Tenascin-R promotes assembly of the extracellular matrix of perineuronal nets via clustering of aggrecan.

Perineuronal nets (PNs) in the brains of tenascin-R-deficient (tn-r(-/-)) mice develop in temporal concordance with those of wild-type (tn-r(+/+)) mice. However, the histological appearance of PNs is abnormal in adult tn-r(-/-) mice. Here, we investigated whether similar defects are also seen in dissociated and organotypic cultures from hippocampus and forebrain of tn-r(-/-) mice and whether the structure of PNs could be normalized. In tn-r(-/-) cultures, accumulations of several extracellular matrix molecules were mostly associated with somata, whereas dendrites were sparsely covered, compared with tn-r(+/+) mice. Experiments to normalize the structure of PNs in tn-r(-/-) organotypic slice cultures by depolarization of neurons, or by co-culturing tn-r(+/+) and tn-r(-/-) brain slices failed to restore a normal PN phenotype. However, formation of dendritic PNs in cultures was improved by the application of tenascin-R protein and rescued by polyclonal antibodies to aggrecan and a bivalent, but not monovalent form of the lectin Wisteria floribunda agglutinin. These results show that tenascin-R and aggrecan are decisive contributors to formation and stabilization of PNs and that tenascin-R may implement these functions by clustering of aggrecan. Proposed approaches for restoration of normal PN structure are noteworthy in the context of PN abnormalities in neurological disorders, such as epilepsy, schizophrenia and addiction.

Erythropoietin enhances cell proliferation and survival of human fetal neuronal progenitors in normoxia.

Extensive data reporting the neurogenerative, neuroprotective and neuroregenerative potential of erythropoietin (EPO), mainly on RNA level, can be found in the literature. However, there is still a poor knowledge on the response of neuronal progenitor cells (NPC) upon stimulation with EPO in terms of the protein species involved. Herein, the effect of EPO on the proliferation of human mesencephalic NPC (hmNPC) under normoxia is monitored using cellular assays and proteomic analysis (two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry). The administration of EPO increased the proliferation of hmNPC within 4 days after application. It positively influenced the cell-cycle progression by affecting the G2 phase of the cell cycle. A proteomic analysis of the protein expression in hmNPC cultures 4 days after EPO treatment identified 8 proteins differentially expressed in EPO-treated cultures. It is likely that one or more of the identified proteins are involved in cellular pathways that promote cell proliferation and differentiation of hmNPC under normoxia. Their further characterization could provide cellular targets for the development of new therapeutic agents to treat CNS injury. Moreover, as EPO signaling is hypoxia-inducible, our findings may also indicate the beneficial effect of EPO to mimic hypoxia, while bypassing its negative effects, to culture human fetal midbrain-derived progenitor cells.

Effect of native and NH3 plasma-functionalized polymeric membranes on the gene expression profiles of primary hepatocytes.

Little is known about how cells respond to different biomaterials at the molecular level. Biomaterials could stimulate specific cellular responses at the molecular level, such as activation of signalling pathways that control gene activity involved in the maintenance, growth and functional regeneration of liver tissue in vitro. This aspect is an important step in liver tissue engineering. Currently, there are no data available concerning the modulation of cellular genomic response by using synthetic membranes in a bioartificial system. For the first time we investigated gene expression profiles of primary hepatocytes cultured on different substrates: collagen sandwich, native and NH(3) plasma-grafted PEEK-WC-PU membranes. Gene expression in cell suspension prepared after cell isolation was used as a control. Generally, microarray data revealed that the expression of the majority of genes remained unchanged compared to the control. Among 31 000 genes, 52 were significantly changed: 20 were upregulated and 32 downregulated. There were similar changes in gene expression of hepatocytes cultured in the membranes and collagen sandwich. However, some genes involved in the cell proliferation and functional metabolic pathways are more expressed in cells cultured on the membranes and especially on the functionalized ones. Both membranes sustained liver functions at the molecular level, demonstrating their suitability for the reconstruction of liver and as a toxicogenomic tool to predict the liver response to novel drugs.

Proteomic analysis to display the effect of low doses of erythropoietin on rat liver regeneration.

AIMS: Several groups found different impact of erythropoietin (EPO) on liver regeneration. Both pro-proliferative as well as anti-proliferative and non-proliferative activities have been reported using high dosage of EPO. Systemic administration of high doses of this cytokine is a clinical concern due to risk of thrombosis. Herein, we applied EPO in low dosages and investigated whether it can stimulate liver regeneration after liver resection. MAIN METHODS: Parameters of liver regeneration were assessed 3 days after 70% hepatectomy by means of immunochemistry and proteomics. EPO was given twice in low dosages (200 and 600 IU/kg BW). KEY FINDINGS: We showed that EPO facilitated hepatic regeneration in rats. Enhanced hepatocyte proliferation (Ki67, BrdU-positive cells) was observed in all EPO-treated groups. By performing Differential Proteomic analysis, we identified two proteins which resulted sensitive to EPO treatment after hepatectomy: Peroxiredoxin-1 and glutathione S-transferase Mu 1. SIGNIFICANCE: Based on our results, low doses of rhEPO increase the hepatic regenerative capacity after partial hepatectomy in rats by enhancing hepatocyte proliferation and acting on antioxidant enzymes. Both proteins identified by proteomic analysis have not previously been associated with liver regeneration and will aid in the understanding of EPO's regenerative response having clinical implications to treat liver failure.

Paracetamol treatment increases telomerase activity in rat embryonic liver cells.

Although paracetamol is known to have a damaging effect, this pharmaceutical is widely applied to pregnant and lactating women. Despite substantial progress in our understanding of its hepatotoxicity, some mechanisms, particularly of its embryonal and developmental toxicity, are still unknown. Thus, cell culture assays that investigate its toxicity are of particular interest. We assessed the effects of acute paracetamol treatment on cell viability (LDH assay, MTT assay), glutathione content (GSH assay), metabolic status (albumin and urea assays) and telomerase activity using rat embryonic liver cells (RLC-18 cells). Incubation with low (6 mmol/l) and high (15 mmol/l) concentrations of toxin for 24 h leads to 20% and 50% cytotoxicity, respectively. Paracetamol exerted its toxicity in a similar pathway (depletion of GSH stores) as in adult liver cells, producing damage at the cellular level. Interestingly, paracetamol treatment significantly enhanced telomerase activity. Mechanisms involved in paracetamol-induced inhibition of cell senescence should be further elucidated. Telomerase activity in RLC-18 cells offers unique opportunities for examining basic biologic mechanisms. Our findings should encourage further studies to investigate a link between telomerase activity and toxicity, implying a role of impaired telomerase activity in human pathology.

The influence of oxygen supply on metabolism of neural cells cultured on a gas-permeable PTFE foil.

The influence of oxygen on neural stem cell proliferation, differentiation, and apoptosis is of great interest for regenerative therapies in neurodegenerative disorders, such as Parkinson's disease. These oxygen depending mechanisms have to been considered for the optimization of neural cell culture conditions. In this study, we used a cell culture system with an oxygen-permeable polytetrafluorethylene (PTFE) foil to investigate the effect of oxygen on metabolism and survival of neural cell lines in vitro. Human glial astrocytoma-derived cells (GOS-3) and rat pheochromacytoma cells (PC12) were cultured on the gas-permeable PTFE foil as well as a conventional non oxygen-permeable cell culture substrate at various oxygen concentrations. Analyses of metabolic activity, gene expression of apoptotic grade, and dopamine synthesis were performed. Under low oxygen partial pressure (2%, 5%) the anaerobic metabolism and apoptotic rate of cultured cells is diminished on PTFE foil when compared with the conventional culture dishes. In contrast, under higher oxygen atmosphere (21%) the number of apoptotic cells on the PTFE foil was enhanced. This culture model demonstrates a suitable model for the improvement of oxygen dependent metabolism under low oxygen conditions as well as for induction of oxidative stress by high oxygen atmosphere without supplementation of neurotoxins.

Telomerase activity and hepatic functions of rat embryonic liver progenitor cell in nanoscaffold-coated model bioreactor.

Presently, there is growing interest on telomerase activity in all cells (somatic cells, stem cells, cancerous cells and others) since this activity is associated with cellular changes such as proliferation, differentiation, immortalization, cell injury and ageing. Telomerase activity is absent in most of the somatic cells but present in over 90% of cancerous cells and other immortalized cell lines. In our present study, we cultured a rat embryonal liver progenitor cell line RLC-18 in a self-assembly nanostructured scaffold-coated bioreactor (NCB), collagen-coated plates (CCP) and uncoated plates (UP), and evaluated changes of telomerase activity by non radioactive techniques (Telo TAGGG Telomerase PCR ELISA, cell proliferation based on mitochondria number by MTT assay and hepatic functions such as albumin secretion, urea metabolism, Cytochrome P450 activity like ethoxyresorufin-O-deethylase (EROD) activity. We found less telomerase activity and less cell proliferation, but more hepatic functions on the NCB than on the CCP and UP. Our data support the concept that cell-scaffold interaction may play a significant in controlling the telomerase activity as well as enhanced hepatic functions. Although our present study does not focus on the exact mechanism of telomerase regulation, our result may provide basic clues on cell differentiation whereby telomerase activity inhibits differentiation of cells as in the rat embryonic liver cell line, may be regulated by cell-scaffold interaction and where there is less proliferation, cells perform enhanced hepatic functions, thereby implying that bioartificial liver support may be possible.

Comparison of uptake and neuroprotective potential of seven zinc-salts.

Zinc plays an important role as an antioxidant in different cells treated with various kinds of oxidative stressors. Although intracellular Zn(2+) is important in many cellular events, little is known about the cellular uptake of this trace metal and the intracellular status that is required for its optimal function. Since previous reports usually employed only one type of zinc-salt, in this work was compared cellular uptake and antioxidative potential of seven zinc-salts in order to discriminate whether different counterions and ligands may influence its function. Oxidative stress was induced by peroxide or iron in neuronal PC12 cells. We compared uptake of zinc-salts into the labile Zn(2+) pool of PC12 cells as well as their effects on the prevention of cell death, glutathione depletion, lipid peroxidation and ROS production. Zinc-salts provided better protection against oxidative stress-induced in PC12 cultures by peroxide than by iron. Preincubations with zinc-salts displayed better neuroprotection in all cases than coincubations. Zinc-histidine complex was shown to be the most potent compound. Our results indicated that protective effect of zinc is not related to its uptake into PC12 cells, what is indicated by the rather low salt concentrations required for the cell protection and by the observation that despite a superior antioxidant effect of zinc-histidine, the uptake of this salt by PC12 cells was remarkably lower in comparison with other zinc-salts. Although zinc-sulfate exerted weak neuroprotective potential, accumulation of Zn(2+) from this salt within cells was significantly higher compared to other salts. The differences in accumulation of zinc-salts were not specific and unique to PC12 cells, since similar results were obtained in rat primary hepatocytes and endothelial HUVEC cells.

Protective effects of flavonoids and two metabolites against oxidative stress in neuronal PC12 cells.

AIMS: Recent interest has focused on plant antioxidants as potentially useful neuroprotective agents. In most studies only the genuine forms of flavonoids were used, although they are rapidly metabolized. Therefore, we have compared protective activities of two flavonoids (luteolin, quercetin) and two of their bioavailable metabolites (3,4-DHPAA and 3,4-DHT) against oxidative stress, induced by peroxides (t-BHP, H(2)O(2)) and iron (FeSO(4)), in neuronal PC12 cells. MAIN METHODS: We have measured their effect on the prevention of cell death (MTT assay), glutathione depletion (GSH assay), lipid peroxidation (MDA assay) and production of ROS (DCF assay). Differentiated PC12 cells were used as a model system of neuronal cells. The compounds (concentration range 6-25 micromol/L) were tested in preincubation and coincubation experiments. KEY FINDINGS: In MTT and DCF assays all tested compounds showed excellent protection. When cells were exposed to peroxides, both metabolites increased GSH levels less efficiently than their parent flavonoids in both types of incubations. Following exposure to iron, only coincubation significantly prevented GSH depletion and the metabolites surprisingly mimicked the suppressive effect of flavonoids. MDA levels induced by all stressors were reduced more potently during coincubation than during preincubation with polyphenols. While the lipophilic metabolite 3,4-DHT exerted excellent antilipoperoxidant activity, the hydrophilic metabolite 3,4-DHPAA was less effective. SIGNIFICANCE: These results demonstrate that most of the protective effects of flavonoids against oxidative stress in PC12 cells are continued despite biodegradation of the parent flavonoids. In general, the lipophilic metabolite 3,4-DHT was more active than the hydrophilic 3,4-DHPAA.

Perineuronal nets are largely unaffected in Alzheimer model Tg2576 mice.

Changes in the molecular organization of the extracellular matrix are key factors in neuropathology. We investigated aggrecan-based perineuronal nets (PNs) in relation to neurodegeneration and activation of glial cells in a transgenic mouse (Tg2576) model of Alzheimer's disease. The formation of amyloid plaques in the cerebral cortex occurred independently of the area-specific distribution of PNs. Matrix components were only affected in the core of plaques in advanced stages of pathology. PNs remained unchanged in the large marginal zone occupied by reactive astrocytic processes. We conclude that the aggrecan-based extracellular matrix of PNs is not enzymatically altered in peripheral plaque territories and is only removed after neuronal death.

Rat embryonic liver cell expansion and differentiation on NH3 plasma-grafted PEEK-WC-PU membranes.

Biomaterials can potentially influence stem and progenitor cell proliferation and differentiation in both a positive and a negative way. Herein, we report on the expansion and differentiation of rat embryonic (E17) liver (RLC-18) cells on new bioactive membrane made of PEEK-WC-PU, whose surface was grafted with nitrogen functionalities by means of NH(3) glow discharges. The performance of the developed membrane was evaluated by analyzing the expression of the liver specific functions of cells cultured in a 6-well gas-permeable bioreactor. It was found that native and NH(3) plasma-grafted PEEK-WC-PU membranes enabled expansion of liver cells in the bioreactor. Liver embryonic cells on the membranes exhibited higher functional activities compared to those cultured on conventional culture dishes as demonstrated by higher albumin and urea production. They showed gene expression of alpha-fetoprotein and albumin in a time-dependent manner of the hepatic differentiation process. LDH assay and SEM analyses revealed that a high number of viable liver stem cells attached to the membranes. Unexpectedly, liver progenitors cultured on membranes had higher telomerase activity than ones in the plates, preventing cell senescence. Thus, membranes are able to sustain in vitro the same in vivo liver functions and to allow the expansion of progenitor cells.

Comparative in vitro toxicity of seven zinc-salts towards neuronal PC12 cells.

Currently much attention has been given to the neurotoxicity of zinc, yet little is known about the influence of the counterions present. Therefore, we investigated the influence of different Zn(2+)-salts (concentrations range 0.05-0.3 mM) on cell viability, ATP and glutathione concentration and caspase activation in differentiated PC12 cells as a model for neuronal cells. Generally, at concentrations of 0.05 mM most Zn(2+)-salts were not cytotoxic except for zinc-citrate. At concentrations between 0.1 and 0.3 mM Zn(2+) a significant decrease in GSH and ATP levels preceded cell death induced by all salts, except of zinc-histidinate. Zinc-citrate and zinc-sulphate turned out to be the most toxic salts particularly at low concentrations. Analyses of caspase 3/7 activity showed that dependent on the concentration and the type of the salt used cell death may show more or less signs of both, necrosis and apoptosis. Interestingly, the uptake of Zn(2+) from zinc-sulphate and zinc-citrate was significantly higher than that of other salts, implicating a correlation between uptake and toxicity. In conclusion, Zn(2+)-salts could be divided into three categories with high (zinc-citrate, zinc-sulphate), moderate (zinc-orotate, zinc-acetate, zinc-chloride(,) zinc-gluconate) and low cytotoxicity (zinc-histidinate).

Organization of brain extracellular matrix in the Chilean fat-tailed mouse opossum Thylamys elegans (Waterhouse, 1839).

We investigated the structural and molecular organization of the extracellular matrix in Thylamys elegans, a marsupial representative of the mammalian order Didelphimorphia. Perineuronal nets (PNs) associated with distinct types of neurons were visualized by detection of chondroitin sulfate proteoglycans and hyaluronan, and by labeling with Wisteria floribunda agglutinin (WFA), a marker for PNs in the mammalian brain. In the neocortex of Thylamys, these methods revealed PNs on pyramidal cells. In contrast, parvalbumin-immunoreactive interneurons in the neocortex and hippocampal formation (displaying robust, WFA-labeled PNs in placental mammals) were ensheathed only with a delicate rim of hyaluronan and proteoglycans not detectable with WFA. The absence of WFA staining was characteristic also of some subcortical regions which contained PNs intensely labeled for chondroitin sulfate proteoglycan and hyaluronan. However, corresponding to placental mammals, numerous subcortical nuclei showed clearly WFA-stained PNs. Similar as in placental mammals, cholinergic basal forebrain neurons and tyrosine hydroxylase-immunoreactive neurons of the substantia nigra and locus coeruleus were devoid of PNs. Together with our earlier study on Monodelphis, the present results reveal that South American opossums show either a particular "marsupial" or "Didelphid" type of extracellular matrix chemoarchitecture, supporting the view that these components may vary phylogenetically as integral parts of neuronal physiology at the systems and single cell level.

Protective effects of ellagic and chlorogenic acids against oxidative stress in PC12 cells.

Following exposure of differentiated neuronal PC12 cells to either t-BHP, hydrogen peroxide (H2O2) or FeSO4 various kinds of reactive oxygen species (ROS) are generated leading to oxidative injury. The protective effects of two plant polyphenols, ellagic (EC) and chlorogenic acid (CGA), as well as of two metabolites, caffeic acid (CA) and ferulic acid (FA), were investigated in preincubation and coincubation experiments with respect to the following parameters: prevention of cell death, GSH depletion, lipid peroxidation and ROS formation. The polyphenols more efficiently suppressed cytotoxicity and loss of GSH caused by peroxides than by iron, particularly in preincubation. Lipid peroxidation which increased much stronger in response to FeSO4 was counteracted completely by the polyphenols. In case of iron, however, only coincubation was effective. EA and CGA and the metabolites CA and FA showed excellent elimination of ROS induced by all stressors. These findings suggest that two dietary antioxidants, EA and CGA, may have protective properties against oxidative stress induced in CNS.