Influence of biological matrix and artificial electrospun scaffolds on proliferation, differentiation and trophic factor synthesis of rat embryonic stem cells.

Abstract Two-dimensional vs three-dimensional culture conditions, such as the presence of extracellular matrix components, could deeply influence the cell fate and properties. In this paper we investigated proliferation, differentiation, survival, apoptosis, growth and neurotrophic factor synthesis of rat embryonic stem cells (RESCs) cultured in 2D and 3D conditions generated using Cultrex® Basement Membrane Extract (BME) and in poly-( l -lactic acid) (PLLA) electrospun sub-micrometric fibres. It is demonstrated that, in the absence of other instructive stimuli, growth, differentiation and paracrine activity of RESCs are directly affected by the different microenvironment provided by the scaffold. In particular, RESCs grown on an electrospun PLLA scaffolds coated or not with BME have a higher proliferation rate, higher production of bioactive nerve growth factor (NGF) and vascular endothelial growth factor (VEGF) compared to standard 2D conditions, lasting for at least 2 weeks. Due to the high mechanical flexibility of PLLA electrospun scaffolds, the PLLA/stem cell culture system offers an interesting potential for implantable neural repair devices

Design and in vitro study of a dual drug-loaded delivery system produced by electrospinning for the treatment of acute injuries of the central nervous system

Vascular and traumatic injuries of the central nervous system are recognized as global health priorities. A polypharmacology approach that is able to simultaneously target several injury factors by the combination of agents having synergistic effects appears to be promising. Herein, we designed a polymeric delivery system loaded with two drugs, ibuprofen (Ibu) and thyroid hormone triiodothyronine (T3) to in vitro release the suitable amount of the anti-inflammation and the remyelination drug. As a production method, electrospinning technology was used. First, Ibuloaded micro (diameter circa 0.95–1.20 µm) and nano (diameter circa 0.70 µm) fibers were produced using poly(L-lactide) PLLA and PLGA with different lactide/glycolide ratios (50:50, 75:25, and 85:15) to select the most suitable polymer and fiber diameter. Based on the in vitro release results and in-house knowledge, PLLA nanofibers (mean diameter = 580 ± 120 nm) loaded with both Ibu and T3 were then successfully produced by a co-axial electrospinning technique. The in vitro release studies demonstrated that the final Ibu/T3 PLLA system extended the release of both drugs for 14 days, providing the target sustained release. Finally, studies in cell cultures (RAW macrophages and neural stem cell-derived oligodendrocyte precursor cells—OPCs) demonstrated the anti-inflammatory and promyelinating efficacy of the dual drug-loaded delivery platform

Low infra red laser light irradiation on cultured neural cells: effects on mitochondria and cell viability after oxidative stress

<p>Abstract</p> <p>Background</p> <p>Considerable interest has been aroused in recent years by the well-known notion that biological systems are sensitive to visible light. With clinical applications of visible radiation in the far-red to near-infrared region of the spectrum in mind, we explored the effect of coherent red light irradiation with extremely low energy transfer on a neural cell line derived from rat pheochromocytoma. We focused on the effect of pulsed light laser irradiation vis-à-vis two distinct biological effects: neurite elongation under NGF stimulus on laminin-collagen substrate and cell viability during oxidative stress.</p> <p>Methods</p> <p>We used a 670 nm laser, with extremely low peak power output (3 mW/cm²) and at an extremely low dose (0.45 mJ/cm²). Neurite elongation was measured over three days in culture. The effect of coherent red light irradiation on cell reaction to oxidative stress was evaluated through live-recording of mitochondria membrane potential (MMP) using JC1 vital dye and laser-confocal microscopy, in the absence (photo bleaching) and in the presence (oxidative stress) of H₂O₂, and by means of the MTT cell viability assay.</p> <p>Results</p> <p>We found that laser irradiation stimulates NGF-induced neurite elongation on a laminin-collagen coated substrate and protects PC12 cells against oxidative stress.</p> <p>Conclusion</p> <p>These data suggest that red light radiation protects the viability of cell culture in case of oxidative stress, as indicated by MMP measurement and MTT assay. It also stimulates neurite outgrowth, and this effect could also have positive implications for axonal protection.</p

Vulnerability of primary neurons derived from Tg2576 Alzheimer mice to oxygen and glucose deprivation: role of intraneuronal amyloid-β accumulation and astrocytes.

Microvascular dysfunction is considered an integral part of Alzheimer disease (AD) pathogenesis, but the possible relationship between amyloid pathology, microvascular dysfunction and cell death is still unclear. In order to investigate the influence of intraneuronal amyloid-β (Aβ) accumulation on vulnerability to hypoxia, we isolated primary cortical neurons from Tg2576 (carrying the amyloid precursor protein APPSwe mutation) and wild-type fetal mice. We first demonstrated that neurons isolated from Tg2576 newborn mice show an increase in VEGFa mRNA expression and a decrease in the expression of the two VEGF receptors, Flt1 and Kdr, compared with wild-type cells. Moreover, APPSwe primary neurons displayed higher spontaneous and glutamate-induced cell death. We then deprived the cultures of oxygen and glucose (OGD) as an in vitro model of hypoxia. After OGD, APPSwe neurons display higher levels of cell death in terms of percentage of pyknotic/fragmented nuclei and mitochondrial depolarization, accompanied by an increase in the intraneuronal Aβ content. To explore the influence of intraneuronal Aβ peptide accumulation, we used the γ-secretase inhibitor LY450139, which showed that the reduction of the intracellular amyloid fully protects APPSwe neurons from OGD-induced degeneration. Conditioned medium from OGD-exposed APPSwe or wild-type astrocytes protected APPswe neurons but not wild-type neurons, during OGD. In conclusion, the presence of the mutated human APP gene, leading to the intracellular accumulation of APP and Aβ fragments, worsens OGD toxicity. Protection of APPSwe neurons can be obtained either using a γ-secretase inhibitor or astrocyte conditioned medium

Iodinated-NPY binding sites: autoradiographic study in the rat brain.

Several authors have described the presence of iodinated neuropeptide-Y binding sites on membranes of the mammalian CNS. In the present study we show a mapping of iodinated-NPY binding sites in the rat brain using receptor autoradiography. The sections were incubated with 125I-Bolton-Hunter coupled NPY (0.5-03 nM), in the absence or presence of 1 microM cold NPY. Some autoradiograms are studied by means of an image analyzer (VDC 501 Tesak) equipped with the host computer PDP 11 Digital, in order to enhance the contrast of the labeling. A very high density of NPY receptors is present in the limbic regions (hippocampus, amygdaloid complex, septal nuclei), in the cortex, and in some thalamic nuclei, while in some hypothalamic regions (paraventricular nucleus and median eminence) we detected a lower amount of NPY receptors. At the mesencephalic level, the substantia nigra presents a very high density of NPY receptors