BBB-endothelial cell response to cerebral cortex demyelination in a mouse model of chronic EAE

Changes in blood-brain barrier (BBB) function have been implicated in demyelinating diseases. This study aimed to investigate the response of cerebral cortex microvessels to nerve fibre demyelination in a chronic model of murine experimental autoimmune encephalomyelitis (EAE) characterized by areas of extensive subpial demyelination along with well-demarcated lesions extended to deeper cortex layers. These cortices showed activation of microglia and astrogliosis with absence of typical perivascular inflammatory infiltrates. On the basis of these data, we have analyzed the expression of two integral proteins of endothelial tight junctions, claudin-5 and occludin, a structural protein of caveolae, caveolin-1, as well as the BBB-specific endothelial transporter, Glut1 in the cerebral cortex of EAE-affected mice by immunofluorescence confocal microscopy. Microvascular endothelial cells showed an increased expression of caveolin-1 and a coincident decrease of both claudin-5 and occludin junctional staining pattern. At a very early disease stage, claudin-5 molecules formed aggregates and vacuoles that also stained for Glut 1, whereas occludin pattern became diffusely cytoplasmic at advanced stages of the disease. Internalization/dismantling and loss of tight junction proteins and impairment of BBB function were confirmed by coexpression of claudin-5 whit the autophagosomal marker MAP1LC3A and by FITC-dextran experiments that showed leakage of the tracer into the perivascular neuropil. Overall, these observations indicate that in the cerebral cortex of EAE mice, during demyelination and independently from the inflammatory involvement of the cortex, a ‘microvascular disease’ characterized by a differential involvement of claudin-5 and occludin occurs, thereby possibly contributing to demyelinating disease progression

Characterization of oligodendrocyte lineage precursor cells in the mouse cerebral cortex: a confocal microscopy approach to demyelinating diseases

The identification of stem cells resident in the adult central nervous system has redirected the focus of research into demyelinating diseases, such as multiple sclerosis, mainly affecting the brain white matter. This immunocytochemical and morphometrical study was carried out by confocal microscopy in the adult mouse cerebral cortex, with the aim of analysing, in the brain grey matter, the characteristics of the oligodendrocyte lineage cells, whose capability to remyelinate is still controversial. The observations demonstrated the presence in all the cortex layers of glial restricted progenitors, reactive to A2B5 marker, oligodendrocyte precursor cells, expressing the NG2 proteoglycan, and pre­oligodendrocytes and pre­myelinating oligodendrocytes, reactive to the specific marker O4. NG2 expressing cells constitute the major immature population of the cortex, since not only oligodendrocyte precursor cells and pre-oligodendrocytes but also a part of the glial restrict progenitors express the NG2 proteoglycan. Together with the popula­ tion of these immature cells, a larger population of mature oligodendrocytes was revealed by the classical oligodendrocyte and myelin markers, 2’,3’-cyclic nucleotide 3’­phosphodiesterase, myelin basic protein and myelin oligodendrocyte glycoprotein. The results indicate that oligodendrocyte precursors committed to differentiate into myelin forming oligodendrocytes are present through all layers of the adult cortex and that their phenotypic features exactly recall those of the oligodendroglial lineage cells during development

Microvascular pericytes involvement in experimental autoimmune encephalomyelitis

In the CNS, pericytes are microvessel wall-encircling cells that, together with endothelial cells, perivascular glial endfeet and basement membrane, form the blood-brain barrier (BBB). Dysfunction of the BBB and migration of autoreactive T lymphocytes into the CNS are histopathological hallmarks of both Multiple Sclerosis (MS), a chronic demyelinating disease, and experimental autoimmune encephalomyelitis (EAE), a widely used MS animal model. The proteoglycan NG2, which has been described to accumulate within MS plaques and at spinal cord (SC) injury sites, is a primary component of pericytes, engaged in pericyte/endothelial cell interaction, proliferation and migration. To explore the role of NG2-expressing pericytes during neuroinflammation and BBB dysfunction, pericyte coverage (pericyte number/vessel length) and density (pericyte number/tissue volume) ratios were studied in brain microvessels by immunohistochemistry and laser confocal microscopy using specific pericyte markers, NG2, RGS5, and CD13. The observations were made in mice affected by MOG-induced chronic EAE with two different genetic C57BL/6 backgrounds: wild type (WT) and homozygous NG2 null (NG2-/-). In literature, NG2-/- mice did not exhibit gross phenotypic or vascular alterations, whereas our results demonstrated an unaltered pericyte density associated with slightly decreased pericyte coverage index and pericyte/endothelial cell ratio. These observations were confirmed in NG2-/- EAE-affected mice, that showed an attenuated disease severity and demyelination, and a milder BBB leakage and leukocyte infiltration, as compared with EAE WT. Taken together these results lend support to the idea of a direct involvement of NG2 proteoglycan in pericyte-endothelial cell interactions essential for the preservation of a proper BBB function

Tight junction protein changes in experimental autoimmune encephalomyelitis models

Experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS), is characterized by vascular changes, particularly endothelial tight junction (TJ) protein (claudin-5 and occludin) alterations. During blood-brain barrier function, the vascular wall components, endothelial cells, pericytes and perivascular astrocytes, engage in crosstalks through cell-associated molecules and soluble factors. Pericyte-associated NG2 is a large transmembrane proteoglycan participating in these interactions, as well as in the control of pericyte proliferation and migration. We have analyzed the role of NG2 on endothelial TJ arrangement in two groups of mice, wild type (WT) and homozygous NG2 null (NG2-/-), affected by MOG-induced EAE. Expression and distribution of the TJ transmembrane proteins claudin-5 and occludin were analyzed in the cerebral cortex microvessels by immunohistochemistry and laser confocal microscopy. In NG2-/-mice, most cortex vessels showed an altered, chain-like claudin-5 staining pattern with aggregates distributed irregularly along the junctional membranes. Unlike the claudin-5 changes, the occludin staining pattern appeared continuous and linear and only a few cortex microvessels showed protein clustering. These TJ protein expression results in NG2-/- mice affected by EAE were compared with our previous results on WT EAE mice sacrificed at 39 days post immunization. In WT EAE both claudin-5 and occludin appeared severely damaged but occludin changes were related to more severe disease stages. Interestingly, in NG2-/- EAE-affected mice, claudin-5 and occludin formed an apparently unaffected linear and continuous junctional staining, suggesting a compensation of TJ damage, with cerebral cortex microvessels showing a restored claudin-5 and occludin junctional pattern. Overall, these observations suggest that absence of NG2 in the brain microvessels of naïve NG2 null mice may affect the normal arrangement of TJ proteins, whereas under inflammatory stimuli these effects seem to be partly reversed

Clinical Features, Cardiovascular Risk Profile, and Therapeutic Trajectories of Patients with Type 2 Diabetes Candidate for Oral Semaglutide Therapy in the Italian Specialist Care

Introduction: This study aimed to address therapeutic inertia in the management of type 2 diabetes (T2D) by investigating the potential of early treatment with oral semaglutide. Methods: A cross-sectional survey was conducted between October 2021 and April 2022 among specialists treating individuals with T2D. A scientific committee designed a data collection form covering demographics, cardiovascular risk, glucose control metrics, ongoing therapies, and physician judgments on treatment appropriateness. Participants completed anonymous patient questionnaires reflecting routine clinical encounters. The preferred therapeutic regimen for each patient was also identified. Results: The analysis was conducted on 4449 patients initiating oral semaglutide. The population had a relatively short disease duration (42%  60% of patients, and more often than sitagliptin or empagliflozin. Conclusion: The study supports the potential of early implementation of oral semaglutide as a strategy to overcome therapeutic inertia and enhance T2D management

Microvascular pericytes involvement in experimental autoimmune encephalomyelitis

In the CNS, pericytes are microvessel wall-encircling cells that, together with endothelial cells, perivascular glial endfeet and basement membrane, form the blood-brain barrier (BBB). Dysfunction of the BBB and migration of autoreactive T lymphocytes into the CNS are histopathological hallmarks of both Multiple Sclerosis (MS), a chronic demyelinating disease, and experimental autoimmune encephalomyelitis (EAE), a widely used MS animal model. The proteoglycan NG2, which has been described to accumulate within MS plaques and at spinal cord (SC) injury sites, is a primary component of pericytes, engaged in pericyte/endothelial cell interaction, proliferation and migration. To explore the role of NG2-expressing pericytes during neuroinflammation and BBB dysfunction, pericyte coverage (pericyte number/vessel length) and density (pericyte number/tissue volume) ratios were studied in brain microvessels by immunohistochemistry and laser confocal microscopy using specific pericyte markers, NG2, RGS5, and CD13. The observations were made in mice affected by MOG-induced chronic EAE with two different genetic C57BL/6 backgrounds: wild type (WT) and homozygous NG2 null (NG2-/-). In literature, NG2-/- mice did not exhibit gross phenotypic or vascular alterations, whereas our results demonstrated an unaltered pericyte density associated with slightly decreased pericyte coverage index and pericyte/endothelial cell ratio. These observations were confirmed in NG2-/- EAE-affected mice, that showed an attenuated disease severity and demyelination, and a milder BBB leakage and leukocyte infiltration, as compared with EAE WT. Taken together these results lend support to the idea of a direct involvement of NG2 proteoglycan in pericyte-endothelial cell interactions essential for the preservation of a proper BBB function