Time-Dependent Internalization of Polymer-Coated Silica Nanoparticles in Brain Endothelial Cells and Morphological and Functional Effects on the Blood-Brain Barrier

Nanoparticle (NP)-assisted procedures including laser tissue soldering (LTS) offer advantages compared to conventional microsuturing, especially in the brain. In this study, effects of polymer-coated silica NPs used in LTS were investigated in human brain endothelial cells (ECs) and blood-brain barrier models. In the co-culture setting with ECs and pericytes, only the cell type directly exposed to NPs displayed a time-dependent internalization. No transfer of NPs between the two cell types was observed. Cell viability was decreased relatively to NP exposure duration and concentration. Protein expression of the nuclear factor k-light-chain-enhancer of activated B cells and various endothelial adhesion molecules indicated no initiation of inflammation or activation of ECs after NP exposure. Differentiation of CD34+ ECs into brain-like ECs co-cultured with pericytes, blood-brain barrier (BBB) characteristics were obtained. The established endothelial layer reduced the passage of integrity tracer molecules. NP exposure did not result in alterations of junctional proteins, BBB formation or its integrity. In a 3-dimensional setup with an endothelial tube formation and tight junctions, barrier formation was not disrupted by the NPs and NPs do not seem to cross the blood-brain barrier. Our findings suggest that these polymer-coated silica NPs do not damage the BBB

Conditioned Medium from Endothelial Progenitor Cells promotes number of dopaminergic neurons and exerts neuroprotection in cultured ventral mesencephalic neuronal progenitor cells.

Transplantation of stem and progenitor cells offers a promising tool for brain repair in the context of neuropathological disorders including Parkinson's disease. There is growing proof that the capacity of adult stem and progenitor cells for tissue regeneration relies rather on the release of paracrine factors than on their cell replacement properties. In line with this notion, we have previously reported that conditioned medium (CM) collected from cultured Endothelial Progenitor Cells (EPC) stimulated survival of striatal neurons. In the present study we investigated whether EPC-CM promotes survival of cultured midbrain progenitor cells. For that purpose primary cultures from fetal rat embryonic ventral mesencephalon (VM) were prepared and grown for 7 days in vitro (DIV). EPC-CM was administered from DIV5-7. First, we found that EPC-CM treatment resulted in significantly increased cell densities of TH-ir neurons. Interestingly, this effect was no longer seen after proteolytic digestion of the EPC-CM. EPC-CM also significantly increased densities of beta-III-tubulin positive neurons and lba-1-ir microglial cells. The effect on dopaminergic neurons was not due to higher cell proliferation as no incorporation of EdU was observed in TH-ir cells. Importantly, EPC-CM exerted neuroprotection against MPP+ induced toxicity as in vitro model of Parkinson's disease. Taken together, our findings identified EPC-CM as a powerful tool to promote survival of cultured VM neurons and further support the importance of paracrine factors in the actions of stem and progenitor cells for brain repair

Restorative neuroscience:concepts and perspectives

There is increasing interest in the search for therapeutic options for diseases and injuries of the central nervous system (CNS), for which currently no effective treatment strategies are available. Replacement of damaged cells and restoration of function can be accomplished by transplantation of cells derived from different sources, such as human foetal tissue, genetically modified cell lines, embryonic or somatic stem cells. Preclinical and clinical trials have shown promising results in neurodegenerative disorders, like Parkinson's and Huntington's disease, but also ischaemic stroke, intracerebral haemorrhage, demyelinating disorders, epilepsy and traumatic lesions of the brain and spinal cord. Other studies have focused on finding new ways to activate and direct endogenous repair mechanisms in the CNS, eg, by exposure to specific neuronal growth factors or by inactivating inhibitory molecules. Neuroprotective drugs may offer an additional tool for improving neuronal survival in acute or chronic CNS diseases. Importantly however, a number of scientific issues need to be addressed in order to permit the introduction of these experimental techniques in the wider clinical setting

Quantitative characterization of phenotypical markers after differentiation of SH-SY5Y cells.

BACKGROUND The human neuroblastoma cell line, SH-SY5Y has been widely used in neuroscience research, especially in studies related to Parkinson's disease. However, differences between clones have been demonstrated, highlighting the importance to characterize the properties of this cell line carefully. OBJECTIVE The aim of this study was to characterize the phenotype of undifferentiated and differentiated SH-SY5Y cells using various differentiation protocols. METHODS A morphological and a quantitative analysis of markers related to dopaminergic and cholinergic neurons, but also other phenotypes, was performed. RESULTS Differentiated cells showed the typical neuronal morphology. Undifferentiated cells expressed low levels of tyrosine hydroxylase (TH) and higher levels of the high-affinity choline transporter (CHT1). Staurosporine (ST)-differentiation resulted in the highest number of TH-immunoreactive cells, followed by phorbol ester phorbol-12-myristate-13-acetat (PMA), whereas differentiation with brain-derived neurotropic factor (BDNF) did not increase TH-immunoreactive cells. TH, dopamine -hydroxylase and vesicular monoamine transporter-2 were also significantly upregulated in ST-differentiated cells compared to both undifferentiated and retinoic acid (RA)-differentiated cells. RA induced the highest number of CHT1-immunoreactive cells while ST- and BDNF-differentiation reduced CHT1-immunoreactive cells, indicating a decrease in the cholinergic phenotype. The presynaptic neuronal protein, αsynuclein, was significantly upregulated in RA- and ST-treated cells compared to undifferentiated cells. Ascorbic acid increased the number of CHT1-immunoreactive cells in all differentiation procedures and ST-differentiated TH-positive cells significantly. CONCLUSIONS Our findings indicate that a quantitative characterization of the phenotype is crucial when using SH-SY5Y cells to study the pathogenesis or evaluate compounds for treatment of neurodegenerative diseases

Effects of gold and PCL- or PLLA-coated silica nanoparticles on brain endothelial cells and the blood–brain barrier

Nanomedicine is a constantly expanding field, facilitating and improving diagnosis and treatment of diseases. As nanomaterials are foreign objects, careful evaluation of their toxicological and functional aspects prior to medical application is imperative. In this study, we aimed to determine the effects of gold and polymer-coated silica nanoparticles used in laser tissue soldering on brain endothelial cells and the blood–brain barrier using rat brain capillary endothelial cells (rBCEC4). All types of nanoparticles were taken up time-dependently by the rBCEC4 cells, albeit to a different extent, causing a time- and concentration-dependent decrease in cell viability. Nanoparticle exposure did not change cell proliferation, differentiation, nor did it induce inflammation. rBCEC4 cells showed blood–brain barrier characteristics including tight junctions. None of the nanoparticles altered the expression of tight junctions or impaired the blood–brain barrier permeability. The findings suggest that effects of these nanoparticles on the metabolic state of cells have to be further characterized before use for medical purposes

Effects of pulse-modulated radiofrequency magnetic field (RF-EMF) exposure on apoptosis, autophagy, oxidative stress and electron chain transport function in human neuroblastoma and murine microglial cells

The use of body-worn wireless devices with different communication protocols and rapidly changing exposure scenarios is still multiplying and the need to identify possible health effects of radiofrequency electromagnetic field (RF-EMF) exposure with extremely low-frequency (ELF) modulation envelops. In this study, effects of ELFmodulated 935 MHz RF-EMF on apoptosis, autophagy, oxidative stress and electron exchange in N9 microglial and SH-SY5Y neuroblastoma cells were investigated. Cells were exposed at 4 W/kg or sham-exposed for 2 and 24 h. RF-EMF exposure of both cell types did not alter apoptosis, the number of living cells nor the apoptosis-inducing factor (AIF), irrespective of the exposure duration. RF-EMF exposure for 24, but not for 2 h, increased protein levels of the autophagy marker ATG5, whereas LC3B-I and II and pERK were not altered in both cell types and exposure times investigated. A transient increase in glutathione (GSH), but not hydrogen peroxide and cytochrome c oxidase was found only in SH-SY5Y cells, indicating that short-time RF-EMF at SAR levels accepted by today's safety guidelines might cause autophagy and oxidative stress with the effect being dependent on cell type and exposure duration. Further studies are needed to evaluate possible underlying mechanisms involved in pulse-modulated RF-EMF exposure

Effects of silica nanoparticle exposure on mitochondrial function during neuronal differentiation

Abstract Background Nanomedicine offers a promising tool for therapies of brain diseases, but potential effects on neuronal health and neuronal differentiation need to be investigated to assess potential risks. The aim of this study was to investigate effects of silica-indocyanine green/poly (ε-caprolactone) nanoparticles (PCL-NPs) engineered for laser tissue soldering in the brain before and during differentiation of SH-SY5Y cells. Considering adaptations in mitochondrial homeostasis during neuronal differentiation, metabolic effects of PCL-NP exposure before and during neuronal differentiation were studied. In addition, kinases of the PI3 kinase (PI3-K/Akt) and the MAP kinase (MAP-K/ERK) pathways related to neuronal differentiation and mitochondrial function were investigated. Results Differentiation resulted in a decrease in the cellular respiration rate and the extracellular acidification rate (ECAR). PCL-NP exposure impaired mitochondrial function depending on the time of exposure. The cellular respiration rate was significantly reduced compared to differentiated controls when PCL-NPs were given before differentiation. The shift in ECAR was less pronounced in PCL-NP exposure during differentiation. Differentiation and PCL-NP exposure had no effect on expression levels and the enzymatic activity of respiratory chain complexes. The activity of the glycolytic enzyme phosphofructokinase was significantly reduced after differentiation with the effect being more pronounced after PCL-NP exposure before differentiation. The increase in mitochondrial membrane potential observed after differentiation was not found in SH-SY5Y cells exposed to PCL-NPs before differentiation. The cellular adenosine triphosphate (ATP) production significantly dropped during differentiation, and this effect was independent of the PCL-NP exposure. Differentiation and nanoparticle exposure had no effect on superoxide levels at the endpoint of the experiments. A slight decrease in the expression of the neuronal differentiation markers was found after PCL-NP exposure, but no morphological variation was observed. Conclusions PCL-NP exposure affects mitochondrial function depending on the time of exposure before and during neuronal differentiation. PCL-NP exposure during differentiation was associated with impaired mitochondrial function, which may affect differentiation. Considering the importance of adaptations in cellular respiration for neuronal differentiation and function, further studies are needed to unravel the underlying mechanisms and consequences to assess the possible risks including neurodegeneration

Co-expression of FA1/dlk1 with calbindin but not parvalbumin in the SN.

<p>Digitalized photomicrographs of FA1/dlk1-ir cells in the ventral mesencephalon of adult rats. Some calbindin (CB)-ir cells (arrowheads) showed co-localization with FA1/dlk1 (open arrowhead, upper row) in the substantia nigra pars compacta, whereas several CB-ir neurons (arrowheads, upper row) and FA1/dlk1-ir cells (arrows, upper row) did not co-localize. As expected from the distribution pattern depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116088#pone.0116088.g001" target="_blank">Fig. 1</a> no co-localization was detected for FA1/dlk1 with parvalbumin (PV) in the substantia nigra pars reticulata (lower row). Scale bar: 50μm.</p