RESUMO
Olfactory ensheathing cells (OECs) are specialized glial cells of the olfactory system, believed to play a role in the continuous production of olfactory neurons and ensheathment of their axons. Although OECs are used in therapeutic applications, little is known about the cellular mechanisms underlying their migratory behavior. Recently, we showed that OEC migration is sensitive to ganglioside blockage through A2B5 and Jones antibody in OEC culture. Gangliosides are common components of lipid rafts, where they participate in several cellular mechanisms, including cell migration. Here, we characterized OEC lipid rafts, analyzing the presence of specific proteins and gangliosides that are commonly expressed in motile neural cells, such as young neurons, oligodendrocyte progenitors, and glioma cells. Our results showed that lipid rafts isolated from OECs were enriched in cholesterol, sphingolipids, phosphatidylcholine, caveolin-1, flotillin-1, gangliosides GM1 and 9-O-acetyl GD3, A2B5-recognized gangliosides, CNPase, α-actinin, and ß1-integrin. Analysis of the actin cytoskeleton of OECs revealed stress fibers, membrane spikes, ruffled membranes and lamellipodia during cell migration, as well as the distribution of α-actinin in membrane projections. This is the first description of α-actinin and flotillin-1 in lipid rafts isolated from OECs and suggests that, together with ß1-integrin and gangliosides, membrane lipid rafts play a role during OEC migration. This study provides new information on the molecular composition of OEC membrane microdomains that can impact on our understanding of the role of OEC lipid rafts under physiological and pathological conditions of the nervous system, including inflammation, hypoxia, aging, neurodegenerative diseases, head trauma, brain tumor, and infection.
Assuntos
Microdomínios da Membrana/metabolismo , Bulbo Olfatório/citologia , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Colesterol/metabolismo , Proteínas do Citoesqueleto/metabolismo , Gangliosídeos/metabolismo , Microdomínios da Membrana/ultraestrutura , Ratos Wistar , Proteínas S100/metabolismoRESUMO
Olfactory ensheathing glia (OEG) are found in the olfactory mucosa, nerve and bulb, and provide in vivo ensheathment for the unmyelinated olfactory axons within the central and peripheral nervous system domains. OEG cells are able to migrate long distances within the neuropil of the central nervous system. Because gangliosides such as 9-O-acetyl GD3 have crucial regulatory roles in neuronal migration during development, we analyzed whether OEG in organotypical cultures are revealed by anti-9-O-acetyl GD3 and/or gangliosides are recognized by the A2B5 antibody (G-A2B5), and whether these gangliosides are involved in OEG migration. Our results showed that all OEG migrating out of a section of olfactory bulb onto a laminin substrate bound to the 9-O-acetyl GD3 and A2B5 antibodies, and that 2',3'-cyclic nucleotide phosphodiesterase (CNPase) colocalized with 9-O-acetyl GD3 and with G-A2B5. Additionally, we showed that the immune blockade of 9-O-acetyl GD3 or G-A2B5 reduced the migration of OEG on laminin, and that 9-O-acetyl GD3 and G-A2B5 colocalized with the ß1-integrin subunit. We also confirmed the phenotype of in-vitro-grown OEG cells derived from adult rats, showing that they express CNPase, and also α-smooth muscle actin, which is not expressed by Schwann cells. Our data showed that the gangliosides 9-O-acetyl GD3 and G-A2B5 participate in the migratory activity of OEG cells, and that the ß1-integrin subunit colocalizes with these gangliosides. These results suggest a new role for ß1-integrin and gangliosides in the polarized migration of OEG cells, and provide new information on the molecules controlling OEG motility and behavior.
Assuntos
Movimento Celular/fisiologia , Gangliosídeos/metabolismo , Integrina beta1/metabolismo , Neuroglia/metabolismo , Bulbo Olfatório/metabolismo , Animais , Neuroglia/citologia , Bulbo Olfatório/citologia , Mucosa Olfatória/citologia , Mucosa Olfatória/metabolismo , Ratos , Ratos Wistar , Células de Schwann/citologia , Células de Schwann/metabolismoRESUMO
OBJECTIVES: Erythropoietin may have neuroprotective potential after ischemia of the central nervous system. Here, we conducted a study to characterize the protective effects of erythropoietin on retinal ganglion cells and gliotic reactions in an experimentally induced oligemia model. METHODS: Rats were subjected to global oligemia by bilateral common carotid artery occlusion and then received either vehicle or erythropoietin via intravitreal injection after 48 h; they were euthanized one week after the injection. The densities of retinal ganglion cells and contents of glial fibrillary acidic protein (astrocytes/Müller cells) and cluster of differentiation 68 clone ED1 (microglia/macrophages), assessed by fluorescence intensity, were evaluated in frozen retinal sections by immunofluorescence and epifluorescence microscopy. RESULTS: Retinal ganglion cells were nearly undetectable one week after oligemia compared with the sham controls; however, these cells were partially preserved in erythropoietin-treated retinas. The contents of glial fibrillary acidic protein and cluster of differentiation 68 clone ED1, markers for reactive gliosis, were significantly higher in retinas after bilateral common carotid artery occlusion than those in both sham and erythropoietin-treated retinas. CONCLUSIONS: The number of partially preserved retinal ganglion cells in the erythropoietin-treated group suggests that erythropoietin exerts a neuroprotective effect on oligemic/ischemic retinas. This effect could be related to the down-modulation of glial reactivity, usually observed in hypoxic conditions, clinically observed during glaucoma or retinal artery occlusion conditions. Therefore, glial reactivity may enhance neurodegeneration in hypoxic conditions, like normal-tension glaucoma and retinal ischemia, and erythropoietin is thus a candidate to be clinically applied after the detection of decreased retinal blood flow.
Assuntos
Eritropoetina/farmacologia , Proteína Glial Fibrilar Ácida/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Lesões das Artérias Carótidas/cirurgia , Artéria Carótida Primitiva/cirurgia , Contagem de Células , Modelos Animais de Doenças , Ectodisplasinas/efeitos dos fármacos , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Masculino , Ratos Wistar , Doenças Retinianas/patologiaRESUMO
OBJECTIVES: Erythropoietin may have neuroprotective potential after ischemia of the central nervous system. Here, we conducted a study to characterize the protective effects of erythropoietin on retinal ganglion cells and gliotic reactions in an experimentally induced oligemia model. METHODS: Rats were subjected to global oligemia by bilateral common carotid artery occlusion and then received either vehicle or erythropoietin via intravitreal injection after 48 h; they were euthanized one week after the injection. The densities of retinal ganglion cells and contents of glial fibrillary acidic protein (astrocytes/Müller cells) and cluster of differentiation 68 clone ED1 (microglia/macrophages), assessed by fluorescence intensity, were evaluated in frozen retinal sections by immunofluorescence and epifluorescence microscopy. RESULTS: Retinal ganglion cells were nearly undetectable one week after oligemia compared with the sham controls; however, these cells were partially preserved in erythropoietin-treated retinas. The contents of glial fibrillary acidic protein and cluster of differentiation 68 clone ED1, markers for reactive gliosis, were significantly higher in retinas after bilateral common carotid artery occlusion than those in both sham and erythropoietin-treated retinas. CONCLUSIONS: The number of partially preserved retinal ganglion cells in the erythropoietin-treated group suggests that erythropoietin exerts a neuroprotective effect on oligemic/ischemic retinas. This effect could be related to the down-modulation of glial reactivity, usually observed in hypoxic conditions, clinically observed during glaucoma or retinal artery occlusion conditions. Therefore, glial reactivity may enhance neurodegeneration in hypoxic conditions, like normal-tension glaucoma and retinal ischemia, and erythropoietin is thus a candidate to be clinically applied after the detection of decreased retinal blood flow.
Assuntos
Animais , Masculino , Células Ganglionares da Retina/efeitos dos fármacos , Eritropoetina/farmacologia , Fármacos Neuroprotetores/farmacologia , Proteína Glial Fibrilar Ácida/efeitos dos fármacos , Doenças Retinianas/patologia , Contagem de Células , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Ratos Wistar , Artéria Carótida Primitiva/cirurgia , Lesões das Artérias Carótidas/cirurgia , Modelos Animais de Doenças , Ectodisplasinas/efeitos dos fármacosRESUMO
The regenerative potential of the peripheral nervous system (PNS) is widely known, but functional recovery, particularly in humans, is seldom complete. Therefore, it is necessary to resort to strategies that induce or potentiate the PNS regeneration. Our main objective was to test the effectiveness of Olfactory Ensheathing Cells (OEC) transplantation into a biodegradable conduit as a therapeutic strategy to improve the repair outcome after nerve injury. Sciatic nerve transection was performed in C57BL/6 mice; proximal and distal stumps of the nerve were sutured into the collagen conduit. Two groups were analyzed: DMEM (acellular grafts) and OEC (1×105/2µL). Locomotor function was assessed weekly by Sciatic Function Index (SFI) and Global Mobility Test (GMT). After eight weeks the sciatic nerve was dissected for morphological analysis. Our results showed that the OEC group exhibited many clusters of regenerated nerve fibers, a higher number of myelinated fibers and myelin area compared to DMEM group. The G-ratio analysis of the OEC group showed significantly more fibers on the most suitable sciatic nerve G-ratio index. Motor recovery was accelerated in the OEC group. These data provide evidence that the OEC therapy can improve sciatic nerve functional and morphological recovery and can be potentially translated to the clinical setting.
Assuntos
Bainha de Mielina/transplante , Regeneração Nervosa/fisiologia , Neuroglia/fisiologia , Animais , Transplante de Células , Camundongos , Camundongos Endogâmicos C57BL , Bainha de Mielina/fisiologia , Fibras Nervosas/fisiologia , Córtex Olfatório , Recuperação de Função Fisiológica/fisiologia , Células de Schwann/transplante , Nervo Isquiático/lesõesRESUMO
Ascidians are interesting neurobiological models because of their evolutionary position as a sister-group of vertebrates and the high regenerative capacity of their central nervous system (CNS). We investigated the degeneration and regeneration of the cerebral ganglion complex of the ascidian Styela plicata following injection of the niacinamide antagonist 3-acetylpyridine (3AP), described as targeting the CNS of several vertebrates. For the analysis and establishment of a new model in ascidians, the ganglion complex was dissected and prepared for transmission electron microscopy (TEM), routine light microscopy (LM), immunohistochemistry and Western blotting, 1 or 10 days after injection of 3AP. The siphon stimulation test (SST) was used to quantify the functional response. One day after the injection of 3AP, CNS degeneration and recruitment of a non-neural cell type to the site of injury was observed by both TEM and LM. Furthermore, weaker immunohistochemical reactions for astrocytic glial fibrillary acidic protein (GFAP) and neuronal ßIII-tubulin were observed. In contrast, the expression of caspase-3, a protein involved in the apoptotic pathway, and the glycoprotein CD34, a marker for hematopoietic stem cells, increased. Ten days after the injection of 3AP, the expression of markers tended toward the original condition. The SST revealed attenuation and subsequent recovery of the reflexes from 1 to 10 days after 3AP. Therefore, we have developed a new method to study ascidian neural degeneration and regeneration, and identified the decreased expression of GFAP and recruitment of blood stem cells to the damaged ganglion as reasons for the success of neuroregeneration in ascidians.
Assuntos
Gânglios dos Invertebrados/fisiopatologia , Regeneração Nervosa/fisiologia , Urocordados/fisiologia , Animais , Antígenos CD34/metabolismo , Células Sanguíneas/fisiologia , Western Blotting , Caspase 3/metabolismo , Gânglios dos Invertebrados/ultraestrutura , Proteína Glial Fibrilar Ácida/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Modelos Animais , Degeneração Neural , Neuroglia/fisiologia , Neuroglia/ultraestrutura , Piridinas , Tubulina (Proteína)/metabolismo , Urocordados/ultraestruturaRESUMO
The neural system appears before the vascular system in the phylogenetic tree. During evolution, vascular system generation takes advantage of the pre-existing vascular endothelial growth factor (VEGF) in order to form its networks. Nevertheless, the role of VEGF in neuronal and glial cells is not yet completely understood. In order to support the hypothesis of a neural role for VEGF, we searched for VEGF- and VEGF receptor (VEGFR)-like immunoreactivities (immunohisto/cytochemistry and Western blotting) in the eyestalk of the invertebrate Ucides cordatus (Crustacea, Brachyura, Ucididae). Our results showed that both neurons and glial cells expressed VEGF-immunoreactivity, and that VEGFR was evidenced in neural cells. This is the first report about the VEGF/VEGFR-like immunoreactivities in the nervous tissue of a crustacean, and enables U. cordatus to be included in the repertoire of animal models used for ascertaining the role of VEGF in the nervous system.
Assuntos
Braquiúros/crescimento & desenvolvimento , Gânglios Sensitivos/crescimento & desenvolvimento , Neurogênese , Neurônios/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/fisiologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Vias Visuais/crescimento & desenvolvimento , Animais , Braquiúros/citologia , Masculino , Neurônios/citologia , Vias Visuais/citologiaRESUMO
Complex carbohydrate structures are essential molecules of infectious bacteria, parasites, and host cells and are involved in cell signaling associated with immune responses, glycoprotein homeostasis, and cell migration. The uptake of mannose-tailed glycans is usually carried out by professional phagocytes to trigger MHC class I- and MHC class II-restricted antigen presentation or, alternatively, to end inflammation. We have detected the mannose receptor (MR) in cultured olfactory ensheathing cells (OECs), so we investigated by flow cytometry whether recently dissociated cells of the olfactory bulb (OB) nerve fiber layer (ONL) could bind a mannosylated ligand (fluorescein conjugate of mannosyl bovine serum albumin; Man/BSA-FITC) in a specific manner. In addition, we estimated the relative proportion of ONL OECs, microglia, and astrocytes, tagged by 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), by the B4 isolectin of Griffonia simplicifonia (IB4), and by glial fibrillary acidic protein (GFAP), respectively, that were Man/BSA-FITC(+) . We also determined by histochemistry and/or immunohistochemistry whether Man/BSA-FITC or an anti-MR antibody (anti-C-terminal MR peptide; anti-cMR) labeled OECs and/or parenchymal microglia. In addition, we confirmed by Western blot with the K1K2 (against the entire MR molecule) antibody that a band of about 180 kDA is expressed in the OB. Our findings are compatible with a prospective sentinel role of OECs against pathogens of the upper airways and/or damage-associated glycidic patterns as well as with homeostasis of OB mannosylated glycoproteins.
Assuntos
Lectinas Tipo C/biossíntese , Lectinas de Ligação a Manose/biossíntese , Neuroglia/metabolismo , Bulbo Olfatório/citologia , Bulbo Olfatório/metabolismo , Receptores de Superfície Celular/biossíntese , Animais , Western Blotting , Citometria de Fluxo , Imuno-Histoquímica , Receptor de Manose , Ratos , Ratos WistarRESUMO
The use of bone marrow mononuclear cells (BMMCs) has been shown as a putative efficient therapy for stroke. However, the mechanisms of therapeutic action are not yet completely known. Mannose receptor (MR) is a subgroup of the C-type lectin receptor superfamily involved in innate immune response in several tissues. Although known primarily for its immune function, MR also has important roles in cell migration, cell debris clearance and tissue remodeling during inflammation and wound healing. Here we analyzed MR expression in brains of rats one week after induction of unilateral focal cortical ischemia by thermocoagulation in blood vessels of sensorimotor cortex. Additionally, we evaluated possible changes in such expression in cortices of rats subjected to ischemia plus treatment with BMMCs. Our results showed high expression of MR in an unknown GFAP(+) cell type and in phagocytic macrophages/microglia within the lesion boundary zone whereas in the non-injured (contralateral) cortical parenchyma, low levels of MR expression were observed. Moreover, therapy with BMMCs induced overexpression of MR in ipsilateral (injured) cortex. Previous studies from our group have shown functional recovery and decreased neurodegeneration in BMMC-treated rats in the same model of focal cortical ischemia. Thus, we suggest that ischemic injury induces large increase in MR expression as part of a mechanism for clearance of damage-associated molecular patterns (DAMPs). In addition, induction of MR overexpression by BMMCs might increase the efficiency of clearance, being one of the protective mechanisms of these cells.
Assuntos
Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Astrócitos/metabolismo , Isquemia Encefálica/terapia , Proteína Glial Fibrilar Ácida/metabolismo , Receptor de Manose , Microglia/metabolismo , Neurônios/metabolismo , Ratos , Ratos Wistar , Receptores Mitogênicos/metabolismoRESUMO
Olfactory ensheathing cells (OECs) are a special glia that ensheath olfactory receptor axons that enter the brain via olfactory phila, thus, providing a potential route for access of pathogens. Streptococcus pneumoniae (Sp), that has a capsule rich in mannosyl residues, is the most common cause of rhinosinusitis that may evolve to meningitis. We have tested whether OECs in vitro express the mannose receptor (MR), and could internalize Sp via MR. Cultures were infected by a suspension of Sp (ATCC 49619), recognized by an anti-Sp antibody, in a 100:1 bacteria:cells ratio. Competition assays, by means of mannan, showed around a 15-fold reduction in the number of internalized bacteria. To verify whether MR could be involved in Sp uptake, OECs were reacted with an antibody against the MR C-terminal peptide (anti-cMR) and bacteria were visualized with Sytox Green. Selective cMR-immunoreaction was seen in perinuclear compartments containing bacteria whereas mannan-treated cultures showed an extremely low percentage of internalized bacteria and only occasional adhered bacteria. Our data suggest the involvement of MR in adhesion of bacteria to OEC surface, and in their internalization. Data are also coherent with a role of OECs as a host cell prior to (and during) bacterial invasion of the brain.