ReN VM spheroids in matrix: A neural progenitor three-dimensional in vitro model reveals DYRK1A inhibitors as potential regulators of radio-sensitivity.

INTRODUCTION: Pre-clinical testing of small molecules for therapeutic development across many pathologies relies on the use of in-vitro and in-vivo models. When designed and implemented well, these models serve to predict the clinical outcome as well as the toxicity of the evaluated therapies. The two-dimensional (2D) reductionist approach where cells are incubated in a mono-layer on hard plastic microtiter plates is relatively inexpensive but not physiologically relevant. In contrast, well developed and applied three dimensional (3D) in vitro models could be employed to bridge the gap between 2D in vitro primary screening and expensive in vivo rodent models by incorporating key features of the tissue microenvironment to explore differentiation, cortical development, cancers and various neuronal dysfunctions. These features include an extracellular matrix, co-culture, tension and perfusion and could replace several hundred rodents in the drug screening validation cascade. METHODS: Human neural progenitor cells from middle brain (ReN VM, Merck Millipore, UK) were expanded as instructed by the supplier (Merck Millipore, UK), and then seeded in 96-well low-attachment plates (Corning, UK) to form multicellular spheroids followed by adding a Matrigel layer to mimic extracellular matrix around neural stem cell niche. ReN VM cells were then differentiated via EGF and bFGF deprivation for 7 days and were imaged at day 7. Radiotherapy was mimicked via gamma-radiation at 2Gy in the absence and presence of selected DYRK1A inhibitors Harmine, INDY and Leucettine 41 (L41). Cell viability was measured by AlamarBlue assay. Immunofluorescence staining was used to assess cell pluripotency marker SOX2 and differentiation marker GFAP. RESULTS: After 7 days of differentiation, neuron early differentiation marker (GFAP, red) started to be expressed among the cells expressing neural stem cell marker SOX2 (green). Radiation treatment caused significant morphology change including the reduced viability of the spheroids. These spheroids also revealed sensitizing potential of DYRK1A inhibitors tested in this study, including Harmine, INDY and L41. DISCUSSION & CONCLUSIONS: Combined with the benefit of greatly reducing the issues associated with in vivo rodent models, including reducing numbers of animals used in a drug screening cascade, cost, ethics, and potential animal welfare burden, we feel the well-developed and applied 3D neural spheroid model presented in this study will provide a crucial tool to evaluate combinatorial therapies, optimal drug concentrations and treatment dosages.

[Effects and mechanisms of electro-acupuncture on proliferation and differentiation of neural stem cells in C57 mice exposed to different doses of X-ray radiation].

The present study was aimed to investigate the effects and mechanisms of electro-acupuncture (EA) on proliferation and differentiation of neural stem cells in the hippocampus of C57 mice exposed to different doses of X-ray radiation. Thirty-day-old C57BL/6J mice were randomly divided into control, irradiation, and EA groups. The control group was not treated with irradiation. The irradiation groups were exposed to different doses of X-ray (4, 8 or 16 Gy) for 10 min. The EA groups were electro-acupunctured at Baihui, Fengfu and bilateral Shenyu for 3 courses of treatment after X-ray radiation. Immunohistochemistry was used to evaluate proliferation and differentiation of the hippocampal neural stem cell. RT-PCR and Western blot were used to detect mRNA and protein expressions of Notch1 and Mash1 in the hippocampus, respectively. The results showed that, compared with the control group, the numbers of BrdU positive cells (4, 8 Gy subgroup) and BrdU/NeuN double-labeling positive cells (3 dose subgroups) were decreased significantly in the irradiation group, but the above changes could be reversed by EA. Compared with the control group, the number of BrdU/GFAP double-labeling positive cells in each dose subgroup of irradiation group was decreased significantly, while EA could reverse the change of 4 and 8 Gy dose subgroups. In addition, compared with the control group, the expression levels of Notch1 mRNA and protein in hippocampus were up-regulated, and the expression levels of Mash1 mRNA and protein were significantly decreased in each dose subgroup of irradiation group. Compared with irradiation group, the expression levels of Notch1 mRNA and protein in hippocampus of EA group were decreased significantly in each dose subgroup, and the expression levels of Mash1 mRNA and protein were increased significantly in 4 and 8 Gy subgroups. These results suggest that irradiation affects the proliferation and differentiation of neural stem cells in hippocampus of mice, whereas EA may significantly increase the proliferation and differentiation of hippocampal neural stem cells via the regulation of Notch signaling pathway.

Dose-dependent short- and long-term effects of ionizing irradiation on neural stem cells in murine hippocampal tissue cultures: neuroprotective potential of resveratrol.

INTRODUCTION: Radiation therapy plays an essential role in the treatment of brain tumors, but neurocognitive deficits remain a significant risk, especially in pediatric patients. In recent trials, hippocampal sparing techniques are applied to reduce these adverse effects. Here, we investigate dose-dependent effects of ionizing radiation (IR) on juvenile hippocampal neurogenesis. Additionally, we evaluate the radioprotective potential of resveratrol, a plant polyphenol recognized for its bifunctional tumor-preventive and anticancer effects. METHODS: Organotypic entorhinal-hippocampal slice cultures from transgenic nestin-CFPnuc C57BL/J6 mice, postnatal days 3-6, were irradiated on a X-ray machine (4.5, 8, 12, and 16 Gy, single doses) after about 2 weeks. Nestin-positive neural stem cells were counted at a confocal live imaging microscope 0, 2, 4, 14, 25, and 42 days after IR. Resveratrol (15 µmol/L) was added 2 hr before and 24 hr after IR. Proliferation and cell death were assessed by BrdU pulse label, 48 hr after and by propidium iodide staining 96 hr after IR. GFAP- and NeuN-positive cells were counted 42 days after IR in cryosectioned immunofluorescence-stained slices. RESULTS: The observed age-related changes of nestin-positive stem cells in the organotypic slice culture model resembled the reduction of neural stem cells in vivo. IR (4.5-16 Gy) led to a dose-dependent damage of the neural stem cell pool in the dentate gyrus. No recovery was seen within 42 days after doses from 4.5 Gy onward. The decline of nestin-positive cells was paralleled by increased cell death and decreased proliferation. The number of GFAP-positive cells was significantly enhanced. No significant change was detected in the overall NeuN-positive cell population, whereas the number of newborn, NeuN/BrdU double-positive neurons was reduced. Resveratrol treatment reversed the irradiation-induced decline of neural stem cells. CONCLUSION: The neuroprotective action of resveratrol on irradiated hippocampal tissue warrants further investigation as a possible supplement to hippocampal sparing procedures.

532 nm Low-Power Laser Irradiation Facilitates the Migration of GABAergic Neural Stem/Progenitor Cells in Mouse Neocortex.

BACKGROUND AND OBJECTIVE: Accumulating evidence has shown that low-power laser irradiation (LLI) affects cell proliferation and survival, but little is known about LLI effects on neural stem/progenitor cells (NSPCs). Here we investigate whether transcranial 532 nm LLI affects NSPCs in adult murine neocortex and in neurospheres from embryonic mice. STUDY DESIGN/MATERIALS AND METHODS: We applied 532 nm LLI (Nd:YVO4, CW, 60 mW) on neocortical surface via cranium in adult mice and on cultured cells from embryonic mouse brains in vitro to investigate the proliferation and migration of NSPCs and Akt expression using immunohistochemical assays and Western blotting techniques. RESULTS: In vivo experiments demonstrated that 532 nm LLI significantly facilitated the migration of GABAergic NSPCs that were induced to proliferate in layer 1 by mild ischemia. In vitro experiments using GABAergic NSPCs derived from embryonic day 14 ganglionic eminence demonstrated that 532 nm LLI for 60 min promoted the migration of GAD67-immunopositive NSPCs with a significant increase of Akt expression. Meanwhile, the LLI induced proliferation, but not migration, of NSPCs that give rise to excitatory neurons. CONCLUSION: It is concluded that 532 nm LLI promoted the migration of GABAergic NSPCs into deeper layers of the neocortex in vivo by elevating Akt expression.

Functional interrogation of adult hypothalamic neurogenesis with focal radiological inhibition.

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

Characterizing low dose and dose rate effects in rodent and human neural stem cells exposed to proton and gamma irradiation.

Past work has shown that exposure to gamma rays and protons elicit a persistent oxidative stress in rodent and human neural stem cells (hNSCs). We have now adapted these studies to more realistic exposure scenarios in space, using lower doses and dose rates of these radiation modalities, to further elucidate the role of radiation-induced oxidative stress in these cells. Rodent neural stem and precursor cells grown as neurospheres and human neural stem cells grown as monolayers were subjected to acute and multi-dosing paradigms at differing dose rates and analyzed for changes in reactive oxygen species (ROS), reactive nitrogen species (RNS), nitric oxide and superoxide for 2 days after irradiation. While acute exposures led to significant changes in both cell types, hNSCs in particular, exhibited marked and significant elevations in radiation-induced oxidative stress. Elevated oxidative stress was more significant in hNSCs as opposed to their rodent counterparts, and hNSCs were significantly more sensitive to low dose exposures in terms of survival. Combinations of protons and γ-rays delivered as lower priming or higher challenge doses elicited radioadaptive changes that were associated with improved survival, but in general, only under conditions where the levels of reactive species were suppressed compared to cells irradiated acutely. Protective radioadaptive effects on survival were eliminated in the presence of the antioxidant N-acetylcysteine, suggesting further that radiation-induced oxidative stress could activate pro-survival signaling pathways that were sensitive to redox state. Data corroborates much of our past work and shows that low dose and dose rate exposures elicit significant changes in oxidative stress that have functional consequences on survival.

Electromagnetic field stimulation potentiates endogenous myelin repair by recruiting subventricular neural stem cells in an experimental model of white matter demyelination.

Electromagnetic fields (EMFs) may affect the endogenous neural stem cells within the brain. The aim of this study was to assess the effects of EMFs on the process of toxin-induced demyelination and subsequent remyelination. Demyelination was induced using local injection of lysophosphatidylcholine within the corpus callosum of adult female Sprague-Dawley rats. EMFs (60 Hz; 0.7 mT) were applied for 2 h twice a day for 7, 14, or 28 days postlesion. BrdU labeling and immunostaining against nestin, myelin basic protein (MBP), and BrdU were used for assessing the amount of neural stem cells within the tissue, remyelination patterns, and tracing of proliferating cells, respectively. EMFs significantly reduced the extent of demyelinated area and increased the level of MBP staining within the lesion area on days 14 and 28 postlesion. EMFs also increased the number of BrdU- and nestin-positive cells within the area between SVZ and lesion as observed on days 7 and 14 postlesion. It seems that EMF potentiates proliferation and migration of neural stem cells and enhances the repair of myelin in the context of demyelinating conditions.