69 research outputs found
Inflammatory reaction in the retina after focal non-convulsive status epilepticus in mice investigated with high resolution magnetic resonance and diffusion tensor imaging
Pathophysiological consequences of focal non-convulsive status epilepticus (fNCSE) have been difficult to demonstrate in humans. In rats fNCSE pathology has been identified in the eyes. Here we evaluated the use of high-resolution 7 T structural T1-weighted magnetic resonance imaging (MRI) and 9.4 T diffusion tensor imaging (DTI) for detecting hippocampal fNCSE-induced retinal pathology ex vivo in mice. Seven weeks post-fNCSE, increased number of Iba1+ microglia were evident in the retina ipsilateral to the hemisphere with fNCSE, and morphologically more activated microglia were found in both ipsi- and contralateral retina compared to non-stimulated control mice. T1-weighted intensity measurements of the contralateral retina showed a minor increase within the outer nuclear and plexiform layers of the lateral retina. T1-weighted measurements were not performed in the ipsilateral retina due to technical difficulties. DTI fractional anisotropy(FA) values were discretely altered in the lateral part of the ipsilateral retina and unaltered in the contralateral retina. No changes were observed in the distal part of the optic nerve. The sensitivity of both imaging techniques for identifying larger retinal alteration was confirmed ex vivo in retinitis pigmentosa mice where a substantial neurodegeneration of the outer retinal layers is evident. With MR imaging a 50 % decrease in DTI FA values and significantly thinner retina in T1-weighted images were detected. We conclude that retinal pathology after fNCSE in mice is subtle and present bilaterally. High-resolution T1-weighted MRI and DTI independently did not detect the entire pathological retinal changes after fNCSE, but the combination of the two techniques indicated minor patchy structural changes
In vivo properties of neural stem cells after transplantation into the rat brain-Studies of phenotypic differentiation and functional integration using cell-specific labelling and electrophysiological techniques
In the present thesis, we have examined the in vivo properties of in vitro expanded human and rat neural stem-and progenitor cells after transplantation into the neonatal and adult rat brain. The survival and differentiation of the grafted cells were assessed using species-specific antisera, and pre-labelling with the reporter gene green fluorescent protein (GFP). Long-term growth factor-expanded human progenitors successfully survived after grafting into the neonatal and adult striatum, subventricular zone (SVZ) and hippocampus. Target-directed migration, and region-specific neuronal differentiation of grafted cells were observed after transplantation into the neurogenic SVZ and hippocampus. Extensive migration of implanted cells identified as glial progenitors, occured within white matter. In the striatum and hippocampus, neuronal and glial differentiation were most pronounced at the graft core, with both neuronal and glial processes extending over long distances. A fraction of non-migratory undifferentiated cells remained at the implantation site. Neurogenic properties of the neural cell line RN33B, carrying the GFP reporter gene, were studied after grafting to the neonatal brain. Large numbers of RN33B cells differentiated into pyramidal neurons in the cortex and hippocampus, with projections to normal target regions, such as the thalamus and contralateral hippocampus, respectively, as revealed by retrograde tracing. Whole-cell patch clamp recordings of grafted cortical pyramidal neurons showed that RN33B cells develop physiological properties of mature neurons and become functionally integrated within host neural circuitry. Our data demonstrate a remarkable capacity of expandable neural precursors for different types of migration, and multipotential differentiation, along neuronal and glial lineages. Along with region-specific neuronal differentiation we observed establishment of appropriate anatomical projections, and functional integration into host circuitry. These results suggest that these cell types are highly useful for further research into the mechanisms responsible for cellular migration, differentiation and integration in the mature central nervous system
Immunocytochemical Profiling of Cultured Mouse Primary Retinal Cells
Primary retinal cell cultures and immunocytochemistry are important experimental platforms in ophthalmic research. Translation of retinal cells from their native environment to the in vitro milieu leads to cellular stress, jeopardizing their in vivo phenotype features. Moreover, the specificity and stability of many retinal immunochemical markers are poorly evaluated in retinal cell cultures. Hence, we here evaluated the expression profile of 17 retinal markers, that is, recoverin, rhodopsin, arrestin, Chx10, PKC, DCX, CRALBP, GS, vimentin, TPRV4, RBPMS, Brn3a, β-tubulin III, NeuN, MAP2, GFAP, and synaptophysin. At 7 and 18 days of culture, the marker expression profiles of mouse postnatal retinal cells were compared with their age-matched in vivo retinas. We demonstrate stable in vitro expression of all markers, except for arrestin and CRALBP. Differences in cellular expression and location of some markers were observed, both over time in culture and compared with the age-matched retina. We hypothesize that these differences are likely culture condition dependent. Taken together, we suggest a thorough evaluation of the antibodies in specific culture settings, before extrapolating the in vitro results to an in vivo setting. Moreover, the identification of specific cell types may require a combination of different genes expressed or markers with structural information
Tailor-Made Electrospun Culture Scaffolds Control Human Neural Progenitor Cell Behavior : Studies on Cellular Migration and Phenotypic Differentiation
In neuroscience research, cell culture systems are essential experimental platforms. It is of great interest to explore in vivo-like culture substrates. We explored how basic properties of neural cells, nuclei polarization, phenotypic differentiation and distribution/migration, were affected by the culture at poly-L-lactic acid (PLLA) fibrous scaffolds, using a multipotent mitogen-expanded human neural progenitor cell (HNPC) line. HNPCs were seeded, at four different surfaces: two different electrospun PLLA (d = 1.2 - 1.3 μm) substrates (parallel or random aligned fibers), and planar PLL- and PLLA surfaces. Nuclei analysis demonstrated a non-directed cellular migration at planar surfaces and random fibers, different from cultures at aligned fibers where HNPCs were oriented parallel with the fibers. At aligned fibers, HNPCs displayed the same capacity for phenotypic differentiation as after culture on the planar surfaces. However, at random fibers, HNPCs showed a significant lower level of phenotypic differentiation compared with cultures at the planar surfaces. A clear trend towards greater neuronal formation at aligned fibers, compared to cultures at random fibers, was noted. We demonstrated that the topography of in vivo-resembling PLLA scaffolds significantly influences HNPC behavior, proven by different migration behavior, phenotypic differentiation potential and nuclei polarization. This knowledge is useful in future exploration of in vivo-resembling neural cell system using electrospun scaffolds
A transplantation study of expanded human embryonic forebrain precursors: evidence for selection of a specific progenitor population.
Gold- and silver nanoparticles affect the growth characteristics of human embryonic neural precursor cells.
Rapid development of nanotechnologies and their applications in clinical research have raised concerns about the adverse effects of nanoparticles (NPs) on human health and environment. NPs can be directly taken up by organs exposed, but also translocated to secondary organs, such as the central nervous system (CNS) after systemic- or subcutaneous administration, or via the olfactory system. The CNS is particularly vulnerable during development and recent reports describe transport of NPs across the placenta and even into brain tissue using in vitro and in vivo experimental systems. Here, we investigated whether well-characterized commercial 20 and 80 nm Au- and AgNPs have an effect on human embryonic neural precursor cell (HNPC) growth. After two weeks of NP exposure, uptake of NPs, morphological features and the amount of viable and dead cells, proliferative cells (Ki67 immunostaining) and apoptotic cells (TUNEL assay), respectively, were studied. We demonstrate uptake of both 20 and 80 nm Au- and AgNPs respectively, by HNPCs during proliferation. A significant effect on the sphere size- and morphology was found for all cultures exposed to Au- and AgNPs. AgNPs of both sizes caused a significant increase in numbers of proliferating and apoptotic HNPCs. In contrast, only the highest dose of 20 nm AuNPs significantly affected proliferation, whereas no effect was seen on apoptotic cell death. Our data demonstrates that both Au- and AgNPs interfere with the growth profile of HNPCs, indicating the need of further detailed studies on the adverse effects of NPs on the developing CNS
Exploration of physical and chemical cues on retinal cell fate
Identification of the key components in the physical and chemical milieu directing donor cells into a desired phenotype is a requirement in the investigation of bioscaffolds for the advancement of cell-based therapies for retinal neurodegeneration. We explore the effect of electrospun poly-ε-caprolactone (PCL) fiber scaffold topography and functionalization and culture medium, on the behavior of mouse retinal cells. Dissociated mouse retinal post-natal cells were seeded on random or aligned oriented fibers, with or without laminin coating and cultured with either basic or neurotrophins enriched medium for 7 days. Addition of laminin in combination with neurotrophins clearly promoted cell– morphology, fate, and neurite extension. Nanotopography per se significantly affected cell morphology, with mainly bipolar profiles on aligned fibers and more multipolar profiles on random fibers. Laminin induced a remarkable 90° switch of neurite orientation. Herewith, we demonstrate that the chemical cue is stronger than the physical cue for the orientation of retinal neurites and describe the requirement of both neurotrophins and extracellular matrix proteins for extended neurite outgrowth and formation of complex retinal neuronal networks. Therefore, tailor-made PCL fiber mats, which can be physically and chemically modified, indeed influence cell behavior and hence motivate further retinal restorative studies using this system
Correction: Gold- and Silver Nanoparticles Affect the Growth Characteristics of Human Embryonic Neural Precursor Cells.
Rapid development of nanotechnologies and their applications in clinical research have raised concerns about the adverse effects of nanoparticles (NPs) on human health and environment. NPs can be directly taken up by organs exposed, but also translocated to secondary organs, such as the central nervous system (CNS) after systemic- or subcutaneous administration, or via the olfactory system. The CNS is particularly vulnerable during development and recent reports describe transport of NPs across the placenta and even into brain tissue using in vitro and in vivo experimental systems. Here, we investigated whether well-characterized commercial 20 and 80 nm Au- and AgNPs have an effect on human embryonic neural precursor cell (HNPC) growth. After two weeks of NP exposure, uptake of NPs, morphological features and the amount of viable and dead cells, proliferative cells (Ki67 immunostaining) and apoptotic cells (TUNEL assay), respectively, were studied. We demonstrate uptake of both 20 and 80 nm Au- and AgNPs respectively, by HNPCs during proliferation. A significant effect on the sphere size- and morphology was found for all cultures exposed to Au- and AgNPs. AgNPs of both sizes caused a significant increase in numbers of proliferating and apoptotic HNPCs. In contrast, only the highest dose of 20 nm AuNPs significantly affected proliferation, whereas no effect was seen on apoptotic cell death. Our data demonstrates that both Au- and AgNPs interfere with the growth profile of HNPCs, indicating the need of further detailed studies on the adverse effects of NPs on the developing CNS
Migration patterns and phenotypic differentiation of long-term expanded human neural progenitor cells after transplantation into the adult rat brain.
We have examined long-term growth-factor expanded human neural progenitors following transplantation into the adult rat brain. Cells, obtained from the forebrain of a 9-week old fetus, propagated in the presence of epidermal growth factor, basic fibroblast growth factor, and leukemia inhibitory factor were transplanted into the striatum, subventricular zone (SVZ), and hippocampus. At 14 weeks, implanted cells were identified using antisera recognizing human nuclei and the reporter gene green fluorescent protein. Different migration patterns of the grafted cells were observed: (i) target-directed migration of doublecortin (DCX, a marker for migrating neuroblasts)-positive cells along the rostral migratory stream to the olfactory bulb and into the granular cell layer following transplantation into the SVZ and hippocampus, respectively; (ii) non-directed migration of DCX-positive cells in the grey matter in striatum and hippocampus, and (iii) extensive migration of above all nestin-positive/DCX-negative cells within white matter tracts. At the striatal implantation site, neuronal differentiation was most pronounced at the graft core with axonal projections extending along the internal capsule bundles. In the hippocampus, cells differentiated primarily into interneurons both in the dentate gyrus and in the CA1-3 regions as well as into granule-like neurons. In the striatum and hippocampus, a significant proportion of the grafted cells differentiated into glial cells, some with long processes extending along white matter tracts. Although the survival time was over 3 months in the present study a large fraction of the grafted cells remained undifferentiated in a stem or progenitor cell stage as revealed by the expression of nestin and/or GFAP
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