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1.
Methods Mol Biol ; 2206: 143-150, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754816

RESUMO

Pericytes are integral part of neurovascular unit and play a role in the maintenance of blood-brain barrier integrity, angiogenesis, and cerebral blood flow regulation. Despite their important functional roles, a univocal phenotypic identification is still emerging also for the lack of a "pan-pericyte" marker. In the present study, we describe in detail the method for performing fluorescence immunohistochemistry on thick free-floating sections from human fetal brain in high resolution laser confocal microscopy. This method enables to obtain three-dimensional images of pericytes and provides insights about their distribution and localization in the microvessels of human developing brain.

2.
Curr Pharm Des ; 26(13): 1428-1437, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32186270

RESUMO

P-Glycoprotein (P-gp) is a 170-kDa transmembrane glycoprotein that works as an efflux pump and confers multidrug resistance (MDR) in normal tissues and tumors, including nervous tissues and brain tumors. In the developing telencephalon, the endothelial expression of P-gp, and the subcellular localization of the transporter at the luminal endothelial cell (EC) plasma membrane are early hallmarks of blood-brain barrier (BBB) differentiation and suggest a functional BBB activity that may complement the placental barrier function and the expression of P-gp at the blood-placental interface. In early fetal ages, P-gp has also been immunolocalized on radial glia cells (RGCs), located in the proliferative ventricular zone (VZ) of the dorsal telencephalon and now considered to be neural progenitor cells (NPCs). RG-like NPCs have been found in many regions of the developing brain and have been suggested to give rise to neural stem cells (NSCs) of adult subventricular (SVZ) neurogenic niches. The P-gp immunosignal, associated with RG-like NPCs during cortical histogenesis, progressively decreases in parallel with the last waves of neuroblast migrations, while 'outer' RGCs and the deriving astrocytes do not stain for the efflux transporter. These data suggest that in human glioblastoma (GBM), P-gp expressed by ECs may be a negligible component of tumor MDR. Instead, tumor perivascular astrocytes may dedifferentiate and resume a progenitor-like P-gp activity, becoming MDR cells and contribute, together with perivascular P-gpexpressing glioma stem-like cells (GSCs), to the MDR profile of GBM vessels. In conclusion, the analysis of Pgp immunolocalization during brain development may contribute to identify the multiple cellular sources in the GBM vessels that may be involved in P-gp-mediated chemoresistance and can be responsible for GBM therapy failure and tumor recurrence.

3.
Neurobiol Dis ; 139: 104821, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32088380

RESUMO

BACKGROUND AND AIM: Patients with Parkinson's disease (PD) are often characterized by functional gastrointestinal disorders. Such disturbances can occur at all stages of PD and precede the typical motor symptoms of the disease by many years. However, the morphological alterations associated with intestinal disturbances in PD are undetermined. This study examined the remodelling of colonic wall in 6-hydroxydopamine (6-OHDA)-induced PD rats. METHODS: 8 weeks after 6-OHDA injection animals were sacrificed. Inflammatory infiltrates, collagen deposition and remodelling of intestinal epithelial barrier and tunica muscularis in the colonic wall were assessed by histochemistry, immunohistochemistry, immunofluorescence and western blot analysis. RESULTS: 6-OHDA rats displayed significant alterations of colonic tissues as compared with controls. Signs of mild inflammation (eosinophil infiltration) and a transmural deposition of collagen fibres were observed. Superficial colonic layers were characterized by severe morphological alterations. In particular, lining epithelial cells displayed a reduced claudin-1 and transmembrane 16A/Anoctamin 1 (TMEM16A/ANO1) expression; goblet cells increased their mucin expression; colonic crypts were characterized by an increase in proliferating epithelial cells; the density of S100-positive glial cells and vimentin-positive fibroblast-like cells was increased as well. Several changes were found in the tunica muscularis: downregulation of α-smooth muscle actin/desmin expression and increased proliferation of smooth muscle cells; increased vimentin expression and proliferative phenotype in myenteric ganglia; reduction of interstitial cells of Cajal (ICCs) density. CONCLUSIONS: A pathological remodelling occurs in the colon of 6-OHDA rats. The main changes include: enhanced fibrotic deposition; alterations of the epithelial barrier; activation of mucosal defense; reduction of ICCs. These results indicate that central nigrostriatal denervation is associated with histological changes in the large bowel at mucosal, submucosal and muscular level. These alterations might represent morphological correlates of digestive symptoms in PD.

4.
Muscle Nerve ; 60(3): 315-327, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31172530

RESUMO

INTRODUCTION: The molecular mechanism of immune-mediated necrotizing myopathy (IMNM) remains unknown. Autophagy impairment, described in autoimmune diseases, is a key process in myofiber protein degradation flux and muscle integrity and has not been studied in IMNM. METHODS: Muscle biopsies from patients with IMNM (n = 40), dermatomyositis (DM; 24), polymyositis (PM; 8), polymyositis with mitochondrial pathology (4), sporadic inclusion body myositis (8), and controls (6) were compared by immunohistochemistry. RESULTS: The proportions of myofibers containing autophagy markers LC3b and p62 were higher in IMNM than in DM or PM and correlated with creatine kinase levels. In IMNM, compartmentalized LC3b puncta were located in regenerating and degenerating myofibers surrounded by major histocompatibility complex type II+ inflammatory cells. Several IMNM myofibers accumulated ubiquitin and misfolded protein. DISCUSSION: The detection of LC3b+ or p62+ myofibers could be used in differentiating IMNM from PM. The identification of autophagy-modifying molecules potentially could improve patients' outcomes. Muscle Nerve, 2019.


Assuntos
Autofagia/imunologia , Músculo Esquelético/patologia , Miosite/imunologia , Miosite/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Biópsia , Dermatomiosite/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miosite de Corpos de Inclusão/patologia , Polimiosite/imunologia , Polimiosite/patologia
5.
PLoS One ; 14(3): e0213508, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30870435

RESUMO

During experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis associated with blood-brain barrier (BBB) disruption, oligodendrocyte precursor cells (OPCs) overexpress proteoglycan nerve/glial antigen 2 (NG2), proliferate, and make contacts with the microvessel wall. To explore whether OPCs may actually be recruited within the neurovascular unit (NVU), de facto intervening in its cellular and molecular composition, we quantified by immunoconfocal morphometry the presence of OPCs in contact with brain microvessels, during postnatal cerebral cortex vascularization at postnatal day 6, in wild-type (WT) and NG2 knock-out (NG2KO) mice, and in the cortex of adult naïve and EAE-affected WT and NG2KO mice. As observed in WT mice during postnatal development, a higher number of juxtavascular and perivascular OPCs was revealed in adult WT mice during EAE compared to adult naïve WT mice. In EAE-affected mice, OPCs were mostly associated with microvessels that showed altered claudin-5 and occludin tight junction (TJ) staining patterns and barrier leakage. In contrast, EAE-affected NG2KO mice, which did not show any significant increase in vessel-associated OPCs, seemed to retain better preserved TJs and BBB integrity. As expected, absence of NG2, in both OPCs and pericytes, led to a reduced content of vessel basal lamina molecules, laminin, collagen VI, and collagen IV. In addition, analysis of the major ligand/receptor systems known to promote OPC proliferation and migration indicated that vascular endothelial growth factor A (VEGF-A), platelet-derived growth factor-AA (PDGF-AA), and the transforming growth factor-ß (TGF-ß) were the molecules most likely involved in proliferation and recruitment of vascular OPCs during EAE. These results were confirmed by real time-PCR that showed Fgf2, Pdgfa and Tgfb expression on isolated cerebral cortex microvessels and by dual RNAscope-immunohistochemistry/in situ hybridization (IHC/ISH), which detected Vegfa and Vegfr2 transcripts on cerebral cortex sections. Overall, this study suggests that vascular OPCs, in virtue of their developmental arrangement and response to neuroinflammation and growth factors, could be integrated among the classical NVU cell components. Moreover, the synchronized activation of vascular OPCs and pericytes during both BBB development and dysfunction, points to NG2 as a key regulator of vascular interactions.


Assuntos
Antígenos/biossíntese , Barreira Hematoencefálica/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Microvasos/metabolismo , Oligodendroglia/metabolismo , Proteoglicanas/biossíntese , Células-Tronco/metabolismo , Animais , Antígenos/genética , Barreira Hematoencefálica/patologia , Movimento Celular/genética , Proliferação de Células/genética , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Claudina-5/genética , Claudina-5/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , Camundongos , Camundongos Knockout , Microvasos/patologia , Oligodendroglia/patologia , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteoglicanas/genética , Células-Tronco/patologia , Junções Íntimas/genética , Junções Íntimas/metabolismo , Junções Íntimas/patologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Fluids Barriers CNS ; 15(1): 28, 2018 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-30290761

RESUMO

BACKGROUND: 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. METHODS: We searched for TNTs by high-resolution immunofluorescence confocal microscopy, applied to the analysis of 20-µm, thick sections from lightly fixed, unembedded samples of both developing cerebral cortex and human glioblastoma (GB), immunolabeled for endothelial, pericyte, and astrocyte markers, and vessel basal lamina molecules. RESULTS: The results revealed the existence of pericyte-derived TNTs, labeled by proteoglycan NG2/CSPG4 and CD146. In agreement with the described heterogeneity of these nanostructures, ultra-long (> 300 µm) and very thin (< 0.8 µm) TNTs were observed to bridge the gap between the wall of distant vessels, or were detected as short (< 300 µm) bridging cables connecting a vessel sprout with its facing vessel or two apposed vessel sprouts. The pericyte origin of TNTs ex vivo in fetal cortex and GB was confirmed by in vitro analysis of brain pericytes, which were able to form and remained connected by typical TNT structures. CONCLUSIONS: None of the multiple roles described for TNTs can be excluded from a possible involvement during the processes of both normal and pathological vessel growth. A possible function, suggested by the pioneering studies made during cerebral cortex vascularization, is in cell searching and cell-to-cell recognition during the processes of vessel collateralization and vascular network formation. According to our results, it is definitely the pericyte-derived TNTs that seem to actively explore the surrounding microenvironment, searching for (site-to-site recognition), and connecting with (pericyte-to-pericyte and/or pericyte-to-endothelial cell communication), the targeted vessels. This idea implies that TNTs may have a primary role in the very early phases of both physiological and tumor angiogenesis in the brain.


Assuntos
Neoplasias Encefálicas/fisiopatologia , Córtex Cerebral/fisiopatologia , Células Endoteliais/fisiologia , Glioblastoma/fisiopatologia , Nanotubos , Neovascularização Patológica , Neovascularização Fisiológica , Pericitos/fisiologia , Adulto , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Comunicação Celular , Células Cultivadas , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/citologia , Células Endoteliais/citologia , Feminino , Glioblastoma/irrigação sanguínea , Glioblastoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Pericitos/citologia
7.
Mol Genet Metab ; 125(4): 322-331, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30145178

RESUMO

Mucopolysaccharidosis (MPS) disorders are caused by deficiencies in lysosomal enzymes, leading to impaired glycosaminoglycan (GAG) degradation. The resulting GAG accumulation in cells and connective tissues ultimately results in widespread tissue and organ dysfunction. The seven MPS types currently described are heterogeneous and progressive disorders, with somatic and neurological manifestations depending on the type of accumulating GAG. Heparan sulfate (HS) is one of the GAGs stored in patients with MPS I, II, and VII and the main GAG stored in patients with MPS III. These disorders are associated with significant central nervous system (CNS) abnormalities that can manifest as impaired cognition, hyperactive and/or aggressive behavior, epilepsy, hydrocephalus, and sleeping problems. This review discusses the anatomical and pathophysiological CNS changes accompanying HS accumulation as well as the mechanisms believed to cause CNS abnormalities in MPS patients. The content of this review is based on presentations and discussions on these topics during a meeting on the brain in MPS attended by an international group of MPS experts.


Assuntos
Encéfalo/anatomia & histologia , Encéfalo/fisiopatologia , Disfunção Cognitiva/etiologia , Epilepsia/etiologia , Heparitina Sulfato/metabolismo , Mucopolissacaridoses/complicações , Disfunção Cognitiva/patologia , Epilepsia/patologia , Humanos
8.
Int J Surg Case Rep ; 48: 34-37, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29783140

RESUMO

INTRODUCTION: Congenital diaphragmatic disease is a quite common condition that usually occurs in the neonatal period, and the diagnosis of congenital diaphragmatic disease in adulthood is rare. CASE PRESENTATION: A 64-years-old Caucasian woman was admitted in emergency at our Department, due to a bowel obstruction and dyspnea. A CT-scan showed a diaphragmatic herniation in the left area, with malposition of dilated transverse and descending colon in the chest. An emergency laparatomy was performed, showing a toxic megacolon, in the absence of a true diaphragmatic hernia, and a left diaphragm and left liver hypoplasia. An intraoperative bronchoscopy revealed concomitant hypoplasia of the left lung. A subtotal colectomy with ileo-rectal anastomosis was performed. The postoperative course was uneventful. Histological examination demonstrated hyperplasia of the muscularis mucosae of the colon and cytoplasmic vacuolization of the Auerbach plexus ganglia. The karyotype genetic analysis excluded concomitant microdeletion or duplication syndromes. DISCUSSION: To our knowledge, this seems to be the first reported case of toxic megacolon in a patient with congenital hypoplasia of the left bronchial-lung system, of the left liver, and of the left diaphragm. CONCLUSION: The correct development of the diaphragm is essential for the neighboring organs. The observed clinical pattern could be related to a partial modification of neural crest cell detachment or migration, which could be responsible for bowel and diaphragm defects, even though it was not included in typical neural crest cell syndromes. Further researches should be performed in order to define the sporadic or syndromic source of these multiorgan defects.

9.
Neuro Oncol ; 19(9): 1173-1182, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28541444

RESUMO

The pericyte, a constitutive component of the central nervous system, is a poorly understood cell type that envelops the endothelial cell with the intended purpose of regulating vascular flow and endothelial cell permeability. Previous studies of pericyte function have been limited to a small number of disease processes such as ischemic stroke and Alzheimer's disease. Recently, publications have postulated a link between glioma stem cell differentiation and pericyte function. These studies suggest that there may be an important interaction of pericytes with tumor cells and other components of the tumor microenvironment in malignant primary glial neoplasms, most notably glioblastoma. This potential cellular interaction underscores the need to pursue more investigations of pericytes in malignant brain tumor biology. In this review, we summarize the functional roles of pericytes, particularly focusing on changes in pericyte biology during response to immune cells, inflammation, and hypoxic conditions. The information presented is based on the available data from studies of pericyte function in other central nervous system diseases but will serve as a foundation for research investigations to further understand the role of pericytes in malignant gliomas.


Assuntos
Barreira Hematoencefálica/patologia , Neoplasias Encefálicas/patologia , Glioma/patologia , Pericitos/patologia , Doença de Alzheimer/patologia , Animais , Humanos , Acidente Vascular Cerebral/patologia
10.
Fluids Barriers CNS ; 13(1): 17, 2016 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-27655189

RESUMO

Annexin A1 is a potent anti-inflammatory molecule that has been extensively studied in the peripheral immune system, but has not as yet been exploited as a therapeutic target/agent. In the last decade, we have undertaken the study of this molecule in the central nervous system (CNS), focusing particularly on the primary interface between the peripheral body and CNS: the blood-brain barrier. In this review, we provide an overview of the role of this molecule in the brain, with a particular emphasis on its functions in the endothelium of the blood-brain barrier, and the protective actions the molecule may exert in neuroinflammatory, neurovascular and metabolic disease. We focus on the possible new therapeutic avenues opened up by an increased understanding of the role of annexin A1 in the CNS vasculature, and its potential for repairing blood-brain barrier damage in disease and aging.

11.
Acta Neuropathol ; 132(1): 23-42, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27026411

RESUMO

In adult CNS, nerve/glial-antigen 2 (NG2) is expressed by oligodendrocyte progenitor cells (OPCs) and is an early marker of pericyte activation in pathological conditions. NG2 could, therefore, play a role in experimental autoimmune encephalomyelitis (EAE), a disease associated with increased blood-brain barrier (BBB) permeability, inflammatory infiltrates, and CNS damage. We induced EAE in NG2 knock-out (NG2KO) mice and used laser confocal microscopy immunofluorescence and morphometry to dissect the effect of NG2 KO on CNS pathology. NG2KO mice developed milder EAE than their wild-type (WT) counterparts, with less intense neuropathology associated with a significant improvement in BBB stability. In contrast to WT mice, OPC numbers did not change in NG2KO mice during EAE. Through FACS and confocal microscopy, we found that NG2 was also expressed by immune cells, including T cells, macrophages, and dendritic cells (DCs). Assessment of recall T cell responses to the encephalitogen by proliferation assays and ELISA showed that, while WT and NG2KO T cells proliferated equally to the encephalitogenic peptide MOG35-55, NG2KO T cells were skewed towards a Th2-type response. Because DCs could be responsible for this effect, we assessed their expression of IL-12 by PCR and intracellular FACS. IL-12-expressing CD11c+ cells were significantly decreased in MOG35-55-primed NG2KO lymph node cells. Importantly, in WT mice, the proportion of IL-12-expressing cells was significantly lower in CD11c+ NG2- cells than in CD11c+ NG2+ cells. To assess the relevance of NG2 at immune system and CNS levels, we induced EAE in bone-marrow chimeric mice, generated with WT recipients of NG2KO bone-marrow cells and vice versa. Regardless of their original phenotype, mice receiving NG2KO bone marrow developed milder EAE than those receiving WT bone marrow. Our data suggest that NG2 plays a role in EAE not only at CNS/BBB level, but also at immune response level, impacting on DC activation and thereby their stimulation of reactive T cells, through controlling IL-12 expression.


Assuntos
Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/imunologia , Animais , Barreira Hematoencefálica/imunologia , Barreira Hematoencefálica/patologia , Células da Medula Óssea/imunologia , Transplante de Medula Óssea , Células Dendríticas/patologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Índice de Gravidade de Doença , Medula Espinal/imunologia , Medula Espinal/patologia , Linfócitos T/imunologia , Linfócitos T/patologia
12.
J Crohns Colitis ; 10(10): 1194-204, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26995183

RESUMO

BACKGROUND AND AIMS: Intestinal fibrosis is a complication of inflammatory bowel disease [IBD]. Although fibrostenosis is a rare event in ulcerative colitis [UC], there is evidence that a fibrotic rearrangement of the colon occurs in the later stages. This is a retrospective study aimed at examining the histopathological features of the colonic wall in both short-lasting [SL] and long-lasting [LL] UC. METHODS: Surgical samples of left colon from non-stenotic SL [≤ 3 years, n = 9] and LL [≥ 10 years, n = 10] UC patients with active disease were compared with control colonic tissues from cancer patients without UC [n = 12] to assess: collagen and elastic fibres by histochemistry; vascular networks [CD31/CD105/nestin] by immunofluorescence; parameters of fibrosis [types I and III collagen, fibronectin, RhoA, alpha-smooth muscle actin [α-SMA], desmin, vimentin], and proliferation [proliferating nuclear antigen [PCNA]] by western blot and/or immunolabelling. RESULTS: Colonic tissue from both SL-UC and LL-UC showed tunica muscularis thickening and transmural activated neovessels [displaying both proliferating CD105-positive endothelial cells and activated nestin-positive pericytes], as compared with controls. In LL-UC, the increased collagen deposition was associated with an up-regulation of tissue fibrotic markers [collagen I and III, fibronectin, vimentin, RhoA], an enhancement of proliferation [PCNA] and, along with a loss of elastic fibres, a rearrangement of the tunica muscularis towards a fibrotic phenotype. CONCLUSIONS: A significant transmural fibrotic thickening occurs in colonic tissue from LL-UC, together with a cellular fibrotic switch in the tunica muscularis. A full-thickness angiogenesis is also evident in both SL- and LL-UC with active disease, as compared with controls.


Assuntos
Colite Ulcerativa/patologia , Colo/patologia , Remodelação Vascular , Doença Aguda , Adulto , Idoso , Biomarcadores/metabolismo , Western Blotting , Estudos de Casos e Controles , Colo/irrigação sanguínea , Colo/metabolismo , Progressão da Doença , Feminino , Fibrose , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Método Simples-Cego
13.
J Cell Mol Med ; 19(2): 485-500, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25521239

RESUMO

Bowel inflammatory fibrosis has been largely investigated, but an integrated assessment of remodelling in inflamed colon is lacking. This study evaluated tissue and cellular changes occurring in colonic wall upon induction of colitis, with a focus on neuromuscular compartment. Colitis was elicited in rats by 2,4-dinitrobenzenesulfonic acid (DNBS). After 6 and 21 days, the following parameters were assessed on paraffin sections from colonic samples: tissue injury and inflammatory infiltration by histology; collagen and elastic fibres by histochemistry; HuC/D, glial fibrillar acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), nestin, substance P (SP), von Willebrand factor, c-Kit and transmembrane 16A/Anoctamin1 (TMEM16A/ANO1) by immunohistochemistry. TMEM16A/ANO1 was also examined in isolated colonic smooth muscle cells (ICSMCs). On day 6, inflammatory alterations and fibrosis were present in DNBS-treated rats; colonic wall thickening and fibrotic remodelling were evident on day 21. Colitis was associated with both an increase in collagen fibres and a decrease in elastic fibres. Moreover, the neuromuscular compartment of inflamed colon displayed a significant decrease in neuron density and increase in GFAP/PCNA-positive glia of myenteric ganglia, enhanced expression of neural SP, blood vessel remodelling, reduced c-Kit- and TMEM16A/ANO1-positive interstitial cells of Cajal (ICCs), as well as an increase in TMEM16A/ANO1 expression in muscle tissues and ICSMCs. The present findings provide an integrated view of the inflammatory and fibrotic processes occurring in the colonic neuromuscular compartment of rats with DNBS-induced colitis. These morphological alterations may represent a suitable basis for understanding early pathophysiological events related to bowel inflammatory fibrosis.


Assuntos
Colite/patologia , Miócitos de Músculo Liso/patologia , Animais , Colo/patologia , Inflamação/patologia , Masculino , Ratos , Ratos Sprague-Dawley
14.
Front Neurosci ; 8: 324, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25360079

RESUMO

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.

15.
J Neuropathol Exp Neurol ; 73(6): 495-506, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24806298

RESUMO

Information on the effects of injury on neovascularization in the immature brain is limited. We investigated the effects of ischemia on cerebral cortex neovascularization after the exposure of fetuses to 30 minutes of cerebral ischemia followed by 48 hours of reperfusion (I/R-48), 30 minutes of cerebral ischemia followed by 72 hours of reperfusion (I/R-72), or sham control treatment (Non-I/R). Immunohistochemical and morphometric analyses of cerebral cortex sections included immunostaining for glial fibrillary acidic protein and collagen type IV (a molecular component of the vascular basal lamina) to determine the glial vascular network in fetal brains and Ki67 as a proliferation marker. Cerebral cortices from I/R-48 and I/R-72 fetuses exhibited general responses to ischemia, including reactive astrocyte morphology, which was not observed in Non-I/R fetuses. Cell bodies of reactive proliferating astrocytes, along with large end-feet, surrounded the walls of cerebral cortex microvessels in addition to the thick collagen type IV-enriched basal lamina. Morphometric analysis of the Non-I/R group with the I/R-48 and I/R-72 groups revealed increased collagen type IV density in I/R-72 cerebral cortex microvessels (p < 0.01), which also frequently displayed a sprouting appearance characterized by growing tip cells and activated pericytes. Increases in cerebral cortex basic fibroblast growth factor were associated with neovascularization. We conclude that increased neovascularization in fetal cerebral cortices occurs within 72 hours of ischemia.


Assuntos
Isquemia Encefálica/complicações , Córtex Cerebral/patologia , Neovascularização Patológica/etiologia , Neovascularização Patológica/patologia , Traumatismo por Reperfusão/complicações , Animais , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Colágeno Tipo IV/metabolismo , Feminino , Feto/metabolismo , Feto/patologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Antígeno Ki-67/metabolismo , Microscopia Confocal , Gravidez , Ovinos , Fatores de Tempo
16.
J Inherit Metab Dis ; 36(3): 455-66, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23344887

RESUMO

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.


Assuntos
Encéfalo/irrigação sanguínea , Encéfalo/embriologia , Comunicação Celular/fisiologia , Quimiocina CXCL12/fisiologia , Receptores CXCR4/fisiologia , Receptores CXCR/fisiologia , Vasos Sanguíneos/embriologia , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiologia , Encéfalo/metabolismo , Encéfalo/patologia , Quimiocina CXCL12/metabolismo , Feto/metabolismo , Feto/patologia , Idade Gestacional , Humanos , Imuno-Histoquímica , Ligantes , Neovascularização Fisiológica/fisiologia , Neuroglia/metabolismo , Neuroglia/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Receptores CXCR/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais/fisiologia
17.
PLoS One ; 8(12): e84883, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24386429

RESUMO

NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV) cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs) directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM) components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS, discriminating between phenotype-specific and neoplastic versus non-neoplastic variants of the PG, thus opening up vistas for more selective immunotherapeutic targeting of brain tumours.


Assuntos
Neoplasias Encefálicas/metabolismo , Encéfalo/metabolismo , Proteoglicanas de Sulfatos de Condroitina/biossíntese , Feto/metabolismo , Glioblastoma/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Neoplasias/biossíntese , Pericitos/metabolismo , Adulto , Animais , Anticorpos Monoclonais Murinos/química , Anticorpos Antineoplásicos/química , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Feminino , Feto/patologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pericitos/patologia , Isoformas de Proteínas/biossíntese
18.
J Neuropathol Exp Neurol ; 71(10): 840-54, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23001217

RESUMO

The 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 extensive subpial 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 experiments that 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.


Assuntos
Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/fisiopatologia , Córtex Cerebral/metabolismo , Córtex Cerebral/fisiopatologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/fisiopatologia , Animais , Barreira Hematoencefálica/patologia , Caveolina 1/metabolismo , Córtex Cerebral/patologia , Claudina-5/metabolismo , Encefalomielite Autoimune Experimental/patologia , Feminino , Transportador de Glucose Tipo 1/biossíntese , Transportador de Glucose Tipo 1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ocludina/metabolismo
19.
Angiogenesis ; 15(4): 761-9, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22886085

RESUMO

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.


Assuntos
Membrana Celular/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/imunologia , Imunofluorescência , Humanos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia
20.
Neurobiol Dis ; 43(3): 678-89, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21679768

RESUMO

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.


Assuntos
Células-Tronco Adultas/patologia , Córtex Cerebral/patologia , Doenças Desmielinizantes/patologia , Encefalomielite Autoimune Experimental/patologia , Oligodendroglia/patologia , Células-Tronco Adultas/metabolismo , Animais , Linhagem da Célula/fisiologia , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Nervosas Mielinizadas/patologia , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/metabolismo
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