Metal bashing: iron deficiency and manganese overexposure impact on peripheral nerves.

Iron (Fe) deficiency (FeD) and manganese (Mn) overexposure (MnOE) may result in several neurological alterations in the nervous system. Iron deficiency produces unique neurological deficits due to its elemental role in central nervous system (CNS) development and myelination, which might persist after normalization of Fe in the diet. Conversely, MnOE is associated with diverse neurocognitive deficits. Despite these well-known neurotoxic effects on the CNS, the influence of FeD and MnOE on the peripheral nervous system (PNS) remains poorly understood. The aim of the present investigation was to examine the effects of developmental FeD and MnOE or their combination on the sciatic nerve of young and adult rats. The parameters measured included divalent metal transporter 1 (DMT1), transferrin receptor (TfR), myelin basic protein (MBP) and peripheral myelin protein 22 (PMP22) expression, as well as Fe levels in the nerve. Our results showed that FeD produced a significant reduction in MBP and PMP22 content at P29, which persisted at P60 after Fe-sufficient diet replenishment regardless of Mn exposure levels. At P60 MnOE significantly increased sciatic nerve Fe content and DMT1 expression. However, the combination of FeD and MnOE produced no marked motor skill impairment. Evidence indicates that FeD appears to hinder developmental peripheral myelination, while MnOE may directly alter Fe homeostasis. Further studies are required to elucidate the interplay between these pathological conditions.

EGFP transgene: a useful tool to track transplanted bone marrow mononuclear cell contribution to peripheral remyelination.

Bone marrow mononuclear cells (BMMC) constitute a heterogeneous population with potential to promote tissue regeneration. For this reason, this cell fraction has recently become a therapeutic alternative to mesenchymal stem cells, as culture is not required and phenotypic transformations can be hence avoided. In this work, and in order to attain long-lasting cell labeling and study longer survival times, we used BMMC isolated from adult transgenic rats expressing GFP to reproduce our wild type model and evaluate their remyelination ability in a reversible model of Wallerian degeneration. RT-PCR and flow cytometry analysis confirmed that cells isolated from the transgenic strain exhibited similar expression levels of markers specific to multipotent progenitors (CD34, CD90 and CD105) and Schwann cells (MPZ, MBP, S100ß and p75NTR) compared to wild type BMMC. BMMC expressing GFP retained their migration capacity, arriving exclusively at the injured nerve. Most importantly, and as detected through long-lasting cell tracking, some of these BMMC settled in the demyelinated area, mingled with endogenous cells, underwent phenotypic changes and colocalized with Schwann cell markers MBP and S100ß. Also worth highlighting, transgenic BMMC replicated wild type BMMC effects in terms of MBP organization and levels. On the basis of these findings, BMMC isolated from transgenic animals constitute a useful tool to evaluate their role in peripheral nervous system demyelination-remyelination and the underlying mechanisms.

All the PNS is a Stage: Transplanted Bone Marrow Cells Play an Immunomodulatory Role in Peripheral Nerve Regeneration.

SUMMARY STATEMENT: Bone marrow cell transplant has proven to be an effective therapeutic approach to treat peripheral nervous system injuries as it not only promoted regeneration and remyelination of the injured nerve but also had a potent effect on neuropathic pain.

Bone marrow mononuclear cells migrate to the demyelinated sciatic nerve and transdifferentiate into Schwann cells after nerve injury: attempt at a peripheral nervous system intrinsic repair mechanism.

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.

Sciatic nerve regeneration after traumatic injury using magnetic targeted adipose-derived mesenchymal stem cells.

Traumatic peripheral nerve injuries constitute a huge concern to public health. Nerve damage leads to a decrease or even loss of mobility of the innervated area. Adult stem cell therapies have shown some encouraging results and have been identified as promising treatment candidates for nerve regeneration. A major obstacle to that approach is securing a sufficient number of cells at the injured site to produce measurable therapeutic effects. The present work tackles this issue and demonstrates enhanced nerve regeneration ability promoted by magnetic targeted cell therapy in an in vivo Wallerian degeneration model. To this end, adipose-derived mesenchymal stem cells (AdMSC) were loaded with citric acid coated superparamagnetic iron oxide nanoparticles (SPIONs), systemically transplanted and magnetically recruited to the injured sciatic nerve. AdMSC arrival to the injured nerve was significantly increased using magnetic targeting and their beneficial effects surpassed the regenerative properties of the stand-alone cell therapy. AdMSC-SPIONs group showed a partially conserved nerve structure with many intact myelinated axons. Also, a very remarkable restoration in myelin basic protein organization, indicative of remyelination, was observed. This resulted in an improvement in nerve conduction, demonstrating functional recovery. In summary, our results demonstrate that magnetically assisted delivery of AdMSC, using a non-invasive and non-traumatic method, is a highly promising strategy to promote cell recruitment and sciatic nerve regeneration after traumatic injury. Last but not least, our results validate magnetic targeting in vivo exceeding previous reports in less complex models through cell magnetic targeting in vitro and ex vivo. STATEMENT OF SIGNIFICANCE: Traumatic peripheral nerve injuries constitute a huge public health concern. They can lead to a decrease or even loss of mobility of innervated areas. Due to their complex pathophysiology, current pharmacological and surgical approaches are only partially effective. Cell-based therapies have emerged as a useful tool to achieve full tissue regeneration. However, a major bottleneck is securing enough cells at injured sites. Therefore, our proposal combining biological (adipose derived mesenchymal stem cells) and nanotechnological strategies (magnetic targeting) is of great relevance, reporting the first in vivo experiments involving "magnetic stem cell" targeting for peripheral nerve regeneration. Using a non-invasive and non-traumatic method, cell recruitment in the injured nerve was improved, fostering nerve remyelination and functional recovery.

Systemic Transplantation of Bone Marrow Mononuclear Cells Promotes Axonal Regeneration and Analgesia in a Model of Wallerian Degeneration.

BACKGROUND: Reinnervation timing after nerve injury is critical for favorable axonal regeneration, remyelination, and clinical improvement. Considering bone marrow mononuclear cells (BMMC) are easily obtained and readily available for transplant, this work analyzed the effect of BMMC systemic administration on nerve repair and pain behavior. METHODS: Adult rats with sciatic nerve crush were immediately and systemically injected BMMC through the caudal artery. Nontreated, sham and naïve rats were also included. Histological, immunohistochemical, biochemical, functional, and behavioral analyses were performed in nerves harvested from each group at different survival times. RESULTS: Axons in BMMC-treated rats exhibited a more conserved morphological appearance than those in nontreated rats, as observed at different survival times both in semithin sections and ultrastructural analysis. BMMC-treated rats also showed a reduction in major myelin protein immunoreactive clusters 7 and 14 days postinjury, as compared with nontreated rats. Electrophysiological analysis showed BMMC treatment to slightly improve the amplitude of compound muscle action potential starting at 14 days postinjury. Finally, mechanical withdrawal threshold revealed a full preventive action against transient mechanical hypersensitivity in BMMC-treated rats. CONCLUSIONS: These data demonstrate the efficiency of BMMC, systemically and noninvasively transplanted, in correcting morphological, functional and behavioral alterations resulting from peripheral nerve injury.

Hypoxic and Reoxygenated Microenvironment: Stemness and Differentiation State in Glioblastoma.

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.

Isolation and Characterization of Ischemia-Derived Astrocytes (IDAs) with Ability to Transactivate Quiescent Astrocytes.

UNLABELLED: 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 tissue IDA show reduced replicative senescence, increased cell division and spontaneous migration IDA potentiate death of oxygen-glucose deprived cortical neurons IDA propagate reactive gliosis on quiescent astrocytes in vitro and in vivo Inhibition of gamma secretases facilitates IDA differentiation to astrocytes.

Phosphatidylinositol 3-Kinase/AKT Pathway Inhibition by Doxazosin Promotes Glioblastoma Cells Death, Upregulation of p53 and Triggers Low Neurotoxicity.

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-3ß 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.

DMT1 iron uptake in the PNS: bridging the gap between injury and regeneration.

Previous studies by our group demonstrated the key role of iron in Schwann cell maturation through an increase in cAMP, PKA activation and CREB phosphorylation. These studies opened the door to further research on non-transferrin-bound iron uptake, which revealed the presence of DMT1 mRNA all along SC progeny, hinting at a constitutive role of DMT1 in ensuring the provision of iron in the PNS. In light of these previous results, the present work evaluates the participation of DMT1 in the remyelination process following a demyelinating lesion promoted by sciatic nerve crush--a reversible model of Wallerian degeneration. DMT1 was observed to colocalize with a SC marker S100ß at all survival times analyzed. In turn, the assessment of DMT1 mRNA expression exhibited an increase 7 days post-injury, while DMT1 protein levels showed an increase 14 days after crush at the lesion site and distal stump; finally, an increase in iron levels became evident as from 14 days post-injury, in parallel with DMT1 values. To sum up, the present work unveils the role of DMT1 in mediating the neuroregenerative action of iron.

DMT1 as a candidate for non-transferrin-bound iron uptake in the peripheral nervous system.

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-100ß (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.