MorphoNeuroNet: an automated method for dense neurite network analysis.

High content cell-based screens are rapidly gaining popularity in the context of neuronal regeneration studies. To analyze neuronal morphology, automatic image analysis pipelines have been conceived, which accurately quantify the shape changes of neurons in cell cultures with non-dense neurite networks. However, most existing methods show poor performance for well-connected and differentiated neuronal networks, which may serve as valuable models for inter alia synaptogenesis. Here, we present a fully automated method for quantifying the morphology of neurons and the density of neurite networks, in dense neuronal cultures, which are grown for more than 10 days. MorphoNeuroNet, written as a script for ImageJ, Java based freeware, automatically determines various morphological parameters of the soma and the neurites (size, shape, starting points, and fractional occupation). The image analysis pipeline consists of a multi-tier approach in which the somas are segmented by adaptive region growing using nuclei as seeds, and the neurites are delineated by a combination of various intensity and edge detection algorithms. Quantitative comparison showed a superior performance of MorphoNeuroNet to existing analysis tools, especially for revealing subtle changes in thin neurites, which have weak fluorescence intensity compared to the rest of the network. The proposed method will help determining the effects of compounds on cultures with dense neurite networks, thereby boosting physiological relevance of cell-based assays in the context of neuronal diseases.

Combined Exposure to Simulated Microgravity and Acute or Chronic Radiation Reduces Neuronal Network Integrity and Survival.

During orbital or interplanetary space flights, astronauts are exposed to cosmic radiations and microgravity. However, most earth-based studies on the potential health risks of space conditions have investigated the effects of these two conditions separately. This study aimed at assessing the combined effect of radiation exposure and microgravity on neuronal morphology and survival in vitro. In particular, we investigated the effects of simulated microgravity after acute (X-rays) or during chronic (Californium-252) exposure to ionizing radiation using mouse mature neuron cultures. Acute exposure to low (0.1 Gy) doses of X-rays caused a delay in neurite outgrowth and a reduction in soma size, while only the high dose impaired neuronal survival. Of interest, the strongest effect on neuronal morphology and survival was evident in cells exposed to microgravity and in particular in cells exposed to both microgravity and radiation. Removal of neurons from simulated microgravity for a period of 24 h was not sufficient to recover neurite length, whereas the soma size showed a clear re-adaptation to normal ground conditions. Genome-wide gene expression analysis confirmed a modulation of genes involved in neurite extension, cell survival and synaptic communication, suggesting that these changes might be responsible for the observed morphological effects. In general, the observed synergistic changes in neuronal network integrity and cell survival induced by simulated space conditions might help to better evaluate the astronaut's health risks and underline the importance of investigating the central nervous system and long-term cognition during and after a space flight.

Mitigation of a radon-rich Belgian dwelling using active subslab depressurization.

In a radon prone area in Belgium, a dwelling with high indoor radon concentrations was identified through a passive measurement. Next, a continuous, active radon monitoring device was installed for one month. A 20-a retrospective radon assessment was also performed. The house was subsequently mitigated through active subslab depressurization with a radial fan. Afterwards the dwelling was actively monitored for several more months to observe the effects of the mitigation and to study the effect of reducing the fan power. Dose evaluations were made to evaluate the health benefit of the mitigation. It was seen that the results of the three measuring techniques before mitigation all yielded between 1700 and 2000 Bq/m3. Clear diurnal radon variations showed up only after mitigation. After mitigation, the average radon concentration fell to less than 200 Bq/m3. The yearly average dose was reduced from potentially 45 mSv/y to less than 4.5 mSv/y through mitigation. Reducing fan power to 50% did not clearly influence the amount of radon entering into the dwelling.

Developmental abnormalities induced by X-irradiation in p53 deficient mice.

In order to assess the influence of p53 inactivation on radiation-induced developmental effects, male mice heterozygous for the wild-type p53 allele (mimicking the human Li-Fraumeni syndrome) were crossed with C57BL females, and their heterozygous p53+/- progeny were mated with each other to obtain p53+/-, p53-/- and p53+/+ embryos. Pregnant females were X-irradiated with 0.5 Gy on days 1 (pre-implantation period), 8 or 11 (organogenesis period) of gestation. Dissection of the pregnant females occurred on day 19 of gestation. The p53 genotype of the foetuses was determined by PCR from small pieces of soft tissues. Exencephaly was the only external malformation found in the control group. It affected essentially p53-/- female foetuses. A number of p53+/- and p53+/- control foetuses also showed dwarfism, or underdevelopment. In the group irradiated on day 1, the frequency of abnormal foetuses was, paradoxically, lower than that found in the control group. As in that group, exencephaly and dwarfism constituted the only anomalies that were found. Exencephaly affected only homozygous p53-/- females, while dwarfism concerned either p53-/- or p53+/- foetuses, with a majority of females. Irradiation on day 8 of gestation induced a significant increase in the frequency of abnormal foetuses, compared to the control group. Various malformations were observed in addition to exencephaly, including gastroschisis, polydactyly, cephalic oedema and cleft palate. All malformed foetuses were either homozygous p53-/- or heterozygous p53+/- while most affected foetuses were females, as was the case for dwarf individuals. Irradiation on day 11 did not cause an increase in the frequency of abnormal foetuses, in comparison with the controls. However, a large spectrum of external malformations was again noticed, as in the group irradiated on day 8. All affected foetuses were homozygous p53-/- and there were slightly more abnormal females than males (3 out of 5). No dwarfs were found in this group. Overall, these results confirm the importance of the p53 tumour-suppressor protein for normal embryonic development. They clearly show that homozygous p53-/- (or heterozygous p53+/- to a lesser extent) foetuses are more at risk for radiation-induction of external malformations during the organogenesis period, and that the risk of developing such malformations is much higher for females than for males. In contrast to results published very recently by others, we found that malformed foetuses resulting from an X-irradiation with a low-dose during the highly sensitive period of gastrulation are able to survive to birth.

Non-conventional apoptotic response to ionising radiation mediated by N-methyl D-aspartate receptors in immature neuronal cells.

During cortical development, N-methyl D-aspartate (NMDA) receptors are highly involved in neuronal maturation and synapse establishment. Their implication in the phenomenon of excitotoxicity has been extensively described in several neurodegenerative diseases due to the permissive entry of Ca2+ ions and massive accumulation in the intracellular compartment, which is highly toxic to cells. Ionising radiation is also a source of stress to the cells, particularly immature neurons. Their capacity to induce cell death has been described for various cell types either by directly damaging the DNA or indirectly through the generation of reactive oxygen species responsible for the activation of a battery of stress response effectors leading in certain cases, to cell death. In this study, in order to determine whether a link exists between NMDA receptors-mediated excitotoxicity and radiation-induced cell death, we evaluated radiation-induced cell death in vitro and in vivo in maturing neurons during the fetal period. Cell death induction was assessed by TUNEL, caspase-3 activity and DNA ladder assays, with or without the administration of dizocilpine (MK-801), a non-competitive NMDA receptor antagonist which blocks neuronal Ca2+ influx. To further investigate the possible involvement of Ca2+-dependent enzyme activation, known to occur at high Ca2+ concentrations, we examined the protective effect of a calpain inhibitor on cell death induced by radiation. Doses ranging from 0.2 to 0.6 Gy of X-rays elicited a clear apoptotic response that was prevented by the injection of dizocilpine (MK-801) or calpain inhibitor. These data demonstrate the involvement of NMDA receptors in radiation-induced neuronal death by the activation of downstream effectors, including calpain-related pathways. An increased apoptotic process elicited by radiation, occurring independently of the normal developmental scheme, may eliminate post-mitotic but immature neuronal cells and deeply impair the establishment of the neuronal network, which in the case of cortical development is critical for cognitive capacities.

Morphological and physiological changes in mature in vitro neuronal networks towards exposure to short-, middle- or long-term simulated microgravity.

One of the objectives of the current international space programmes is to investigate the possible effects of the space environment on the crew health. The aim of this work was to assess the particular effects of simulated microgravity on mature primary neuronal networks and specially their plasticity and connectivity. For this purpose, primary mouse neurons were first grown for 10 days as a dense network before being placed in the Random Positioning Machine (RPM), simulating microgravity. These cultures were then used to investigate the impact of short- (1 h), middle- (24 h) and long-term (10 days) exposure to microgravity at the level of neurite network density, cell morphology and motility as well as cytoskeleton properties in established two-dimensional mature neuronal networks. Image processing analysis of dense neuronal networks exposed to simulated microgravity and their subsequent recovery under ground conditions revealed different neuronal responses depending on the duration period of exposure. After short- and middle-term exposures to simulated microgravity, changes in neurite network, neuron morphology and viability were observed with significant alterations followed by fast recovery processes. Long exposure to simulated microgravity revealed a high adaptation of single neurons to the new gravity conditions as well as a partial adaptation of neuronal networks. This latter was concomitant to an increase of apoptosis. However, neurons and neuronal networks exposed for long-term to simulated microgravity required longer recovery time to re-adapt to the ground gravity. In conclusion, a clear modulation in neuronal plasticity was evidenced through morphological and physiological changes in primary neuronal cultures during and after simulated microgravity exposure. These changes were dependent on the duration of exposure to microgravity.

Different responsiveness of endothelial cells to vascular endothelial growth factor and basic fibroblast growth factor added to culture media under gravity and simulated microgravity.

When incubated under simulated microgravity (s-microg), endothelial cells (EC) form tubular structures that resemble vascular intimas. This delayed formation of 3D EC structures begins between the 5th and 7th day of culturing EC under conditions of s-microg, when double-row cell assemblies become visible. With the aim of learning about this initial phase of tubular structure formation, we found that NFkappaBp65 protein content was similar in all cell populations, but gene and protein expression of phosphokinase A catalytic subunit, phosphokinase Calpha, and extracellular signal-regulated kinases 1 and 2 was altered in cells cultured under s-microg. Apoptosis remained below 30% in all EC cultures. In contrast to controls, the 7-day-old s-microg cultures contained 3D aggregates with proliferating cells, enhanced numbers of necrotic cells, and osteopontin-negative EC as well as supernatants with reduced quantities of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), soluble TNFRSF5, TNFSF5, intercellular adhesion molecule-1, tumor necrosis factor receptor 2, IL-18, complement C3, and von Willebrand factor. VEGF and/or bFGF (10 ng/mL) application influenced the accumulation of proteins in supernatants more profoundly under 1 g than under s-microg. These findings provide evidence that phosphokinase Calpha plays a key role in tube formation. Improving the interaction of VEGF and/or bFGF with EC under s-microg could enhance the engineering of vascular intimas.