A revised concept for deriving reference values for internal exposures to chemical substances and its application to population-representative biomonitoring data in German children and adolescents 2014-2017 (GerES V).

HBM reference values, in contrast to toxicologically derived values, are statistically derived values that provide information on the exposure of the population. The exceedance frequency (if applicable for individual population groups) is often a first assessment standard for the local exposure situation for municipalities. More than 25 years have passed since the German Human Biomonitoring Commission (HBMC) formulated the first recommendations for the derivation of population-based reference values (HBM reference values, RV95) for substance concentrations based on HBM studies. A fundamental revision is timely, for several reasons. There have been considerable advances in relevant statistical methods, which meant that previously time-consuming and inaccessible procedures and calculations are now widely available. Furthermore, not all steps for the derivation of HBM reference values were clearly elaborated in the first recommendations. With this revision we intended to achieve a rigorous standardization of the entire process of deriving HBM reference values, also to realise a higher degree of transparency. In accordance with established international practice, it is recommended to use the 95th percentile of the reference distribution as the HBM reference value. To this end, the empirical 95th percentile of a suitable sample should be rounded, ensuring that the rounded value is within the two-sided 95% confidence interval of the percentile. All estimates should be based on distribution-free methods, and the confidence interval should be estimated using a bootstrap approach, if possible, according to the BCa ("bias-corrected and accelerated bootstrap"). A minimum sample size of 80 observations is considered necessary. The entire procedure ensures that the derived HBM reference value is robust against at least two extreme values and can also be used for underlying mixed distributions. If it is known in advance that certain subgroups (different age groups, smokers, etc.) show differing internal exposures, it is recommended that group-specific HBM reference values should be derived. Especially when the sample sizes for individual subgroups are too small, individual datasets with potential outliers can be excluded in advance to homogenize the reference value population. In the second part, new HBM reference values based on data of the German Environmental Survey for Children and Adolescents (GerES V, 2014-2017) were derived in accordance with the revised recommendations. The GerES V is the most recent population-representative monitoring of human exposure to pollutants in Germany on children and adolescents aged 3-17 years (N = 2294). RV95 for GerES V are reported for four subgroups (males/females and 3-11/12-17 years) for 108 different substances including phthalates and alternative plasticisers, metals, organochlorine pesticides, polychlorinated biphenyls (PCB), per- and polyfluoroalkyl substances (PFAS), parabens, aprotic solvents, chlorophenols, polycyclic aromatic hydrocarbons (PAH) and UV filter, in total 135 biomarkers. Algorithms implemented in R were used for the statistics and the determination of the HBM reference values. To facilitate a quality control of the study data, the corresponding R source code is given, together with graphical representations of results. The HBM reference values listed in this article replace earlier RV95 values derived by the HBMC for children and adolescents from data of precedent GerES studies (e.g. published in Apel et al., 2017).

Concept for the Evaluation of Carcinogenic Substances in Population-Based Human Biomonitoring.

The Human Biomonitoring (HBM) Commission at the German Environment Agency holds the opinion that for environmental carcinogens for which no exposure levels can be assumed and are harmless to health, health-based guidance values corresponding to the classical definition of the HBM-I or HBM-II value cannot be established. Therefore, only reference values have been derived so far for genotoxic carcinogens from exposure data of the general population or subpopulations. The concept presented here opens up the possibility of performing health risk assessments of carcinogenic substances in human biomonitoring, and thus goes decisively beyond the purely descriptive statistical reference value concept. Using the presented method, quantitative dose descriptors of internal exposure can be derived from those of external exposure, provided that sufficient toxicokinetic information is available. Dose descriptors of internal exposure then allow the simple estimate of additional lifetime cancer risks for measured biomarker concentrations or, conversely, of equivalent concentrations for selected risks, such as those considered as tolerable for the general population. HBM data of chronic exposures to genotoxic carcinogens can thus be used to assess the additional lifetime cancer risk referring to the general population and to justify and prioritize risk management measures.

Polychlorinated dioxins and dibenzofurans (PCDD/F), polybrominated dioxins and dibenzofurans (PBDD/F), polychlorinated biphenyls (PCB), polybrominated diphenyl ethers (PBDE), and per- and polyfluoroalkyl substances (PFAS) in German breast milk samples (LUPE 8).

Most organic pollutants (POP) are persistent in the environment, accumulate in fatty tissues, and so a transfer through the food chain is probably, thereby causing various health effects. We quantified PCDD/F, PBDD/F, PCB, PBDE, perfluorinated substances, and ADONA in breast milk samples collected in two German federal states and breast milk and blood samples from subjects additionally exposed to PFOA. The median (95th percentile) concentrations were 2.43 (6.58) pgWHO2005TEQ/g l.w. for PCDD/F, 2.45 (4.82) pgWHO2005TEQ/g l.w. for dioxin-like PCB (dl-PCB), and 0.62 (2.69) pgWHO2005TEQ/g l.w. for PBDD/F. The relative contributions of the median values of PCDD/F, dl-PCB, and PBDD/F to the total-TEQ were approximately 41%, 42%, and 11%, respectively. Nondioxin-like PCB (ndl-PCB) concentrations were clearly dominated by the higher chlorinated PCB congeners, with medians of 23.2 ng/g l.w. for PCB 153, 13.9 ng/g l.w. for PCB 138, and 13.0 ng/g l.w. for PCB 180. The sum of the 3 congeners (PCB 138, 153, and 180) were multiplied with 1.64 (total PCB) and showed a median of 82.16 ng/g l.w. and a 95th percentile of 173.3 ng/g l.w. Only PFOA and PFOS could be quantified in 29% and 17% of in total 180 samples with 95th percentiles of 53 ng/l and 33 ng/l, respectively. Milk samples (n = 13) from subjects living on PFOA contaminated sites showed higher levels between 33 and 854 ng/l PFOA (mean: 199 ng/l), whilst PFOS could be quantified only in three samples. The sum of 17 PBDE congeners showed medians (95th percentile) of 1737 pg/g l.w. (22,806 pg/g l.w.), with the highest medians of 422 pg/g l.w. for BDE 209 and 378 pg/g l.w. for BDE 153. Overall, our study confirms the declining contamination level in breast milk during the last decade, but points out the need to further reduce the environmental contamination with persistent substances and subsequently the exposure in childhood.

Long-term simulation of lead concentrations in agricultural soils in relation to human adverse health effects.

Lead (Pb) exposure of consumers and the environment has been reduced over the past decades. Despite all measures taken, immission of Pb onto agricultural soils still occurs, with fertilizer application, lead shot from hunting activities, and Pb from air deposition representing major sources. Little is known about the intermediate and long-term consequences of these emissions. To gain more insight, we established a mathematical model that considers input from fertilizer, ammunition, deposition from air, uptake of Pb by crops, and wash-out to simulate the resulting Pb concentrations in soil over extended periods. In a further step, human oral exposure by crop-based food was simulated and blood concentrations were derived to estimate the margin of exposure to Pb-induced toxic effects. Simulating current farming scenarios, a new equilibrium concentration of Pb in soil would be established after several centuries. Developmental neurotoxicity represents the most critical toxicological effect of Pb for humans. According to our model, a Pb concentration of ~ 5 mg/kg in agricultural soil leads to an intake of approximately 10 µg Pb per person per day by the consumption of agricultural products, the dose corresponding to the tolerable daily intake (TDI). Therefore, 5 mg Pb/kg represents a critical concentration in soil that should not be exceeded. Starting with a soil concentration of 0.1 mg/kg, the current control level for crop fields, our simulation predicts periods of ~ 50 and ~ 175 years for two Pb immission scenarios for mass of Pb per area and year [scenario 1: ~ 400 g Pb/(ha × a); scenario 2: ~ 175 g Pb/(ha × a)], until the critical concentration of ~ 5 mg/kg Pb in soil would be reached. The two scenarios, which differ in their Pb input via fertilizer, represent relatively high but not unrealistic Pb immissions. From these scenarios, we calculated that the annual deposition of Pb onto soil should remain below ~ 100 g/(ha × a) in order not to exceed the critical soil level of 5 mg/kg. We propose as efficient measures to reduce Pb input into agricultural soil to lower the Pb content of compost and to use alternatives to Pb ammunition for hunting.

Dietary Manganese Exposure in the Adult Population in Germany-What Does it Mean in Relation to Health Risks?

Manganese is both an essential nutrient and a potential neurotoxicant. Therefore, the question arises whether the dietary manganese intake in the German population is on the low or high side. Results from a pilot total diet study in Germany presented here reveal that the average dietary manganese intake in the general population in Germany aged 14-80 years is about 2.8 mg day-1 for a person of 70 kg body weight. This exposure level is within the intake range of 2-5 mg per person and day as recommended by the societies for nutrition in Germany, Austria, and Switzerland. No information on the dietary exposure of children in Germany can be provided so far. Although reliable information on health effects related to oral manganese exposure is limited, there is no indication from the literature that these dietary intake levels are associated with adverse health effects either by manganese deficiency or excess. However, there is limited evidence that manganese taken up as a highly bioavailable bolus, for example, uptake via drinking water or food supplements, could pose a potential risk to human health-particularly in certain subpopulations-when certain intake amounts, which are currently not well defined, are exceeded.

High exposure to inorganic arsenic by food: the need for risk reduction.

Arsenic is a human carcinogen that occurs ubiquitously in soil and water. Based on epidemiological studies, a benchmark dose (lower/higher bound estimate) between 0.3 and 8 µg/kg bw/day was estimated to cause a 1 % increased risk of lung, skin and bladder cancer. A recently published study by EFSA on dietary exposure to inorganic arsenic in the European population reported 95th percentiles (lower bound min to upper bound max) for different age groups in the same range as the benchmark dose. For toddlers, a highly exposed group, the highest values ranged between 0.61 and 2.09 µg arsenic/kg bw/day. For all other age classes, the margin of exposure is also small. This scenario calls for regulatory action to reduce arsenic exposure. One priority measure should be to reduce arsenic in food categories that contribute most to exposure. In the EFSA study the food categories 'milk and dairy products,' 'drinking water' and 'food for infants' represent major sources of inorganic arsenic for infants and also rice is an important source. Long-term strategies are required to reduce inorganic arsenic in these food groups. The reduced consumption of rice and rice products which has been recommended may be helpful for a minority of individuals consuming unusually high amounts of rice. However, it is only of limited value for the general European population, because the food categories 'grain-based processed products (non rice-based)' or 'milk and dairy products' contribute more to the exposure with inorganic arsenic than the food category 'rice.' A balanced regulatory activity focusing on the most relevant food categories is required. In conclusion, exposure to inorganic arsenic represents a risk to the health of the European population, particularly to young children. Regulatory measures to reduce exposure are urgently required.

Manufactured nanomaterials: categorization and approaches to hazard assessment.

Nanotechnology offers enormous potential for technological progress. Fortunately, early and intensive efforts have been invested in investigating toxicology and safety aspects of this new technology. However, despite there being more than 6,000 publications on nanotoxicology, some key questions still have to be answered and paradigms need to be challenged. Here, we present a view on the field of nanotoxicology to stimulate the discussion on major knowledge gaps and the critical appraisal of concepts or dogma. First, in the ongoing debate as to whether nanoparticles may harbour a specific toxicity due to their size, we support the view that there is at present no evidence of 'nanospecific' mechanisms of action; no step-change in hazard was observed so far for particles below 100 nm in one dimension. Therefore, it seems unjustified to consider all consumer products containing nanoparticles a priori as hazardous. Second, there is no evidence so far that fundamentally different biokinetics of nanoparticles would trigger toxicity. However, data are sparse whether nanoparticles may accumulate to an extent high enough to cause chronic adverse effects. To facilitate hazard assessment, we propose to group nanomaterials into three categories according to the route of exposure and mode of action, respectively: Category 1 comprises nanomaterials for which toxicity is mediated by the specific chemical properties of its components, such as released ions or functional groups on the surface. Nanomaterials belonging to this category have to be evaluated on a case-by-case basis, depending on their chemical identity. Category 2 focuses on rigid biopersistent respirable fibrous nanomaterials with a specific geometry and high aspect ratio (so-called WHO fibres). For these fibres, hazard assessment can be based on the experiences with asbestos. Category 3 focuses on respirable granular biodurable particles (GBP) which, after inhalation, may cause inflammation and secondary mutagenicity that may finally lead to lung cancer. After intravenous, oral or dermal exposure, nanoscaled GBPs investigated apparently did not show 'nanospecific' effects so far. Hazard assessment of GBPs may be based on the knowledge available for granular particles. In conclusion, we believe the proposed categorization system will facilitate future hazard assessments.

Toxicity of functional nano-micro zinc oxide tetrapods: impact of cell culture conditions, cellular age and material properties.

With increasing production and applications of nanostructured zinc oxide, e.g., for biomedical and consumer products, the question of safety is getting more and more important. Different morphologies of zinc oxide structures have been synthesized and accordingly investigated. In this study, we have particularly focused on nano-micro ZnO tetrapods (ZnO-T), because their large scale fabrication has been made possible by a newly introduced flame transport synthesis approach which will probably lead to several new applications. Moreover, ZnO-T provide a completely different morphology then classical spherical ZnO nanoparticles. To get a better understanding of parameters that affect the interactions between ZnO-T and mammalian cells, and thus their biocompatibility, we have examined the impact of cell culture conditions as well as of material properties on cytotoxicity. Our results demonstrate that the cell density of fibroblasts in culture along with their age, i.e., the number of preceding cell divisions, strongly affect the cytotoxic potency of ZnO-T. Concerning the material properties, the toxic potency of ZnO-T is found to be significantly lower than that of spherical ZnO nanoparticles. Furthermore, the morphology of the ZnO-T influenced cellular toxicity in contrast to surface charges modified by UV illumination or O2 treatment and to the material age. Finally, we have observed that direct contact between tetrapods and cells increases their toxicity compared to transwell culture models which allow only an indirect effect via released zinc ions. The results reveal several parameters that can be of importance for the assessment of ZnO-T toxicity in cell cultures and for particle development.

Huge increase of therapeutic window at a bioactive silver/titania nanocomposite coating surface compared to solution.

A silver containing coating used in the human body, e.g., on an implant should be both effectively antimicrobial and non-cytotoxic to human cells. It is generally believed that the biologic effect originates from silver ions released from the coating. Nanocomposites with well controlled Ag filling factor were prepared by co-sputtering, and the silver surface concentration and the silver release were determined by XPS and ICP-MS, respectively. Here we show that only a small therapeutic window exists for dissolved silver but the therapeutic window is largely increased at the surface. While the toxicity observed for mammalian cells in contact with the bioactive Ag/TiO2 nanocomposite surface and for silver ions in solution is rather similar the antimicrobial activity is drastically enhanced at the surface. A model is proposed to explain the strong increase of the antimicrobial activity at the surface. The present results not only question well-established tests for antimicrobial activity but they are also important for the design of antimicrobial coatings, e.g., for biomedical devices.

Inhibition of bacterial adhesion to live human cells: activity and cytotoxicity of synthetic mannosides.

Bacterial adhesion to glycosylated surfaces is a key issue in human health and disease. Inhibition of bacterial adhesion by suitable carbohydrates could lead to an anti-adhesion therapy as a novel approach against bacterial infections. A selection of five α-mannosides has been evaluated as inhibitors of bacterial adhesion to the polysaccharide mannan, as well as to the surface of live human HT-29 cells. Cell toxicity studies were performed to identify the therapeutic window for a potential in vivo-application of the tested carbohydrates. A previously published mannosidic squaric acid diamide was shown to be exceptionally effective as inhibitor of the bacterial lectin FimH.

Virostatic potential of micro-nano filopodia-like ZnO structures against herpes simplex virus-1.

Herpes simplex virus type-1 (HSV-1) entry into target cell is initiated by the ionic interactions between positively charged viral envelop glycoproteins and a negatively charged cell surface heparan sulfate (HS). This first step involves the induction of HS-rich filopodia-like structures on the cell surface that facilitate viral transport during cell entry. Targeting this initial first step in HSV-1 pathogenesis, we generated different zinc oxide (ZnO) micro-nano structures (MNSs) that were capped with multiple nanoscopic spikes mimicking cell induced filopodia. These MNSs were predicted to target the virus to compete for its binding to cellular HS through their partially negatively charged oxygen vacancies on their nanoscopic spikes, to affect viral entry and subsequent spread. Our results demonstrate that the partially negatively charged ZnO-MNSs efficiently trap the virions via a novel virostatic mechanism rendering them unable to enter into human corneal fibroblasts - a natural target cell for HSV-1 infection. The anti-HSV-1 activity of ZnO MNSs was drastically enhanced after creating additional oxygen vacancies under UV-light illumination. Our results provide a novel insight into the significance of ZnO MNSs as the potent HSV-1 inhibitor and rationalize their development as a novel topical agent for the prevention of HSV-1 infection.

Effects of methylprednisolone and glatiramer acetate on nitric oxide formation of cytokine-stimulated cells from the rat oligodendroglial cell line OLN-93.

OBJECTIVES: In multiple sclerosis, an autoimmune inflammatory disease, oligodendroglia are primarily affected and play an important role in the onset and process of the degeneration of neuronal axons. High-dose therapy with glucocorticoids like 6alpha-methylprednisolone (MP) as well as the application of immunomodulatory agents like glatiramer acetate (GA) are commonly used in the treatment of MS. The purpose of our study was to examine, in an adequate cell culture model, the effect of MP and GA on oligodendroglial activation induced by pro-inflammatory stimuli. METHODS: In the present study, we measured the mRNA (real-time RT-PCR) and protein (Western blot) expression of inducible nitric oxide synthase (iNOS) and the release of nitric oxide (NO; Griess reagent) of rat oligodendroglial progenitor cell line OLN-93 after pro-inflammatory stimulation, and searched for influences of MP and GA on these parameters. OLN-93 cells were treated either with a combination of TNF-alpha and IFN-gamma alone, or additionally with MP or GA. Cell viability and cell protein contents were determined in parallel. RESULTS: Our results show that TNF-alpha and IFN-gamma increased iNOS mRNA and protein expression and the NO production in OLN-93 cells. The elevated production of NO and iNOS protein was reduced in the presence of MP, whereas under treatment with GA, the cytokine-induced overproduction of NO did not change significantly. CONCLUSIONS: The presented data suggest an active role of oligodendroglial cells in inflammatory processes like multiple sclerosis and indicate different properties of MP and GA regarding immunosuppression.

Mechanisms involved in the modulation of astroglial resistance to oxidative stress induced by activated microglia: antioxidative systems, peroxide elimination, radical generation, lipid peroxidation.

Microglia and astrocytes are the cellular key players in many neurological disorders associated with oxidative stress and neuroinflammation. Previously, we have shown that microglia activated by lipopolysaccharides (LPS) induce the expression of antioxidative enzymes in astrocytes and render them more resistant to hydrogen peroxide (H2O2). In this study, we examined the mechanisms involved with respect to the cellular action of different peroxides, the ability to detoxify peroxides, and the status of further antioxidative systems. Astrocytes were treated for 3 days with medium conditioned by purified quiescent (microglia-conditioned medium, MCM[-]) or LPS-activated (MCM[+]) microglia. MCM[+] reduced the cytotoxicity of the organic cumene hydroperoxide in addition to that of H2O2. Increased peroxide resistance was not accompanied by an improved ability of astrocytes to remove H2O2 or an increased expression/activity of peroxide eliminating antioxidative enzymes. Neither peroxide-induced radical generation nor lipid peroxidation were selectively affected in MCM[+] treated astrocytes. The glutathione content of peroxide resistant astrocytes, however, was increased and superoxide dismutase and heme oxygenase were found to be upregulated. These changes are likely to contribute to the higher peroxide resistance of MCM[+] treated astrocytes by improving their ability to detoxify reactive oxygen radicals and oxidation products. For C6 astroglioma cells a protective effect of microglia-derived factors could not be observed, underlining the difference of primary cells and cell lines concerning their mechanisms of oxidative stress resistance. Our results indicate the importance of microglial-astroglial cell interactions during neuroinflammatory processes.

Activated microglia modulate astroglial enzymes involved in oxidative and inflammatory stress and increase the resistance of astrocytes to oxidative stress in vitro.

Neuropathological processes in the central nervous system are commonly accompanied by an activation of microglia and astrocytes. The involvement of both cell populations in the onset and progress of neurological disorders has been widely documented, implicating both beneficial and detrimental influences on the neural tissue. Nevertheless, little is known about the interplay of these glial cell populations, especially under diseased conditions. To examine the effects of activated microglia on astrocytes purified rat astroglial cell cultures were treated with medium conditioned by purified quiescent (MCM[-]) or lipopolysaccharide (LPS)-activated rat microglia (MCM[+]) and subjected to a comparative proteome analysis based on two-dimensional gel electrophoresis. No significant down regulation of proteins was observed. The majority of the 19 proteins identified by means of nano HPLC/ESI-MS/MS in the 12 most prominent protein spots significantly overexpressed (> or =2-fold) in MCM[+] treated astrocytes are involved in inflammatory processes and oxidative stress response: superoxide dismutases (Sod), peroxiredoxins, glutathione S-transferases (Gst), nucleoside diphosphate kinase B, argininosuccinate synthase (Ass), and cellular retinol-binding protein I (Rbp1). Sod2, Rbp1, Gstp1, and Ass were also significantly increased on the mRNA level determined by quantitative RT-PCR. The upregulation of antioxidative enzymes in astrocytes was accompanied by a higher resistance to oxidative stress induced by H2O2. These results show that activated microglia change the expression of antioxidative proteins in astrocytes and protect them against oxidative stress, which might be an effective way to increase the neuroprotective potential of astrocytes under pathological conditions associated with oxidative stress and inflammation.

Impact of fraud on the mean-field dynamics of cooperative social systems.

The evolution of costly cooperation between selfish individuals seems to contradict Darwinian selection, as it reduces the fitness of a cooperating individual. However, several mechanisms such as repeated interactions or spatial structure can lead to the evolution of cooperation. One such mechanism for the evolution of cooperation, in particular among humans, is indirect reciprocity, in which individuals base their decision to cooperate on the reputation of the potential receiver, which has been established in previous interactions. Cooperation can evolve in these systems if individuals preferably cooperate with those that have shown to be cooperative in the past. We analyze the impact of fake reputations or fraud on the dynamics of reputation and on the success of the reputation system itself, using a mean-field description for evolutionary games given by the replicator equation. This allows us to classify the qualitative dynamics of our model analytically. Our results show that cooperation based on indirect reciprocity is robust with respect to fake reputations and can even be enhanced by them. We conclude that fraud per se does not necessarily have a detrimental effect on social systems.

The effect of activated microglia on astrogliosis parameters in astrocyte cultures.

In the diseased central nervous system, astrogliosis is accompanied by microglial activation. Depending on the context of their activation, reactive astrocytes are involved in neuronal survival and regeneration in an either protective or impedimental way. Major reactive changes of astrocytes in vivo are the upregulation of the intermediate filaments GFAP (glial fibrillary acidic protein) and vimentin with accompanying cellular hypertrophy and/or hyperplasia. To examine the involvement of activated microglia in the onset and maintenance of astrogliosis, we used an in vitro model of purified cultures of astrocytes and assessed as parameters for astrogliosis GFAP, vimentin, astroglial hypertrophy and cell growth after treatment with medium conditioned by LPS (lipopolysaccarides)-stimulated microglia. Furthermore, IL-6 as a typically upregulated cytokine in proinflammatory processes in the brain was determined in treated astrocytes. GFAP, the classical marker for astrogliosis, was downregulated on its protein and in parallel with vimentin on its mRNA level. The expression of actin, another cytoskeleton protein used as control, remained unchanged. Ultrastructural studies of astroglial intermediate filaments supported these findings. No hypertrophy was found. Nevertheless, LPS-activated microglia stimulated astrocytes as demonstrated by an increased cell number and an enhanced mRNA expression of IL-6. Resting microglia did not change any of the determined parameters. Our results suggest that the role of activated microglia in astrogliotic processes following injury of the brain has to be reevaluated, as microglia in their activated state might support the onset of astrogliosis on the one hand, but might delay or reduce subsequent glial scar formation on the other hand.

Microglia is activated by astrocytes in trimethyltin intoxication.

Microglia participates in most acute and chronic neuropathologies and its activation appears to involve interactions with neurons and other glial cells. Trimethyltin (TMT)-induced brain damage is a well-characterized model of neurodegeneration, in which microglial activation occurs before neuronal degeneration. The aim of this in vitro study was to investigate the role of astroglia in TMT-induced microgliosis by using nitric oxide (NO), inducible NO synthase (iNOS), and morphological changes as parameters for microglial activation. Our investigation discusses (a) whether microglial cells can be activated directly by TMT; (b) if astroglial cells are capable of triggering or modulating microglial activation; (c) how the morphology and survival of microglia and astrocytes are affected by TMT treatment; and (d) whether microglial-astroglial interactions depend on direct cell contact or on soluble factors. Our results show that microglia are more vulnerable to TMT than astrocytes are and cannot be activated directly by TMT with regard to the examined parameters. In bilayer coculture with viable astroglial cells, microglia produce NO in significant amounts at subcytotoxic concentrations of TMT (20 micromol/l). At these TMT concentrations, microglial cells in coculture convert into small round cells without cell processes, whereas flat, fibroblast-like astrocytes convert into thin process bearing stellate cells with a dense and compact cell body. We conclude that astrocytes trigger microglial activation after treatment with TMT, although the mechanisms of this interaction remain unknown.

Developmental changes of parameters for astrogliosis during cultivation of purified cerebral astrocytes from newborn rats.

Astrogliosis is a common phenomenon seen in most neuropathological changes of the central nervous system. Several in vitro models have been used to study the mechanisms and conditions for the induction of astrogliosis, however many do not take into account that the metabolic and structural characteristics of astrocytes change with time in culture. Thus, it appears difficult to attribute changes of, e.g., GFAP to the normal change in vitro as opposed to additional changes due to an astrogliotic reaction. The present study was therefore undertaken to characterize these developmental changes in purified astroglial secondary cultures during cultivation to provide a basis for further investigations of astrogliosis in vitro. During 6 weeks of cultivation (3-43 days) GFAP (ELISA) increased much more (22-fold) than the cell number (2.5-fold) and the total protein (3.5-fold). The GFAP/protein ratio increased during the first 4 weeks of cultivation and reached a plateau thereafter, which was accompanied by a significant increase of GFAP mRNA (Northern blot). At the ultrastructural level (transmission electron microscopy) gliofilaments in the perinuclear region as well as in the cell processes of 4-day-old astrocytes showed a dispersed pattern, whereas an accumulation of gliofilaments was found in 39-day-old cells, which formed large aggregated bundles localized mostly in the cell processes. Our results show that in vitro astrocytes undergo developmental changes in their accumulation of GFAP and intermediate filaments which reach a stable steady state after 4 weeks in culture. These 'normal' developmental changes will have to be taken into account, when experiments with variations of the level of GFAP are performed. Stable culture conditions for experimentation appear to be present after 4 weeks in culture.

Erythropoietin and VEGF promote neural outgrowth from retinal explants in postnatal rats.

PURPOSE: Recent studies have reported neuroprotective effects of erythropoietin (EPO) and vascular endothelial growth factor (VEGF). The purpose of the present study was to clarify their influence on neurite outgrowth and regeneration of rat retinal ganglion cells (RGCs) in vitro and to elucidate the expression of corresponding receptors in the rat retina in vivo. METHODS: Retinal explants from postnatal rats were stimulated with VEGF alone; VEGF in combination with anti-VEGF-receptor (VEGF-R)-2 antibody or T-type Ca2+ channel blocker ethosuximide (ESX); EPO alone; or EPO in combination with anti-EPO-receptor antibody or ESX. The presence of the corresponding receptors in the rat retina was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and by immunohistochemistry. RESULTS: EPO induced a stable improvement of neurite outgrowth of RGCs in a dose-dependent manner (5 x 10(-15) M to 5 x 10(-13) M) up to 169% (P < 0.05). Treatment of the explants with anti-EPO-R antibody (1:80 dilution) and with ESX (5 microM) totally inhibited EPO-mediated effects on RGCs. In comparison, VEGF (50 ng/mL), induced neurite outgrowth of retina explants up to 167% (P < 0.05), which again was inhibited in the presence of anti-VEGF-R2 antibody or ESX. Transcripts of EPO-R, VEGF-R1, and VEGF-R2 were detected by RT-PCR. Intense immunoreactivity for VEGF-R1, VEGF-R2, and EPO-R were found in the RGC layer of the retina. CONCLUSIONS: The data demonstrate for the first time that EPO and VEGF have a significant and specific biological effect on neurite regrowth of axotomized RGCs. Therefore, these results imply that EPO and VEGF have not only a neuroprotective but also a neuroregenerative role in ischemic retinal conditions.