In the present work, we analyzed whether endogenous and/or transplanted bone marrow mononuclear cells (BMMC) migrate spontaneously to the crushed sciatic nerve and whether they transdifferentiate into Schwann cells (SC) in order to help repair the damaged tissue. We also studied both the immunohistochemical evolution of myelin proteins MBP and P(0) and the myelin composition of both the proximal and distal stumps of the crushed sciatic nerve to determine the demyelination-remyelination period. Immunohistochemical analysis of crushed animals showed that the degeneration process consists of loss of nerve fiber integrity accompanied by degradation of myelin basic proteins MBP and P(0) , which is anticipated by protein cluster formation. The remyelination process appears as a recovery in nerve fiber structure as well as in MBP and P(0) immunoreactivity; results obtained studying isolated myelin from the crushed sciatic nerve show a strong correlation between them. As opposed to demyelination, axonal damage is observed for a short period of time and takes place mostly in the crush area and the segments adjacent to the lesion. Evidence of spontaneous migration of endogenous or intravascularly transplanted BMMC (CD34(+) and vimentin(+) ) is found during the demyelination period exclusively to the injured sciatic nerve. Once migration takes place, transdifferentiation to SC is observed. Such migration and transdifferentiation processes might be inferred to constitute a spontaneous repair mechanism after nerve injury.
Reactive gliosis involving activation and proliferation of astrocytes and microglia, is a widespread but largely complex and graded glial response to brain injury. Astroglial population has a previously underestimated high heterogeneity with cells differing in their morphology, gene expression profile, and response to injury. Here, we identified a subset of reactive astrocytes isolated from brain focal ischemic lesions that show several atypical characteristics. Ischemia-derived astrocytes (IDAs) were isolated from early ischemic penumbra and core. IDA did not originate from myeloid precursors, but rather from pre-existing local progenitors. Isolated IDA markedly differ from primary astrocytes, as they proliferate in vitro with high cell division rate, show increased migratory ability, have reduced replicative senescence and grow in the presence of macrophages within the limits imposed by the glial scar. Remarkably, IDA produce a conditioned medium that strongly induced activation on quiescent primary astrocytes and potentiated the neuronal death triggered by oxygen-glucose deprivation. When re-implanted into normal rat brains, eGFP-IDA migrated around the injection site and induced focal reactive gliosis. Inhibition of gamma secretases or culture on quiescent primary astrocytes monolayers facilitated IDA differentiation to astrocytes. We propose that IDA represent an undifferentiated, pro-inflammatory, highly replicative and migratory astroglial subtype emerging from the ischemic microenvironment that may contribute to the expansion of reactive gliosis.Main Points:Ischemia-derived astrocytes (IDA) were isolated from brain ischemic tissueIDA show reduced replicative senescence, increased cell division and spontaneous migrationIDA potentiate death of oxygen-glucose deprived cortical neuronsIDA propagate reactive gliosis on quiescent astrocytes in vitro and in vivoInhibition of gamma secretases facilitates IDA differentiation to astrocytes
Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Hypoxia is a distinct feature in GBM and plays a significant role in tumor progression, resistance to treatment, and poor outcome. However, there is lack of studies relating type of cell death, status of Akt phosphorylation on Ser473, mitochondrial membrane potential, and morphological changes of tumor cells after hypoxia and reoxygenation. The rat glioma C6 cell line was exposed to oxygen deprivation (OD) in 5 % fetal bovine serum (FBS) or serum-free media followed by reoxygenation (RO). OD induced apoptosis on both 5 % FBS and serum-free groups. Overall, cells on serum-free media showed more profound morphological changes than cells on 5 % FBS. Moreover, our results suggest that OD combined with absence of serum provided a favorable environment for glioblastoma dedifferentiation to cancer stem cells, since nestin, and CD133 levels increased. Reoxygenation is present in hypoxic tumors through microvessel formation and cell migration to oxygenated areas. However, few studies approach these phenomena when analyzing hypoxia. We show that RO caused morphological alterations characteristic of cells undergoing a differentiation process due to increased GFAP. In the present study, we characterized an in vitro hypoxic microenvironment associated with GBM tumors, therefore contributing with new insights for the development of therapeutics for resistant glioblastoma.
Glioblastoma is the most frequent and malignant brain tumor. Treatment includes chemotherapy with temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide, as well as the human glioblastoma cell line U138-MG. We investigated doxazosin’s (an antihypertensive drug) activity against glioblastoma cells (C6 and U138-MG) and its neurotoxicity on primary astrocytes and organoptypic hippocampal cultures. For this study, the following methods were used: citotoxicity assays, flow cytometry, western-blotting and confocal microscopy. We showed that doxazosin induces cell death on C6 and U138-MG cells. We observed that doxazosin’s effects on the PI3K/Akt pathway were similar as LY294002 (PI3K specific inhibitor). In glioblastoma cells treated with doxasozin, Akt levels were greatly reduced. Upon examination of activities of proteins downstream of Akt we observed upregulation of GSK-3β and p53. This led to cell proliferation inhibition, cell death induction via caspase-3 activation and cell cycle arrest at G0/G1 phase in glioblastoma cells. We used in this study Lapatinib, a tyrosine kinase inhibitor, as a comparison with doxazosin because they present similar chemical structure. We also tested the neurocitotoxicity of doxazosin in primary astrocytes and organotypic cultures and observed that doxazosin induced cell death on a small percentage of non-tumor cells. Aggressiveness of glioblastoma tumors and dismal prognosis require development of new treatment agents. This includes less toxic drugs, more selective towards tumor cells, causing less damage to the patient. Therefore, our results confirm the potential of doxazosin as an attractive therapeutic antiglioma agent.
Iron, either in its chelated form or as holotransferrin (hTf), prevents the dedifferentiation of Schwann cells (SC), cells responsible for the myelination of the peripheral nervous system (PNS). This dedifferentiation is promoted by serum deprivation through cAMP release, PKA activation, and CREB phosphorylation. Since iron elicits its effect in a transferrin (Tf)-free environment, in this work we postulate that non-transferrin-bound iron (NTBI) uptake must be involved. Divalent metal transporter 1(DMT1) has been widely described in literature as a key player in iron metabolism, but never before in the PNS context. The presence of DMT1 was demonstrated in nerve homogenate, isolated adult-rat myelin, and cultured SC by Western Blot (WB) analysis and confirmed through its colocalization with S-100β (SC marker) by immunocytochemical and immunohistochemical analyses. Furthermore, the existence of its mRNA was verified in sciatic nerve homogenate by RT-PCR and throughout SC maturational stages. Finally, we describe DMT1's subcellular location in the plasma membrane by confocal microscopy of SC and WB of different subcellular fractions. These data allow us to suggest the participation of DMT1 as part of a Tf independent iron uptake mechanism in SC and lead us to postulate a crucial role for iron in SC maturation and, as a consequence, in PNS myelination.
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