesion. The main results are: (i) a different gene expression regulation is observed in the Spinal Cord (SC) segments rostral and caudal to the lesion; (ii) long lasting changes in the SC includes the extracellular matrix (ECM) enzymes Timp1, transcription regulators (Egr, Nr4a1), second messenger associated proteins (Gna1, Ywhaq); (iii) long-lasting changes in the Motor Cortex includes transcription regulators (Cebpd), neurotransmitters/neuromodulators and receptors (Cnr1, Gria1, Nos1), growth factors and related receptors (Igf1, Ntf3, Ntrk2), second messenger associated proteins (Mapk1); long lasting changes in Basal Ganglia and Cerebellum include ECM protein (Reln), growth factors (Ngf, Bdnf), transcription regulators (Egr, Cebpd), neurotransmitter receptors (Grin2c). These data suggest the molecular mapping as a useful tool to investigate the brain and SC reorganization after SCI.

Time-Course Changes of Extracellular Matrix Encoding Genes Expression Level in the Spinal Cord Following Contusion Injury-A Data-Driven Approach.

The involvement of the extracellular matrix (ECM) in lesion evolution and functional outcome is well recognized in spinal cord injury. Most attention has been dedicated to the "core" area of the lesion and scar formation, while only scattered reports consider ECM modification based on the temporal evolution and the segments adjacent to the lesion. In this study, we investigated the expression profile of 100 genes encoding for ECM proteins at 1, 8 and 45 days post-injury, in the spinal cord segments rostral and caudal to the lesion and in the scar segment, in a rat model. During both the active lesion phases and the lesion stabilization, we observed an asymmetric gene expression induced by the injury, with a higher regulation in the rostral segment of genes involved in ECM remodeling, adhesion and cell migration. Using bioinformatic approaches, the metalloproteases inhibitor Timp1 and the hyaluronan receptor Cd44 emerged as the hub genes at all post-lesion times. Results from the bioinformatic gene expression analysis were then confirmed at protein level by tissue analysis and by cell culture using primary astrocytes. These results indicated that ECM regulation also takes place outside of the lesion area in spinal cord injury.

Differential effects of glucose deprivation on the survival of fetal versus adult neural stem cells-derived oligodendrocyte precursor cells.

Impaired myelination is a key feature in neonatal hypoxia/ischemia (HI), the most common perinatal/neonatal cause of death and permanent disabilities, which is triggered by the establishment of an inflammatory and hypoxic environment during the most critical period of myelin development. This process is dependent on oligodendrocyte precursor cells (OPCs) and their capability to differentiate into mature oligodendrocytes. In this study, we investigated the vulnerability of fetal and adult OPCs derived from neural stem cells (NSCs) to inflammatory and HI insults. The resulting OPCs/astrocytes cultures were exposed to cytokines to mimic inflammation, or to oxygen-glucose deprivation (OGD) to mimic an HI condition. The differentiation of both fetal and adult OPCs is completely abolished following exposure to inflammatory cytokines, while only fetal-derived OPCs degenerate when exposed to OGD. We then investigated possible mechanisms involved in OGD-mediated toxicity: (a) T3-mediated maturation induction; (b) glutamate excitotoxicity; (c) glucose metabolism. We found that while no substantial differences were observed in T3 intracellular content regulation and glutamate-mediated toxicity, glucose deprivation lead to selective OPC cell death and impaired differentiation in fetal cultures only. These results indicate that the biological response of OPCs to inflammation and demyelination is different in fetal and adult cells, and that the glucose metabolism perturbation in fetal central nervous system (CNS) may significantly contribute to neonatal pathologies. An understanding of the underlying molecular mechanism will contribute greatly to differentiating myelination enhancing and neuroprotective therapies for neonatal and adult CNS white matter lesions.

A dynamic culture platform enhances the efficiency of the 3D HUVEC-based tube formation assay.

Cell-based in vitro biological models traditionally use monolayer cell cultures grown over plastic surfaces bathing in static media. Higher fidelity to a natural biological tissue is expected to result from growing the cells in a three-dimensional (3D) matrix. However, due to the decreased rate of diffusion inherent to increased distances within a tridimensional space, proper fluidic conditions are needed in this setting to better approximate a physiological environment. To this aim, we here propose a prototypal dynamic cell culture platform for the automatic medium replacement, via periodic perfusion flow, in a human umbilical vein endothelial cell (HUVECs) culture seeded in a Geltrex™ matrix. A state-of-the-art angiogenesis assay performed in these dynamic conditions showed sizable effects with respect to conventional static control cultures, with significantly enhanced pro-(dual antiplatelet therapy [DAPT]) and anti-(EDTA) angiogenic compound activity. In particular, dynamic culture conditions (a) enhance the 3D-organization of HUVECs into microtubule structure; (b) accelerate and improve endothelial tube formation by HUVECs in the presence of DAPT; (c) are able to completely revert the blocking effects of EDTA. These evidence emphasize the need of setting proper fluidic conditions for a better approximation of a physiological environment as an appropriate evolution of current cell culture paradigms.

Thyroid Hormone Signaling in Embryonic Stem Cells: Crosstalk with the Retinoic Acid Pathway.

While the role of thyroid hormones (THs) during fetal and postnatal life is well-established, their role at preimplantation and during blastocyst development remains unclear. In this study, we used an embryonic stem cell line isolated from rat (RESC) to study the effects of THs and retinoic acid (RA) on early embryonic development during the pre-implantation stage. The results showed that THs play an important role in the differentiation/maturation processes of cells obtained from embryoid bodies (EB), with thyroid hormone nuclear receptors (TR) (TRα and TRß), metabolic enzymes (deiodinases 1, 2, 3) and membrane transporters (Monocarboxylate transporters -MCT- 8 and 10) being expressed throughout in vitro differentiation until the Embryoid body (EB) stage. Moreover, thyroid hormone receptor antagonist TR (1-850) impaired RA-induced neuroectodermal lineage specification. This effect was significantly higher when cells were treated with retinoic acid (RA) to induce neuroectodermal lineage, studied through the gene and protein expression of nestin, an undifferentiated progenitor marker from the neuroectoderm lineage, as established by nestin mRNA and protein regulation. These results demonstrate the contribution of the two nuclear receptors, TR and RA, to the process of neuroectoderm maturation of the in vitro model embryonic stem cells obtained from rat.

Early CSF Biomarkers and Late Functional Outcomes in Spinal Cord Injury. A Pilot Study.

Although, biomarkers are regarded as an important tool for monitoring injury severity and treatment efficacy, and for predicting clinical evolution in many neurological diseases and disorders including spinal cord injury, there is still a lack of reliable biomarkers for the assessment of clinical course and patient outcome. In this study, a biological dataset of 60 cytokines/chemokines, growth factorsm and intracellular and extracellular matrix proteins, analyzed in CSF within 24 h of injury, was used for correlation analysis with the clinical dataset of the same patients. A heat map was generated of positive and negative correlations between biomarkers and clinical rating scale scores at discharge, and between biomarkers and changes in clinical scores during the observation period. Using very stringent statistical criteria, we found 10 molecules which correlated with clinical scores at discharge, and five molecules, which correlated with changes in clinical scores. The proposed methodology may be useful for generating hypotheses regarding "predictive" and "treatment effectiveness" biomarkers, thereby suggesting potential candidates for disease-modifying therapies using a "bed-to-bench" approach.

White Matter and Neuroprotection in Alzheimer's Dementia.

Myelin is the main component of the white matter of the central nervous system (CNS), allowing the proper electrical function of the neurons by ensheathing and insulating the axons. The extensive use of magnetic resonance imaging has highlighted the white matter alterations in Alzheimer's dementia (AD) and other neurodegenerative diseases, alterations which are early, extended, and regionally selective. Given that the white matter turnover is considerable in the adulthood, and that myelin repair is currently recognized as being the only true reparative capability of the mature CNS, oligodendrocyte precursor cells (OPCs), the cells that differentiate in oligodendrocyte, responsible for myelin formation and repair, are regarded as a potential target for neuroprotection. In this review, several aspects of the OPC biology are reviewed. The histology and functional role of OPCs in the neurovascular-neuroglial unit as described in preclinical and clinical studies on AD is discussed, such as the OPC vulnerability to hypoxia-ischemia, neuroinflammation, and amyloid deposition. Finally, the position of OPCs in drug discovery strategies for dementia is discussed.

Potential biomarkers for neuroinflammation and neurodegeneration at short and long term after neonatal hypoxic-ischemic insult in rat.

BACKGROUND: Hypoxic-ischemic (HI) encephalopathy causes life-long morbidity and premature mortality in term neonates. Therapies in addition to whole-body cooling are under development to treat the neonate at risk for HI encephalopathy, but are not a quickly measured serum inflammatory or neuronal biomarkers to rapidly and accurately identify brain injury in order to follow the efficacy of therapies. METHODS: In order to identify potential biomarkers for early inflammatory and neurodegenerative events after neonatal hypoxia-ischemia, both male and female Wistar rat pups at postnatal day 7 (P7) were used and had their right carotid artery permanently doubly occluded and exposed to 8% oxygen for 90 min. Sensory and cognitive parameters were assessed by open field, rotarod, CatWalk, and Morris water maze (MWM) test. Plasma and CSF biomarkers were investigated on the acute (24 h and 72 h) and chronic phase (4 weeks). Brains were assessed for gene expression analysis by quantitative RT-PCR Array. RESULTS: We found a delay of neurological reflex maturation in HI rats. We observed anxiolytic-like baseline behavior in males more than females following HI injury. HI rats held on the rotarod for a shorter time comparing to sham. HI injury impaired spatial learning ability on MWM test. The CatWalk assessment demonstrated a long-term deficit in gait parameters related to the hind paw. Proinflammatory biomarkers such as IL-6 in plasma and CCL2 and TNF-α in CSF showed an upregulation at 24 h after HI while other cytokines, such as IL-17A and CCL5, were upregulated after 72 h in CSF. At 24 h post-injury, we observed an increase of Edn1, Hif1-α, and Mmp9 mRNA levels in the ipsilateral vs the contralateral hemisphere of HI rats. An upregulation of genes involved with clotting and hematopoietic processes was observed 72 h post-injury. CONCLUSIONS: Our work showed that, in the immature brain, the HI injury induced an early increased production of several proinflammatory mediators detectable in plasma and CSF, followed by tissue damage in the hypoxic hemisphere and short-term as well as long-lasting neurobehavioral deficits.

Constitutive and LPS-stimulated secretome of porcine Vascular Wall-Mesenchymal Stem Cells exerts effects on in vitro endothelial angiogenesis.

BACKGROUND: MSCs secretome is under investigation as an alternative to whole-cell-based therapies, since it is enriched of bioactive molecules: growth factors, cytokines and chemokines. Taking into account the translational value of the pig model, the leading aim of the present paper was to characterize the secretome of porcine Vascular Wall-Mesenchymal Stem Cells (pVW-MSCs) and its change in presence of LPS stimulation. Moreover, considering the importance of angiogenesis in regenerative mechanisms, we analysed the effect of pVW-MSCs secretome on in vitro angiogenesis. RESULTS: Our results demonstrated that conditioned medium from unstimulated pVW-MSCs contained high levels of IL-8, GM-CSF, IFN-γ and other immunomodulatory proteins: IL-6 IL-18 IL-4 IL-2 IL-10. LPS modulates pVW-MSCs gene expression and secretome composition, in particular a significant increase of IL-6 and IL-8 was observed; conversely, the amount of GM-CSF, IFN-γ, IL-2, IL-4, IL-10 and IL-18 showed a significant transient decrease with the LPS stimulation. Conditioned medium from unstimulated pVW-MSCs induced in vitro endothelial angiogenesis, which is more evident when the conditioned medium was from LPS stimulated pVW-MSCs. CONCLUSIONS: The lines of evidence here presented shed a light on possible future application of secretome derived by pVW-MSCs on research studies in translational regenerative medicine.

Long-term effect of neonatal inhibition of APP gamma-secretase on hippocampal development in the Ts65Dn mouse model of Down syndrome.

Neurogenesis impairment is considered a major determinant of the intellectual disability that characterizes Down syndrome (DS), a genetic condition caused by triplication of chromosome 21. Previous evidence obtained in the Ts65Dn mouse model of DS showed that the triplicated gene APP (amyloid precursor protein) is critically involved in neurogenesis alterations. In particular, excessive levels of AICD (amyloid precursor protein intracellular domain) resulting from APP cleavage by gamma-secretase increase the transcription of Ptch1, a Sonic Hedgehog (Shh) receptor that keeps the mitogenic Shh pathway repressed. Previous evidence showed that neonatal treatment with ELND006, an inhibitor of gamma-secretase, reinstates the Shh pathway and fully restores neurogenesis in Ts65Dn pups. In the framework of potential therapies for DS, it is extremely important to establish whether the positive effects of early intervention are retained after treatment cessation. Therefore, the goal of the current study was to establish whether early treatment with ELND006 leaves an enduring trace in the brain of Ts65Dn mice. Ts65Dn and euploid pups were treated with ELND006 in the postnatal period P3-P15 and the outcome of treatment was examined at ~one month after treatment cessation. We found that in treated Ts65Dn mice the pool of proliferating cells in the hippocampal dentate gyrus (DG) and total number of granule neurons were still restored as was the number of pre- and postsynaptic terminals in the stratum lucidum of CA3, the site of termination of the mossy fibers from the DG. Accordingly, patch-clamp recording from field CA3 showed functional normalization of the input to CA3. Unlike in field CA3, the number of pre- and postsynaptic terminals in the DG of treated Ts65Dn mice was no longer fully restored. The finding that many of the positive effects of neonatal treatment were retained after treatment cessation provides proof of principle demonstration of the efficacy of early inhibition of gamma-secretase for the improvement of brain development in DS.

Inflammation severely alters thyroid hormone signaling in the central nervous system during experimental allergic encephalomyelitis in rat: Direct impact on OPCs differentiation failure.

Differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes is severely impaired by inflammatory cytokines and this could lead to remyelination failure in inflammatory/demyelinating diseases. Due to the role of thyroid hormone in the maturation of OPCs and developmental myelination, in this study we investigated (i) the possible occurrence of dysregulation of thyroid hormone signaling in the CNS tissue during experimental neuroinflammation; (ii) the possible impact of inflammatory cytokines on thyroid hormone signaling and OPCs differentiation in vitro. The disease model is the experimental allergic encephalomyelitis in female Dark-Agouti rats, whereas in vitro experiments were carried out in OPCs derived from neural stem cells. The main results are the following: (i) a strong upregulation of cytokine mRNA expression level was found in the spinal cord during experimental allergic encephalomyelitis; (ii) thyroid hormone signaling in the spinal cord (thyroid hormone receptors; deiodinase; thyroid hormone membrane transporter) is substantially downregulated, due to the upregulation of the thyroid hormone inactivating enzyme deiodinase 3 and the downregulation of thyroid hormone receptors, as investigated at mRNA expression level; (iii) when exposed to inflammatory cytokines, deiodinase 3 is upregulated in OPCs as well, and OPCs differentiation is blocked; (iv) deiodinase 3 inhibition by iopanoic acid recovers OPCs differentiation in the presence on inflammatory cytokines. These data suggest that cellular hypothyroidism occurs during experimental allergic encephalomyelitis, possibly impacting on thyroid hormone-dependent cellular processes, including maturation of OPCs into myelinating oligodendrocytes. GLIA 2016;64:1573-1589.

Cytokine and chemokine alterations in tissue, CSF, and plasma in early presymptomatic phase of experimental allergic encephalomyelitis (EAE), in a rat model of multiple sclerosis.

BACKGROUND: Experimental allergic encephalomyelitis (EAE) is the most commonly used experimental animal model for human multiple sclerosis (MS) that has been used so far to study the acute and remission-relapsing phases of the disease. Despite the vast literature on neuroinflammation onset and progression in EAE, important questions are still open regarding in particular the early asymptomatic phase between immunization and clinical onset. METHODS: In this study, we performed a time-course investigation of neuroinflammation and demyelination biomarkers in the spinal cord (SC), cerebrospinal fluid (CSF), and blood in EAE induced in dark agouti (DA) female rats compared to the controls and adjuvant-injected rats, using high-throughput technologies for gene expression and protein assays and focusing on the time-course between immunization, clinical onset (1, 5, 8 days post-immunization (DPI)), and progression (11 and 18 DPI). The expression profile of 84 genes related to T cell activation/signaling, adaptive immunity, cytokine/chemokine inflammation, demyelination, and cellular stress were analyzed in the tissue; 24 cytokines were measured in the CSF and plasma. RESULTS: The macrophage colony-stimulating factor (CSF1) was the first up-regulated protein as far as 1 DPI, not only in blood but also in CSF and SC. A treatment with GW2580, a selective CSF1R inhibitor, slowed the disease progression, significantly reduced the severity, and prevented the relapse phase. Moreover, both pro-inflammatory (IL-1ß, TNF-α) and anti-inflammatory cytokines (IL-5, IL-10, VEGF) were up-regulated starting from 8 DPI. Myelin genes were down-regulated starting from 8 DPI, especially MAL, MBP, and PMP22 while an opposite expression profile was observed for inflammation-related genes, such as CXCL11 and CXCL10. CONCLUSIONS: This early cytokine and chemokine regulation indicates that novel biomarkers and therapeutic options could be explored in the asymptomatic phase of EAE. Overall, our findings provide clear evidence that CSF1R signaling regulates inflammation in EAE, supporting therapeutic targeting of CSF1R in MS.