Angiogenesis, the formation of new vessels, is found in Multiple Sclerosis (MS) demyelinating lesions following Vascular Endothelial Growth Factor (VEGF) release and the production of several other angiogenic molecules. The increased energy demand of inflammatory cuffs and damaged neural cells explains the strong angiogenic response in plaques and surrounding white matter. An angiogenic response has also been documented in an experimental model of MS, experimental allergic encephalomyelitis (EAE), where blood – brain barrier disruption and vascular remodelling appeared in a pre-symptomatic disease phase. In both MS and EAE, VEGF acts as a pro-inflammatory factor in the early phase but its reduced responsivity in the late phase can disrupt neuroregenerative attempts, since VEGF naturally enhances neuron resistance to injury and regulates neural progenitor proliferation, migration, differentiation and oligodendrocyte precursor cell (OPC) survival and migrati on to demyelinated lesions. An giogenesis, neurogenesis and oligodendroglia maturation are closely intertwined in the neurovascular niches of the subventricular zone, one of the preferential locations of inflammatory lesions in MS, and in all the other temporary vascular niches where the mutual fostering of angiogenesis and OPC maturation occurs. Angiogenesis, induced either by CNS inflammation or by hypoxic stimuli related to neurovascular uncoupling, appears to be ineffective in chronic MS due to a counterbalancing effect of vasoconstrictive mechanisms determined by the reduced axonal activity, astrocyte dysfunction, microglia secretion of free radical species and mitochondrial abnormalities. Thus, angiogenesis, that supplies several trophic factors, should be promoted in therapeutic neuroregeneration efforts to combat the progressive, degenerative phase of MS.

he pathophysiology of cerebral cortical lesions in multiple sclerosis (MS) is not understood. We investigated cerebral cortex microvessels during immune-mediated demyelination in the MS model chronic murine experimental autoimmune encephalomyelitis (EAE) by immunolocalization of the endothelial cell tight junction (TJ) integral proteins claudin-5 and occludin, a structural protein of caveolae, caveolin-1, and the blood-brain barrier-specific endothelial transporter, Glut 1. In EAE-affected mice, there were areas of extensivesubpial demyelination and well-demarcated lesions that extended to deeper cortical layers. Activation of microglia and absence of perivascular inflammatory infiltrates were common in these areas. Microvascular endothelial cells showed increased expression of caveolin-1 and a coincident loss of both claudin-5 and occludin normal junctional staining patterns. At a very early disease stage, claudin-5 molecules tended to cluster and form vacuoles that were also Glut 1 positive; the initially preserved occludin pattern became diffusely cytoplasmic at more advanced stages. Possible internalization of claudin-5 on TJ dismantling was suggested by its coexpression with the autophagosomal marker MAP1LC3A. Loss of TJ integrity was confirmed by fluorescein isothiocyanate-dextran experimentsthat showed leakage of the tracer into the perivascular neuropil. These observations indicate that, in the cerebral cortex of EAE-affected mice, there is a microvascular disease that differentially targets claudin-5 and occludin during ongoing demyelination despite only minimal inflammation.

Experimentally induced autoimmune encephalomyelitis (EAE) in mice provides an animal model that shares many features with human demyelinating diseases such as multiple sclerosis (MS). To what extent the cerebral cortex is affected by the process of demyelination and how the corollary response of the oligodendrocyte lineage is explicated are still not completely known aspects of EAE. By performing a detailed in situ analysis of expression of myelin and oligodendrocyte markers we have identified areas of subpial demyelination in the cerebral cortex of animals with conventionally induced EAE conditions. On EAE-affected cerebral cortices, the distribution and relative abundance of cells of the oligodendrocyte lineage were assessed and compared with control mouse brains. The analysis demonstrated that A2B5(+) glial restricted progenitors (GRPs) and NG2(+)/PDGFR-α(+) oligodendrocyte precursor cells (OPCs) were increased in number during "early" disease, 20 days post MOG immunization, whereas in the "late" disease, 39 days post-immunization, they were strongly diminished, and there was an accompanying reduction in NG2(+)/O4(+) pre-oligodendrocytes and GST-π mature oligodendrocytes. These results, together with the observed steady-state amount of NG2(-)/O4(+) pre-myelinating oligodendrocytes, suggested that oligodendroglial precursors attempted to compensate for the progressive loss of myelin, although these cells appeared to fail to complete the last step of their differentiation program. Our findings confirm that this chronic model of EAE reproduces the features of neocortex pathology in progressive MS and suggest that, despite the proliferative response of the oligodendroglial precursors, the failure to accomplish final differentiation may be a key contributing factor to the impaired remyelination that characterizes these demyelinating conditions.

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 pre-oligodendrocytes and pre-myelinating 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 population 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.

F3/contactin, a cell-adhesion molecule belonging to the immunoglobulin supergene family, is involved in several aspects of neural development including synapse building, maintenance and functioning. Here we examine F3/contactin function in adult hippocampal neurogenesis, synaptic plasticity and memory, using as a model TAG/F3 transgenic mice, where F3/contactin overexpression was induced under control of regulatory sequences from the human TAG-1 (TAX-1) gene. Transgenic mice aged 5 (M5) and 12 (M12) months exhibited an increase in hippocampal size, which correlated with positive effects on precursor proliferation and NeuN expression, these data suggesting a possible role for F3/contactin in promoting adult hippocampal neurogenesis. On the functional level, TAG/F3 mice exhibited increased CA1 long-term potentiation and improved spatial and object recognition memory, notably at 12 months of age. Interestingly, these mice showed an increased expression of the phosphorylated transcription factor CREB, which may represent the main molecular correlate of the observed morphological and functional effects. Altogether, these findings indicate for the first time that F3/contactin plays a role in promoting adult hippocampal neurogenesis and that this effect correlates with improved synaptic function and memory.

AIMS: Sporadic inclusion body myositis (s-IBM) is characterized by rimmed vacuole formation and misfolded protein accumulation. Intracellular protein aggregates are cleared by autophagy. When autophagy is blocked aggregates accumulate, resulting in abnormal rimmed vacuole formation. This study investigated the autophagy-lysosome pathway contribution to rimmed vacuole accumulation. METHODS: Autophagy was studied in muscle biopsy specimens obtained from eleven s-IBM patients, one suspected hereditary IBM patient, nine patients with other inflammatory myopathies and nine non-myopathic patients as controls. The analysis employed morphometric methods applied to immunohistochemistry using the endosome marker Clathrin, essential proteins of the autophagic cascade such as AuTophaGy-related protein ATG5, splicing variants of microtubule-associated protein light chain 3a (LC3a) and LC3b, compared with Beclin 1, the major autophagy regulator of both the initiation phase and late endosome/lysosome fusion of the autophagy-lysosome pathway. RESULTS: In muscle biopsies of s-IBM patients, an increased expression of Clathrin, ATG5, LC3a, LC3b and Beclin 1 was shown. Moreover, the inflammatory components of the disease, essentially lymphocytes, were preferentially distributed around the Beclin 1(+) myofibres. These affected myofibres also showed a moderate sarcoplasmic accumulation of SMI-31(+) phospho-tau paired helical filaments. CONCLUSION: The overexpression of autophagy markers linked to the decreased clearance of misfolded proteins, including SMI-31, and rimmed vacuoles accumulation may exhaust cellular resources and lead to cell death.

During human foetal brain vascularization, activated CD31(+)/CD105(+) endothelial cells are characterized by the emission of filopodial processes which also decorate the advancing tip of the vascular sprout. Together with filopodia, both the markers also reveal a number of plasma membrane-derived microvesicles (MVs) which are concentrated around the tip cell tuft of processes. At this site, MVs appear in tight contact with endothelial filopodia and follow these long processes, advancing into the surrounding neuropil to a possible cell target. These observations suggest that, like shedding vesicles of many other cell types that deliver signalling molecules and play a role in cell-to-cell communication, MVs sent out from endothelial tip cells could be involved in tip cell guidance and/or act on target cells, regulating cell-to-cell mutual recognition during vessel sprouting and final anastomosis. The results also suggest a new role for tip cell filopodia as conveyor processes for transporting MVs far from the cell of origin in a controlled microenvironment. Additional studies focused on the identification of MV content are needed to ultimately clarify the significance of tip cell MVs during human brain vascularization.

OBJECTIVES: Aim of this work is to show the effectiveness of a protocol involving the use of platelet-rich plasma (PRP) as a grafting material in bone regeneration before dental implant rehabilitation.MATERIALS AND METHODS: 127 patients, requiring maxillary sinus lift, were enrolled in a follow-up study plan, which established clinical and radiological examinations on the day after surgery and six months later. PRP, in combination with autogenous bone, anorganic bone material and organic bone substitutes, was used before implant-prosthetic rehabilitation.RESULTS AND CONCLUSIONS: After implant placement, 63 patients, previously treated with PRP, reached a statistically significant improvement in implant-prosthetic rehabilitation, established by primary stability and radiographic integration criteria, in comparison with the other 64 patients receiving implant-prosthetic rehabilitation without PRP treatment.

This study was conducted on human developing brain by laser confocal and transmission electron microscopy (TEM) to make a detailed analysis of important features of blood-brain barrier (BBB) microvessels and possible control mechanisms of vessel growth and differentiation during cerebral cortex vascularization. The BBB status of cortex microvessels was examined at a defined stage of cortex development, at the end of neuroblast waves of migration, and before cortex lamination, with BBB-endothelial cell markers, namely tight junction (TJ) proteins (occludin and claudin-5) and influx and efflux transporters (Glut-1 and P-glycoprotein), the latter supporting evidence for functional effectiveness of the fetal BBB. According to the well-known roles of astroglia cells on microvessel growth and differentiation, the early composition of astroglia/endothelial cell relationships was analyzed by detecting the appropriate astroglia, endothelial, and pericyte markers. GFAP, chemokine CXCL12, and connexin 43 (Cx43) were utilized as markers of radial glia cells, CD105 (endoglin) as a marker of angiogenically activated endothelial cells (ECs), and proteoglycan NG2 as a marker of immature pericytes. Immunolabeling for CXCL12 showed the highest level of the ligand in radial glial (RG) fibers in contact with the growing cortex microvessels. These specialized contacts, recognizable on both perforating radial vessels and growing collaterals, appeared as CXCL12-reactive en passant, symmetrical and asymmetrical, vessel-specific RG fiber swellings. At the highest confocal resolution, these RG varicosities showed a CXCL12-reactive dot-like content whose microvesicular nature was confirmed by ultrastructural observations. A further analysis of RG varicosities reveals colocalization of CXCL12 with Cx43, which is possibly implicated in vessel-specific chemokine signaling.

This study investigates glio-vascular interactions in human fetal brain at midgestation, specifically examining the expression and immunolocalization of the CXCL12/CXCR4/CXCR7 ligand-receptor axis and its possible role in the vascular patterning of the developing brain. At midgestation, the telencephalic vesicles are characterized by well developed radial glia cells (RGCs), the first differentiated astrocytes and a basic vascular network mainly built of radial vessels. RGCs have been recognized to contribute to cerebral cortex neuro-vascular architecture and have also been demonstrated to act as a significant source of neural cells (Rakic, Brain Res 33:471-476, 1971; Malatesta et al, Development 127:5253-5263, 2000). According to our hypothesis CXCL12, a potent migration and differentiation chemokine released by RGCs, may act as a linking factor coordinating neuroblast migration with vessel growth and patterning through the activation of different ligand/receptor axes. The obtained results support this hypothesis showing that together with CXCR4/CXCR7-reactive neuroblasts, which migrate in close association with CXCL12 RGCs, layer-specific subsets of CXCL12 RGCs and astrocytes specifically contact the microvessel wall. Moreover, the CXCL12/CXCR4/CXCR7 system appears to be directly involved in microvessel growth, its members being differentially expressed in angiogenically activated microvessels and vascular sprouts

Nanotubular structures, denoted tunneling nanotubes (TNTs) have been described in recent times as involved in cell-to-cell communication between distant cells. Nevertheless, TNT-like, long filopodial processes had already been described in the last century as connecting facing, growing microvessels during the process of cerebral cortex vascularization and collateralization. Here we have investigated the possible presence and the cellular origin of TNTs during normal brain vascularization and also in highly vascularized brain tumors.

Background The aim of this study was to assess the distribution of key SNARE proteins in glutamatergic and GABAergic synapses of the adult rat cerebellar cortex using light microscopy immunohistochemical techniques. Analysis was made of co-localizations of vGluT-1 and vGluT-2, vesicular transporters of glutamate and markers of glutamatergic synapses, or GAD, the GABA synthetic enzyme and marker of GABAergic synapses, with VAMP-2, SNAP-25A/B and syntaxin-1. Results The examined SNARE proteins were found to be diffusely expressed in glutamatergic synapses, whereas they were rarely observed in GABAergic synapses. However, among glutamatergic synapses, subpopulations which did not contain VAMP-2, SNAP-25A/B and syntaxin-1 were detected. They included virtually all the synapses established by terminals of climbing fibres (immunoreactive for vGluT-2) and some synapses established by terminals of parallel and mossy fibres (immunoreactive for vGluT-1, and for vGluT-1 and 2, respectively). The only GABA synapses expressing the SNARE proteins studied were the synapses established by axon terminals of basket neurons. Conclusion The present study supplies a detailed morphological description of VAMP-2, SNAP-25A/B and syntaxin-1 in the different types of glutamatergic and GABAergic synapses of the rat cerebellar cortex. The examined SNARE proteins characterize most of glutamatergic synapses and only one type of GABAergic synapses. In the subpopulations of glutamatergic and GABAergic synapses lacking the SNARE protein isoforms examined, alternative mechanisms for regulating trafficking of synaptic vesicles may be hypothesized, possibly mediated by different isoforms or homologous proteins. --------------------------------------------------------------------------------