Embryonic neural stem cells in a 3D bioassay for trophic stimulation studies.

Progenitors were discovered in the corpus striatum several years ago, but little is known about their proliferation and differentiation. The aim of this study was to analyze embryonic progenitor cells from the corpus striatum using a bioassay with trophic stimulation. Primary cells obtained from brains of rat embryos at E13-14 were dissected from striatum niches and cultured in stem cell media. These floating dispersed cells clumped together to forming floating bodies like irregular spheres (spheroids), which were placed in type I collagen gel and cultured under basal conditions or with the addition of NGF, NT-3, or NTN. Optimum growth of neurites was obtained, and after 24 and 48 h, they were measured for number and length. The expression of proliferation markers such as PCNA and Ki67, and of neural progenitor markers such as GFAP, nestin, vimentin, O4, A2B5, Pax6, S100, TubIII, and NeuN, was then analyzed. The initial behavior in cell cultures showed distinguishable spheroids that, when placed in 3D gels and with trophic support, generated neurites. A similar effect was observed in glial cell outgrowth from the spheroids. Our assay showed high reproducibility, short culture time, and high resolution for tracing neuron-neurite outgrowth or visualizing glial outgrowth in a few hours.

Morphometric analysis of brain lesions in rat fetuses prenatally exposed to low-level lead acetate: correlation with lipid peroxidation.

The effect of prenatal lead acetate exposure was studied microscopically together with the concentration of lead and lipid fluorescent products (LFP) in the brain of rat fetuses. Wistar rats were intoxicated with a lead solution containing either 160 or 320 ppm of lead acetate solution during 21 days through drinking water. The control group (ten rats) received deionized water for the same period. The rats were killed on gestation day 21 and fetuses were obtained; the placenta, umbilical cord and parietal cortex (Cx), striatum (St), thalamus (Th) and cerebellum (Ce) were collected for measuring tissue lead concentration, LFP as an index of lipid peroxidation and histopathologic examination. Lead contents were increased in placenta, umbilical cord, St, Th and Cx in both lead-exposed groups. Lead exposure increased (LFP) in placenta and umbilical cord, St, Th and Ce as compared to the control group. Histopathological examination showed severe vascular congestion in placenta, the Cx, St, Th and Ce with hyperchromatic and shrunken cells. Interstitial oedema was found in all regions studied of both lead exposed groups. The morphometric evaluation of the studied brain regions showed an absolute decrease in total cell number and increased number of damaged cells and interstitial oedema. Our results show that morphological changes in rat brain are correlated with increased lipid peroxidation, and the lead levels of the umbilical cord, however it is not clear whether oxidative stress is the cause or the consequence of these neurotoxic effects of lead.

Characterization of proliferation and differentiation of EGF-responsive striatal and septal precursor cells.

The epigenetic manipulation of precursors may provide data to elucidate the potential interactions among these cells in different brain regions. However, the response to epigenetic signals is modulated by the environment in which the cells are manipulated. Therefore, data regarding the action of a particular factor must be considered in the light of a specific system. To compare septal and striatal precursors, we have tested the effect of nerve growth factor (NGF) on the proliferation and neuronal differentiation of epidermal growth factor (EGF)-responsive cells from these brain regions. Precursors were cultivated as 'neuropheres' in serum free medium (SFM) to which NGF was added. NGF did not support the proliferation of EGF-generated precursors so that no differences in the cell magnitude with respect to control cultures were observed. Differentiation of precursors in SFM plus 1% fetal bovine serum (FBS) on poly-D-lysine showed that the neuron number was increased two-fold in septal cultures treated with NGF but not in those from striatum. A quantitative evaluation of the soma surface and the number of primary neurites showed differences between both populations of precursor-generated neurons. In addition, we also observed no influence of NGF on these parameters of cellular morphology. Thus, taken together these results seem to indicate that at this developmental stage in which these populations of precursors were isolated, heterogeneities exist between them, which is probably related to their origin and/or functional roles in vivo.

[Epidermal growth factor-responsive precursors with similar properties isolated from embryonic rat septum and striatum]. / Características similares de los precursores sensibles al factor de crecimiento epidérmico aislados del septo y del estriado de rata embrionaria.

INTRODUCTION: The potential correspondence of precursor cells isolated from different brain regions is partially unknown. Since models and culture conditions used in several studies vary, comparison of precursor characteristics has been limited. OBJECTIVE: In this paper epidermal growth factor (EGF)-responsive precursors from the striatum and septum were isolated and their growth pattern in vitro were determined. We also evaluated the influence of fibroblast growth factor (FGF) and nerve growth factor (NGF) on the proliferation of these cells. MATERIAL AND METHODS: Dissociated cells from rat septum and striatum were cultivated in suspension with 20 ng/ml of EGF. Total cells quantification, immunocytochemical staining and neuron counts were used to evaluate cell proliferation and cellular phenotypes produced by EGF-generated cells. RESULTS: Considering both culture evolution and cellular growth we demonstrated a similar growth pattern of septal and striatal EGF-responsive precursors. Furthermore, the proliferation of both cells populations was supported by FGF. On the contrary, NGF neither had a proliferative effect nor affected cell survival. Upon differentiation, a small proportion of precursor cells differentiated into neuronal phenotype.

Heterochronous maturation of regional brain astroglia: neuronal modulation of striatal glial cells differentiation ex vivo.

Subcultured astroglial cells from striatum, cerebral cortex and ventral mesencephalon obtained from primary cultures of fetal (E14, E17 and E21) or postnatal (days 5-6) rats showed different regional, age-dependent morphological response (stellation) to cyclic AMP. While most of the cerebral cortex and ventral mesencephalic astroglial cell population was responsive at all ages tested, striatal cells at E14 and E17 were not. At age E21 striatal astroglia showed a significant shift toward a mature-like type of response to cyclic AMP. Postnatal striatal astroglia responded to cyclic AMP as the cortical and ventral mesencephalic astroglia did, with generalized stellation. Prenatal striatal astroglia was characterized immunocytochemically as A2B5+, fibronectin+, vimentin+, S-100+ and GFAP-. Failure of early prenatal (E14, E17) striatal astroglia to differentiate in response to cyclic AMP, was overcome by previous (5-7 days) co-culture with primary cell dissociates from postnatal-, but not from prenatal donors, from all brain regions tested including a non-target region for striatal cells, such as septum. This effect was duplicated when striatal astroglia was co-cultured with cell populations enriched in neurons through Percoll gradients. Only cell-to-cell contact co-cultures were able to induce a change in the studied response. Dead neuron-enriched populations obtained following various types of physical treatments were also able to change significantly striatal cell response toward cyclic AMP. Enriched astroglial populations from postnatal donors did not change striatal astroglial response toward cyclic AMP, except for ventral mesencephalic astroglia which induced a comparatively reduced but significant increase in striatal cell responsiveness. It is concluded that astroglial maturation and potential for phenotype expression during brain development proceeds with regional heterochrony. Also, that maturation of prenatal striatal astroglia responsiveness toward cyclic AMP is inducible by non-diffusible factors, probably of neuronal origin, expressed in live or dead primary cultures from various, homotopic and heterotopic, postnatal brain regions. It is further suggested that striatal afferents and/or mature local striatal neurons express membrane associated molecules that regulate responsiveness for phenotype expression of striatal glial cells, thus reinforcing the concept of a highly interactive, continuous neuron-glial developmental process that takes place during brain organization.