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.

Background: Epileptic seizures are associated with an immune response in the brain. However, it is not known whether it can extend to remote areas of the brain, such as the eyes. Hence, we investigated whether epileptic seizures induce inflammation in the retina.

Methods: Adult rats underwent electrically induced temporal status epilepticus, and the eyes were studied 6 h, 1, and 7 weeks later with biochemical and immunohistochemical analyses. An additional group of animals received CX3CR1 antibody intracerebroventricularly for 6 weeks after status epilepticus.

Results: Biochemical analyses and immunohistochemistry revealed no increased cell death and unaltered expression of several immune-related cytokines and chemokines as well as no microglial activation, 6 h post-status epilepticus compared to non-stimulated controls. At 1 week, again, retinal cytoarchitecture appeared normal and there was no cell death or micro- or macroglial reaction, apart from a small decrease in interleukin-10. However, at 7 weeks, even if the cytoarchitecture remained normal and no ongoing cell death was detected, the numbers of microglia were increased ipsi- and contralateral to the epileptic focus. The microglia remained within the synaptic layers but often in clusters and with more processes extending into the outer nuclear layer. Morphological analyses revealed a decrease in surveying and an increase in activated microglia. In addition, increased levels of the chemokine KC/GRO and cytokine interleukin-1β were found. Furthermore, macroglial activation was noted in the inner retina. No alterations in numbers of phagocytic cells, infiltrating macrophages, or vascular pericytes were observed. Post-synaptic density-95 cluster intensity was reduced in the outer nuclear layer, reflecting seizure-induced synaptic changes without disrupted cytoarchitecture in areas with increased microglial activation. The retinal gliosis was decreased by a CX3CR1 immune modulation known to reduce gliosis within epileptic foci, suggesting a common immunological reaction.

Conclusions: Our results are the first evidence that epileptic seizures induce an immune response in the retina. It has a potential to become a novel non-invasive tool for detecting brain inflammation through the eyes.

Background Disease progression in retinal neurodegeneration is strongly correlated to immune cell activation, which may have either a neuroprotective or neurotoxic effect. Increased knowledge about the immune response profile and retinal neurodegeneration may lead to candidate targets for treatments. Therefore, we have used the explanted retina as a model to explore the immune response and expression of the immune modulator galectin-3 (Gal-3), induced by the cultivation per se and after additional immune stimulation with lipopolysaccharide (LPS), and how this correlates with retinal neurotoxicity. Methods Post-natal mouse retinas were cultured in a defined medium. One group was stimulated with LPS (100 ng/ml, 24 h). Retinal architecture, apoptotic cell death, and micro- and macroglial activity were studied at the time of cultivation (0 days in vitro (DIV)) and at 3, 4 and 7 DIV using morphological staining, biochemical- and immunohistochemical techniques. Results Our results show that sustained activation of macro- and microglia, characterized by no detectable cytokine release and limited expression of Gal-3, is not furtherinducing apoptosis additional to the axotomy-induced apoptosis in innermost nuclear layer. An elevated immune response was detected after LPS stimulation, as demonstrated primarily by release of immune mediators (i.e. interleukin 2 (IL-2), IL-6, KC/GRO (also known as CLCX1) and tumour necrosis factor-α (TNF-α)), increased numbers of microglia displaying morphologies of late activation stages as well as Gal-3 expression. This was accompanied with increased apoptosis in the two additional nuclear layers, and damage to retinal gross architecture. Conclusion We demonstrate that an immune response characterized by sustained and increased release of cytokines, along with an increase in Gal-3 expression, is accompanied by significant increased neurotoxicity in the explanted retina. Further investigations using the current setting may lead to increased understanding on the mechanisms involved in neuronal loss in retinal neurodegenerations. © 2016 Bauer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

As previously reported, the brain-derived precursor cell line RN33B has a great capacity to migrate when transplanted to adult brain or retina. This cell line is immortalized with the SV40 large T-antigen and carries the reporter gene LacZ and the green fluorescent protein GFP. In the present study, the precursor cells were transplanted to the subretinal space of adult rats and investigated early after grafting. The purpose was to demonstrate the migration of the grafted cells from the subretinal space into the retina and the glial cell response of the host retina. Detachment caused by the transplantation method was persistent up to 4 days after transplantation, and then reattachment occurred. The grafted cells were shown to migrate in between the photoreceptor cells before entering into the plexiform layers. Molecules involved in migration of immature neuronal cells as the polysialylated neural cell adhesion molecule (PSA-NCAM) and the collapsing response-mediated protein 4 (TUC-4) was found in the plexiform layers of the host retina, but not in the grafted cells. The expression of the intermediate filaments GFAP, vimentin, and nestin was intensely upregulated immediately after transplantation. A less pronounced upregulation was observed on sham-operated animals. In summary, the RN33B cell line migrated promptly posttransplantation and settled preferably into the plexiform layers of the retina, the same layers where the migration cues PSA-NCAM and TUC-4 were established. In addition, both the transplantation method per se and the implanted cells caused an intense glial cell response by the host retina.

Multipotent stem/progenitor cells derived from human first trimester forebrain can be expanded as free-floating aggregates, so called neurospheres. These cells can differentiate into neurons, astrocytes and oligodendrocytes. In vitro differentiation protocols normally yield γ-aminobutyric acid-immunoreactive neurons, whereas only few tyrosine hydroxylase (TH) expressing neurons are found. The present report describes conditions under which 4-10% of the cells in the culture become TH immunoreactive (ir) neurons within 24 h. Factors including acidic fibroblast growth factor (aFGF) in combination with agents that increase intracellular cyclic AMP and activate protein kinase C, in addition to a substrate that promotes neuronal differentiation appear critical for efficient TH induction. The cells remain THir after trypsinization and replating, even when their subsequent culturing takes place in the absence of inducing factors. Consistent with a dopaminergic phenotype, mRNAs encoding aromatic acid decarboxylase, but not dopamine-β-hydroxylase were detected by quantitative real time RT-PCR. Ten weeks after the cells had been grafted into the striatum of adult rats with unilateral nigrostriatal lesions, only very few of the surviving human neurons expressed TH. Our data suggest that a significant proportion of expandable human neural progenitors can differentiate into TH-expressing cells in vitro and that they could be useful for drug and gene discovery. Additional experiments, however, are required to improve the survival and phenotypic stability of these cells before they can be considered useful for cell replacement therapy in Parkinson's disease. 

Peripheral nerve injuries are difficult to treat, and the clinical outcome after surgical repair and reconstruction is still insufficient, particularly concerning recovery of sensory function. To improve the clinical treatment strategies, experimental models are used to systematically examine the mechanisms behind nerve regeneration and assess the improvement of nerve regeneration by introduction of new surgical nerve repair and reconstruction methods (e.g., novel devices made by bioartificial materials). Rat models, where the sciatic nerve has essentially a similar size as a human digital nerve, are widely used to evaluate nerve regeneration with the inherent advantages and disadvantages of the experimental models. Estimations revealing that a large number of diabetic patients will eventually suffer from peripheral nerve injury have motivated development of suitable experimental diabetes models for studying the nerve regeneration process and novel treatment approaches. We have successfully used the Goto-Kakizaki rat model, which shows moderately increased blood sugar closely resembling type 2 diabetes, for assessing the surgical peripheral nerve regeneration potential with and without artificial scaffolds. In order to improve outcome after repair and reconstruction of nerve injuries, one has to have a clear concept concerning how to evaluate novel repair and reconstruction techniques in experimental models before clinical studies can be initiated in an accurate way. 

Forskning om stamceller har fått mycket uppmärksamhet de senaste åren. Mycket av intresset avspeglar möjligheten att använda denna typ av celler som en terapi för att bota olika sjukdomar. En stam cell från hjärnan är en cell som har förmåga att dela sig symmetriskt, varvid två kopior bildas eller asymmetriskt, vilket ger upphov till en ny stam cell samt en progenitorcell. En progenitorcell har ett förutbestämt öde, att utvecklas till en nervcell eller en stödjecell (gliacell), samt förmågan att dela sig under en kortare period. Stamceller som har isolerats från den embryonala och vuxna hjärnan, kan odlas i cellkultur under specifika betingelser. Egenskaperna hos de isolerade stam- och progenitorcellerna för nerv- och gliaceller (tillsammans kallade neurala celler) har studerats främst i cellodling (in vitro) och efter transplantation (in vivo), och resultaten har varit betydelsefulla för kartläggandet av neurobiologiska förlopp under utvecklingen. Då neurala stam-och progenitor celler kan mångfaldigas och modifieras i cellodling betraktas de också som en möjlig celltyp för framtida klinisk tillämpning med celltransplantation, till exempel för patienter med Parkinsons sjukdom. En eventuell användning av stamceller i denna typ av applikation kräver grundläggande experimentella försök för att dokumentera cellernas egenskaper och framförallt förmåga att funktionellt integrera med värdhjärnan efter transplantation.

Mitt avhandlingsarbete handlar om karakterisering av sådana embryonala neurala stam- och progenitor celler efter transplantation till hjärnan på råtta. Ett antal olika metoder användes för att detektera cellerna efter implantationen, bland annat reporter genen Green Fluorescent Protein (GFP). I det första delarbetet optimerades metoden för inmärkning av neurala celler med reporter genen, och då primärt i syfte att uttryckas efter transplantation. Då GFP är distribuerad i hela cytoplasman, visualiseras hela morfologin av cellerna. I de två följande delarbetena beskrivs hur humana neurala cell linjer etablerade från embryonal vävnad, kan odlas som så kallade neurosfärer och expanderas upp till ett år i närvaro av mitogena tillväxtfaktorer, med bibehållen förmåga att mogna till nerv- och gliaceller. Dessa cell linjer överlever väl upp till över ett år efter transplantation till hjärnan på nyfödda och vuxna råttor. Cellerna analyserades in vivo med human-specifika generella och phenotypiska markörer, samt med hjälp av reporter genen GFP. Efter transplantation till de neurogena områden, d v s den subventrikulära zonen och hippocampus, migrerar de humana cellerna och differentierar till region-specifika neuron likt värdhjärnans nervcells-progenitorer. Efter implantation i det icke-neurogena striatum migrerar cellerna, identifierade som gliaprogenitorer, över långa distanser i vit vävnad. I striatum utvecklades en signifikant del av de transplanterade cellerna till nervceller, med projicerar till korrekta målområden. En del av de transplanterade humana cellerna mognade till astrocyter och oligodendrocyter, vilket innebär att de humana celllinjerna är multipotenta även in vivo. GFP-positiva nervceller hade morfologiska egenskaper som är karakteristiska för mogna nervceller, såsom rikt förgrenade dendriter med spines. Trots kapacitet att migrera och differentiera till nerv och- gliaceller efter transplantation, kvarstår frågan till vilken grad de humana progenitorerna kan integrera funktionellt med värdhjärnan.

I de två sista delarbetena studerades den immortaliserade neurala cellinjen RN33B, som är genererad från embryonala hjärnstamsceller i råtta. Den temperaturkänsliga immortaliseringsgenen gör att cellerna delar sig vid 33°C i cell odling, men vid 37°C mognar cellerna och då främst till nervceller in vitro. RN33B- cellerna märktes med reporter genen GFP (se ovan) in vitro, och sedan studerades cellernas kapacitet att utvecklas till regionspecifika nervceller samt att integrera funktionellt med värdhjärnan studerades morfologiskt efter transplantation till hjärnan på nyfödda råttor. RN33B cellerna överlevde väl upp till 4 månader efter transplantationen och bildade både nervceller och gliaceller. Vidare visade den morfologiska analysen att RN33B-cellerna har en exceptionell förmåga att bilda pyramidala nervceller i cortex och hippocampus, med projektioner till korrekta målområden. Projektionerna studerades genom injektioner av Fluorogold (FG), som transporteras från projektionerna i målområdet till cellkroppen. Slutligen visade vi, med hjälp av så kallad patch-clamp teknik, att transplanterade GFP-positiva RN33B pyramidceller i cortex har normala elektrofysiologiska egenskaper samt tar emot information från omkringliggande celler i värdhjärnan. Sammanfattningsvis visar studierna i denna avhandling att neurala stamceller överlever transplantation, och utmognar till nerv- och gliaceller. Vidare så utvecklade nervcellerna projektioner som nådde till korrekta målområden, samt integrerade funktionellt med värdhjärnan. Dessa egenskaper i kombination med möjligheten att mångfaldiga de neurala celltyperna in vitro är mycket intressanta för fortsatta studier rörande funktionell integration efter transplantation och utveckling av celler för transplantation i djurmodeller av neurodegenerativa sjukdomar.

The ability to isolate neural stem and precursor cells and expand them in culture has provided researchers a new tool, not only assisting studies of neural development but also providing a new source of defined and expandable cells for in vivo studies using transplantation. The purposes of this chapter are, first, to review available protocols for in vitro expansion of neural precursor cells, either epigenetically using growth factors or genetically by inserting immortalizing genes; and, second, to discuss the in vivo properties of in vitro-propagated neural stem and progenitor cells, as assessed by grafting to the developing or adult rodent brain. This discussion will focus on our own recent studies using growth factor-expanded neurosphere cells of mouse and human origin and a particularly interesting, conditionally immortalized neural cell line, RN33B.

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.

The pig is becoming an increasingly used non-primate model in experimental studies of human retinal diseases and disorders. The anatomy, size, and vasculature of the porcine eye and retina closely resemble their human counterparts, which allows for application of standard instrumentation and diagnostics used in the clinic. Despite many reports that demonstrate immunohistochemistry as a useful method for exploring neuropathological changes in the mammalian central nervous system, including the pig, the porcine retina has been sparsely described. Hence, to facilitate further immunohistochemical analysis of the porcine retina, we report on the successful use of a battery of antibodies for staining of paraformaldehyde-fixed cryosectioned retina. The following antibodies were evaluated for neuronal cells and structures: recoverin (cones and rods), Rho4D2 (rods), transducin-γ (cones), ROM-1 (photoreceptor outer segments), calbindin (horizontal cells), PKC-α (bipolar cells), parvalbumin (amacrine and displaced amacrine cells), and NeuN (ganglion cells and displaced amacrines). For detecting synaptic connections in fiber layers, we used an antibody against synaptobrevin. For detecting retinal pigment epithelium, we studied antibodies against cytokeratin and RPE65, respectively. The glial cell markers used were bFGF (Müller cells and displaced amacrine cells), GFAP (Müller cells and astrocytes), and vimentin (Müller cells). Each staining effect was evaluated with regard to its specificity, sensitivity, and reproducibility in the identification of individual cells, specific cell structures, and fiber layers, respectively. The markers parvalbumin and ROM-1 were tested here for the first time for the porcine retina. All antibodies tested resulted in specific staining of high quality. In conclusion, all immunohistochemical protocols presented here will be applicable in fixed, cryosectioned pig retina.

A major obstacle in ex vivo gene transfer has been the loss of transgene expression soon after implantation of the grafted transduced cells. Recently, a lentiviral vector system has been developed which has proven to express high levels of transgenes in vivo after direct injection into the tissue. In this study, we have investigated the use of such a vector for ex vivo gene transfer to the brain. A number of neural cell types were found to be permissive to transduction by the lentiviral vector in vitro and a majority of them expressed the transgene after transplantation to the rat brain. Transgene expression was detected up to 8 weeks post-grafting. These findings suggest that recombinant lentiviral vectors may be used for further development of ex vivo gene therapy protocols to the CNS.

Here we examined the ability of human neural progenitors from the embryonic forebrain, expanded for up to a year in culture in the presence of growth factors, to respond to environmental signals provided by the developing rat brain. After survival times of up to more than a year after transplantation into the striatum, the hippocampus, and the subventricular zone, the cells were analyzed using human-specific antisera and the reporter gene green fluorescent protein (GFP). From grafts implanted in the striatum, the cells migrated extensively, especially within white matter structures. Neuronal differentiation was most pronounced at the striatal graft core, with axonal projections extending caudally along the internal capsule into mesencephalon. In the hippocampus, cells migrated throughout the entire hippocampal formation and into adjacent white matter tracts, with differentiation into neurons both in the dentate gyrus and in the CA1–3 regions. Directed migration along the rostral migratory stream to the olfactory bulb and differentiation into granule cells were observed after implantation into the subventricular zone. Glial differentiation occurred at all three graft sites, predominantly at the injection sites, but also among the migrating cells. A lentiviral vector was used to transduce the cells with the GFP gene prior to grafting. The reporter gene was expressed for at least 15 weeks and the distribution of the gene product throughout the entire cytoplasmic compartment of the expressing cells allowed for a detailed morphological analysis of a portion of the grafted cells. The extensive integration and differentiation of in vitro-expanded human neural progenitor cells indicate that multipotent progenitors are capable of responding in a regionally specific manner to cues present in the developing rat brain.

Different types of progenitor and stem cells have been shown to provide neuroprotection in animal models of photoreceptor degeneration. The present study was conducted to investigate whether human neural progenitor cells (HNPCs) have neuroprotective properties on retinal explants models with calpain- and caspase-3-dependent photoreceptor cell death. In the first experiments, HNPCs in a feeder layer were co-cultured for 6 days either with postnatal rd1 mouse or normal rat retinas. Retinal histological sections were used to determine outer nuclear layer (ONL) thickness, and to detect the number of photoreceptors with labeling for calpain activity, cleaved caspase-3 and TUNEL. The ONL thickness of co-cultured rat and rd1 retinas was found to be almost 10% and 40% thicker, respectively, compared to controls. Cell counts of calpain activity, cleaved caspase-3 and TUNEL labeled photoreceptors in both models revealed a 30-50% decrease when co-cultured with HNPCs. The results represent significant increases of photoreceptor survival in the co-cultured retinas. In the second experiments, for an identification of putative survival factors, or a combination of them, a growth factor profile was performed on conditioned medium. The relative levels of various growth factors were analyzed by densitometric measurements of growth factor array membranes. Following growth factors were identified as most potential survival factors; granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), insulin-like growth factor II (IGF-II), neurotrophic factor 3 (NT-3), placental growth factor (PIGF), transforming growth factors (TGF-beta1 and TGF-beta2) and vascular endothelial growth factor (VEGF-D). HNPCs protect both against calpain- and caspase-3-dependent photoreceptor cell death in the rd1 mouse and against caspase-3-dependent photoreceptor cell death in normal rat retinas in vitro. The protective effect is possibly achieved by a variety of growth factors secreted from the HNPCs.

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.

Electrospinning can be used to mimic the architecture of an acellular nerve graft, combining microfibers for guidance, and pores for cellular infiltration. We made electrospun nerve guides, from polycaprolactone (PCL) or poly-L-lactic acid (PLLA), with aligned fibers along the insides of the channels and random fibers around them. We bridged a 10 mm rat sciatic nerve defect with the guides, and, in selected groups, added a cell transplant derived from autologous stromal vascular fraction (SVF). For control, we compared to hollow silicone tubes; or autologous nerve grafts. PCL nerve guides had a high degree of autotomy (8/43 rats), a negative indicator with respect to future usefulness, while PLLA supported axonal regeneration, but did not outperform autologous nerve grafts. Transplanted cells survived in the PLLA nerve guides, but axonal regeneration was not enhanced as compared to nerve guides alone. The inflammatory response was partially enhanced by the transplanted cells in PLLA nerve grafts; Schwann cells were poorly distributed compared to nerve guide without cells. Tailor-made electrospun nerve guides support axonal regeneration in vivo, and can act as vehicles for co-transplanted cells. Our results motivate further studies exploring novel nerve guides and the effect of stromal cell-derived factors on nerve generation.

New nanomaterials are constantly developed with applications in everything from cosmetics to high tech electronics. Assessing their biological impact has been done by analysis of their adsorbed protein corona, in vitro cell assays, and larger scale ecotoxicological studies. This has proved to be a huge challenge due to the wide range of available nanomaterials and their unpredictable behaviour in different environments. Furthermore, the enormous number of experimental variables make comparisons difficult. Concentration is one of these variables and can vary greatly depending on the aim of the study. When analysing the protein corona, concentrations are often higher than in cell assays. Using a combination of complementary techniques, we have characterised 20 nm gold nanoparticles in a concentration level commonly used in cell studies. We compare their behaviour in a commonly used, protein rich medium and one protein poor medium over 24 hours. Under these conditions, the NPs were stable in protein rich environment but underwent gradual aggregation in protein poor medium. We characterise the biomolecular corona in both media. In protein poor medium, we can describe the often overlooked aggregation. The aggregates’ morphology is confirmed by cryo-TEM. Finally, in the protein poor medium, by infrared spectroscopy, we have identified the amino acid arginine in the biomolecular corona which drives the aggregation.

Retinal ischemia arises from circulatory failure. As the retinal blood vessels are key organs in circulatory failure, our aim was to study the retinal vasculature separately from the neuroretina to elucidate the role of hypoxia-inducible factor (HIF) 1α and 1β and vascular endothelial growth factor (VEGF) in retinal ischemia. Retinal ischemia was induced in porcine eyes by applying an intraocular pressure, followed by 12 h of reperfusion. HIF-1α mRNA expression was not affected by ischemia, while immunofluorescence staining was higher after ischemia in the neuroretina. HIF-1β immu-noreactivity and mRNA expression were unaffected. VEGF protein levels in the vitreous humor and VEGF staining in the neuroretina were more pronounced in eyes subjected to ischemia than in the sham eyes. VEGF may be activated downstream of HIF-1 and is known to stimulate retinal neovascularization, which causes sight-threatening complications. These results emphasize the need for pharmacological treatment to block the HIF and VEGF signaling pathways in retinal ischemia. 

Three-dimensional neuronal culture systems such as spheroids, organoids, and assembloids constitute a branch of neuronal tissue engineering that has improved our ability to model the human brain in the laboratory. However, the more elaborate the brain model, the more difficult it becomes to study functional properties such as electrical activity at the neuronal level, similar to the challenges of studying neurophysiology in vivo. We describe a simple approach to generate self-assembled three-dimensional neuronal spheroid networks with defined human cell composition on microelectrode arrays. Such spheroid networks develop a highly three-dimensional morphology with cell clusters up to 60 μm in thickness and are interconnected by pronounced bundles of neuronal fibers and glial processes. We could reliably record from up to hundreds of neurons simultaneously per culture for ≤90 d. By quantifying the formation of these three-dimensional structures over time, while regularly monitoring electrical activity, we were able to establish a strong link between spheroid morphology and network activity. In particular, the formation of cell clusters accelerates formation and maturation of correlated network activity. Astrocytes both influence electrophysiological network activity as well as accelerate the transition from single cell layers to cluster formation. Higher concentrations of astrocytes also have a strong effect of modulating synchronized network activity. This approach thus represents a practical alternative to often complex and heterogeneous organoids, providing easy access to activity within a brain-like 3D environment.

We demonstrate an artificial three-dimensional (3D) electrical active human neuronal network system, by the growth of brain neural progenitors in highly porous low density electrospun poly-ε-caprolactone (PCL) fiber scaffolds. In neuroscience research cell-based assays are important experimental instruments for studying neuronal function in health and disease. Traditional cell culture at 2D-surfaces induces abnormal cell–cell contacts and network formation. Hence, there is a tremendous need to explore in vivo-resembling 3D neural cell culture approaches. We present an improved electrospinning method for fabrication of scaffolds that promote neuronal differentiation into highly 3D integrated networks, formation of inhibitory and excitatory synapses and extensive neurite growth. Notably, in 3D scaffolds in vivo-resembling intermixed neuronal and glial cell network were formed, whereas in parallel 2D cultures a neuronal cell layer grew separated from an underlying glial cell layer. Hence, the use of the 3D cell assay presented will most likely provide more physiological relevant results.

Objectives. To study possible nerve regeneration of a damaged auditory nerve by the use of stem cell transplantation. Methods. We transplanted HNPCs to the rat AN trunk by the internal auditory meatus (IAM). Furthermore, we studied if addition of BDNF affects survival and phenotypic differentiation of the grafted HNPCs. A bioactive nanofiber gel (PA gel), in selected groups mixed with BDNF, was applied close to the implanted cells. Before transplantation, all rats had been deafened by a round window niche application of β-bungarotoxin. This neurotoxin causes a selective toxic destruction of the AN while keeping the hair cells intact. Results. Overall, HNPCs survived well for up to six weeks in all groups. However, transplants receiving the BDNF-containing PA gel demonstrated significantly higher numbers of HNPCs and neuronal differentiation. At six weeks, a majority of the HNPCs had migrated into the brain stem and differentiated. Differentiated human cells as well as neurites were observed in the vicinity of the cochlear nucleus. Conclusion. Our results indicate that human neural precursor cells (HNPC) integration with host tissue benefits from additional brain derived neurotrophic factor (BDNF) treatment and that these cells appear to be good candidates for further regenerative studies on the auditory nerve (AN).

Current putative regeneration oriented studies express possible role of stem cell based implantation strategy in the restoration of fundamental perception of hearing. The present work utilizes arat auditory nerve (AN) directed transplantation of human neural progenitor cells (HNPCs) as acell replacement therapy for impaired auditory function. Groups of β-bungarotoxin induced auditory function compromised female rats were used to transplant HNPCs in the nerve trunk. In thetreatment groups, brain derived neurotrophic factor (BDNF), peptide amphiphile nanofiber bioactive gel (Bgel) and Chondroitinase ABC (ChABC), a digestive enzyme that cleaves the core of chondroitin sulphate proteoglycans, were added along with HNPCs while the control groups were withPA inert gel (Igel) and devoid of ChABC. Six weeks post transplantation survival, migration, and differentiation of HNPCs were studied and compared. The groups treated with BDNF and Bgelshowed improved survival and differentiation of transplanted HNPCs while the ChABC treatedgroup showed significant migration of HNPCs along the AN and elongation of neuronal fibers alongthe nerve towards the cochlear nucleus (CN) which was characterized by immunocytochemicalmarkers for human Nuclei (HuN), human mitochondria (HuM) and neuronal β-tubulin (Tuj1).These findings show that addition of BDNF and ChABC consisted Bgel environment facilitatedHNPC survival, migration and differentiation along the transplanted rat AN towards the CN. Thistransplantation strategy provides unique experimental validation for futuristic role of cell basedbiomaterial consisted neurotrophic factor application in clinically transferable treatment of sensorineural hearing loss (SNHL) along with cochlear implants (CI).

This study investigates whether intravitreal administration of glial cell line-derived neurotrophic factor (GDNF) enhances survival of NeuN positive retinal cells in a porcine model of retinal ischemia. 16 pigs were subjected to an ischemic insult where intraocular pressure was maintained at 5 mmHg below mean arterial blood pressure for 2 h. The mean IOP during the ischemic insult was 79.5 mmHg (s.e.m. 2.1 mmHg, n = 15). Three days after the insult the pigs received an intravitreal injection of GDNF microspheres or blank microspheres. The pigs were evaluated by way of multifocal electroretinography (mfERG), quantification of NeuN positive cells and evaluation of the degree of retinal perivasculitis and inflammation 6 weeks after the insult. In the post-injection eyes (days 14, 28 and 42), the ratios of the iN1 and the iP2 amplitudes were 0.10 (95% CI: 0.05-0.15) and 0.09 (95% CI: 0.04-0.16) in eyes treated with blank microspheres, and 0.24 (95% CI: 0.18-0.32) and 0.23 (95% CI: 0.15-0.33) in eyes treated with GDNF microspheres. These differences were statistically significant (P < 0.05). The number of NeuN positive cells in the area of the visual streak area was significantly higher in eyes injected with GDNF microspheres compared to eyes injected with blank microspheres. In eyes injected with GDNF microspheres the ganglion cell count was 9.5/field (s.e.m.: 2.1, n = 8), in eyes injected with blank microspheres it was 3.5/field (s.e.m.: 1.2, n = 7). This difference was statistically significant (P < 0.05). There was also a significant difference (P < 0.01) in the degree of perivasculiitis between GDNF treated eyes (median perivasculitis score 1.5) and blank treated eyes (median perivasculitis score 3.0). In conclusion, injection of GDNF microspheres 3 days after an ischemic insult results in functional and morphological rescue of NeuN positive cells in a porcine model of acute ocular ischemia.

The rat neural cell line RN33B has a remarkable ability to undergo region-specific neuronal differentiation after transplantation into the CNS. To further study its neurogenic properties in vivo, we used a recombinant lentiviral vector to genetically label the cells with the Green Fluorescent Protein (GFP) gene before implantation into the striatum/cortex, hippocampus, or mesencephalon of newborn rats. Three weeks after implantation, about 1-2% of the GFP-expressing cells had developed morphologies typical of neurons, astrocytes, or oligodendrocytes, the rest remained as either immature or undifferentiated nestin-positive cells. At 15-17 weeks postgrafting, the immature cells had disappeared in most graft recipients and only cells with neuronal or glial morphologies remained in similar numbers as at 3 weeks. The GFP distributed throughout the expressing cells, revealing fine morphological details, including dendrites with spines and extensive axonal projections. In all forebrain regions, the grafted cells differentiated into neurons with morphologies characteristic for each site, including large numbers of pyramidal-like cells in the cortex and the hippocampus, giving rise to dense projections to normal cortical target regions and to the contralateral hippocampus, respectively. In lower numbers, it was also possible to identify GFP-positive granulelike cells in the hippocampus, as well as densely spiny neurons in the striatum. In the mesencephalon by contrast, cells with astrocytic features predominated. The ability of the grafted RN33B cells to undergo region-specific differentiation into highly specialized types of forebrain projection neurons and establish connections with appropriate targets suggests that cues present in the microenvironment of the neonatal rat brain can effectively guide the development of immature progenitors, also in the absence of ongoing neurogenesis.

Objective - Disturbances in hippocampal neurogenesis may be involved in the pathophysiology of depression and it has been argued that an increase in the generation of new nerve cells in the hippocampus is involved in the mechanism of action of antidepressants.

Materials and Methods - Adult Wistar rats were treated with fluoxetine (10 mg/kg) 1 h, daily for 5 (subchronic) or 28 days (chronic) before the Novelty Suppressed Feeding test was performed. Cell proliferation and neurogenesis were analysed using the markers 5-bromo-deoxy-2′-uridine, Ki-67, and doublecortin.

Results - A significant behavioural effect was found after 28 days of fluoxetine administration. However, no behavioural improvement was demonstrated after acute and subchronic treatment with fluoxetine. We further demonstrate that chronic antidepressant treatment increases cell proliferation as well as neurogenesis in the dentate gyrus, here using Wistar rats.

Conclusions - In further development of antidepressants, neurogenesis may serve as an important parameter to examine the efficacy and mechanism of action of novel drugs.

Hearing function lost by degeneration of inner ear spiral ganglion neurons (SGNs) in the auditory nervous system could potentially be compensated by cellular replacement using suitable donor cells. Donor cell-derived neuronal development with functional synaptic formation with auditory neurons of the cochlear nucleus (CN) in the brainstem is a prerequisite for a successful transplantation. Here a rat auditory brainstem explant culture system was used as a screening platform for donor cells. The explants were co-cultured with human neural precursor cells (HNPCs) to determine HNPCs developmental potential in the presence of environmental cues characteristic for the auditory brainstem region in vitro. We explored effects of pharmacological inhibition of GTPase Rho with its effector Rho-associated kinase (ROCK) and epidermal growth factor receptor (EGFR) signaling on the co-cultures. Pharmacological agents ROCK inhibitor Y27632 and EGFR blocker PD168393 were tested. Effect of the treatment on explant penetration by green fluorescent protein (GFP)-labeled HNPCs was evaluated based on the following criteria: number of GFP-HNPCs located within the explant; distance migrated by the GFP-HNPCs deep into the explant; length of the GFP+/neuronal class III β-tubulin (TUJ1)+ processes developed and phenotypes displayed. In a short 2-week co-culture both inhibitors had growth-promoting effects on HNPCs, prominent in neurite extension elongation. Significant enhancement of migration and in-growth of HNPCs into the brain slice tissue was only observed in Y27632-treated co-cultures. Difference between Y27632- and PD168393-treated HNPCs acquiring neuronal fate was significant, though not different from the fates acquired in control co-culture. Our data suggest the presence of inhibitory mechanisms in the graft–host environment of the auditory brainstem slice co-culture system with neurite growth arresting properties which can be modulated by administration of signaling pathways antagonists. Therefore the co-culture system can be utilized for screens of donor cells and compounds regulating neuronal fate determination.

Congenital or acquired hearing loss is often associated with a progressive degeneration of the auditory nerve (AN) in the inner ear. The AN is composed of processes and axons of the bipolar spiral ganglion neurons (SGN), forming the connection between the hair cells in the inner ear cochlea and the cochlear nuclei (CN) in the brainstem (BS). Therefore, replacement of SGNs for restoring the AN to improve hearing function in patients who receive a cochlear implantation or have severe AN malfunctions is an attractive idea. A human neural precursor cell (HNPC) is an appropriate donor cell to investigate, as it can be isolated and expanded in vitro with maintained potential to form neurons and glia. We recently developed a post-natal rodent in vitro auditory BS slice culture model including the CN and the central part of the AN for initial studies of candidate cells. Here we characterized the survival, distribution, phenotypic differentiation, and integration capacity of HNPCs into the auditory circuitry in vitro. HNPC aggregates (spheres) were deposited adjacent to or on top of the BS slices or as a monoculture (control). The results demonstrate that co-cultured HNPCs compared to monocultures (1) survive better, (2) distribute over a larger area, (3) to a larger extent and in a shorter time-frame form mature neuronal and glial phenotypes. HNPC showed the ability to extend neurites into host tissue. Our findings suggest that the HNPC-BS slice co-culture is appropriate for further investigations on the integration capacity of HNPCs into the auditory circuitry.

Neural stem and progenitor cells can be expanded under growth factor stimulation in vitro. It is likely that different mitogens and different culturing techniques selectively expand specific subclasses of cells, but this selection has not been well studied. We have expanded human cells isolated from the lateral ganglionic eminence (LGE) of a 10-week-old embryo in the presence of serum and epidermal growth factor. We provide evidence that culturing in this manner favors expansion of cells with characteristics similar to a subpopulation of LGE cells, the olfactory bulb progenitors, as revealed by their expression of Er81 in vitro. After transplantation into neonatal rats, the cells displayed similar properties to endogenous olfactory bulb progenitors when exposed to local cues present in the subventricular zone (SVZ) and rostral migratory stream (RMS). However, the human LGE cells do not migrate or undergo region-specific differentiation when placed outside the SVZ and RMS.

The risk of Parkinson's disease (PD) is associated with a lower intake of caffeine, a non-selective adenosine A2A antagonist. In agreement, genetic or pharmacological inactivation of adenosine A2A receptors in animal models of PD has demonstrated both symptomatic and neuroprotective effects. These findings and the lack of disease modifying therapies have led to intense research on adenosine A2A antagonists as a novel treatment for PD. In the present study the neuroprotective effect of the A2A receptor antagonist KW-6002 was investigated using different models of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice, which induced dopaminergic terminal and or dopaminergic cell loss and inflammation. Treatment with KW-6002 prevented the loss of dopaminergic striatal terminals and nigral cell bodies and inhibited the nigral microglia activation. Our results confirm previous findings that pharmacological inactivation of A2A receptors inhibits MPTP-induced dopaminergic damage at the level of striatum. In addition, we demonstrate for the first time that, after MPTP treatment in mice, an A 2A antagonist is neuroprotective, and has anti-inflammatory effects, at the level of the substantia nigra. Thus, our data further support the use of A2A receptor antagonists as a novel neuroprotective therapy for PD

The complex network of neuronal cells in the retina makes it a potential target of neuronal toxicity - a risk factor for visual loss. With growing use of nanoparticles (NPs) in commercial and medical applications, including ophthalmology, there is a need for reliable models for early prediction of NP toxicity in the eye and retina. Metal NPs, such as gold and silver, gain much of attention in the ophthalmology community due to their potential to cross the barriers of the eye. Here, NP uptake and signs of toxicity were investigated after exposure to 20 and 80 nm Ag- and AuNPs, using an in vitro tissue culture model of the mouse retina. The model offers long-term preservation of retinal cell types, numbers and morphology and is a controlled system for delivery of NPs, using serum-free defined culture medium. AgNO3-treatment was used as control for toxicity caused by silver ions. These end-points were studied; gross morphological organization, glial activity, microglial activity, level of apoptosis and oxidative stress, which are all well described as signs of insult to neural tissue. TEM analysis demonstrated cellular- and nuclear uptake of all NP types in all neuronal layers of the retina. Htx-eosin staining showed morphological disruption of the normal complex layered retinal structure, vacuole formation and pyknotic cells after exposure to all Ag- and AuNPs. Significantly higher numbers of apoptotic cells as well as an increased number of oxidative stressed cells demonstrated NP-related neuronal toxicity. NPs also caused increased glial staining and microglial cell activation, typical hallmarks of neural tissue insult. This study demonstrates that low concentrations of 20 and 80 nm sized Ag- and AuNPs have adverse effects on the retina, using an organotypic retina culture model. Our results motivate careful assessment of candidate NP, metallic or-non-metallic, to be used in neural systems for therapeutic approaches.

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.

The ability of in vitro-expanded neural precursor cells or cell lines to differentiate following transplantation has significant implications for current research on central nervous system repair. Recently, interest has been focussed on grafts of such neural precursors implanted also into the eye or retina. Here, we demonstrate with a non-traumatizing subretinal transplantation method, that grafts of the two immortalized brain-derived cell lines C 17-2 (from postnatal mouse cerebellum) and RN33B (from the embryonic rat medullary raphe) survive for at least up to four weeks, after implantation into the adult normal rat retina. For both cell lines, implanted cells gradually integrate into all major retinal cell layers, including the retinal pigment epithelium, and judged by the morphology differentiate into both glial- and neuron-like cells, as shown by thymidine autoradiography, mouse-specific in situ hybridization, and using immunohistochemistry to detect the reporter gene LacZ. Our results suggest that these and other similar neural cell lines could be very useful in the continuos experiments in models of retinal disorders to further assess both the cell replacement and ex vivo gene therapy approaches.

The potential use of in vitro-expanded precursor cells or cell lines in repair includes transplantation of such cells for cell replacement purposes and the activation of host cells to provide "self-repair." Recently, we have reported that cells from the brain-derived cell line RN33B (derived from the embryonic rat medullary raphe and immortalized through retroviral transduction of the temperature-sensitive mutant of the simian virus 40 ([SV40] large T-antigen) survive for at least 4 weeks, integrate, and differentiate after subretinal grafting to normal adult rats. Here, we demonstrate that grafts of these cells survive for at least 4 months after subretinal transplantation to adult, normal immunosuppressed rats. Implanted cells integrate into the retinal pigment epithelium and the inner retinal layers, and the anterior part of the optic nerve. In addition, the RN33B cells migrate within the retina, occupying the whole retina from one eccentricity to the other. A large fraction of the grafted cells differentiate into glial cells, as shown by double labeling of the reporter genes LacZ or green fluorescent protein, and several glial markers, including oligodendrocytes. However, the cells did not differentiate into retinal neurons, judging from their lack of expression of retinal neuronal phenotypic markers. A significant number of the implanted cells in the host retina were in a proliferative stage, judging from proliferative cell nuclear antigen and SV40 large T-antigen immunohistochemistry. To conclude, the cells survived, integrated, and migrated over long distances within the host. Therefore, our results may be advantageous for future design of therapeutic strategies, since such cells may have the potential of being a source of, for example, growth factor delivery in experimental models of retinal degeneration.

Neural precursor cells transplanted to adult retina can integrate into the host. This is especially true when the neural precursor rat cell line RN33B is used. This cell line carries the reporter genes LacZ and green fluorescent protein (GFP). In grafted rat eyes, RN33B cells are localized from one eccentricity to the other of the host retina. In the present study, whole-mounted retinas were analyzed to obtain a more appropriate evaluation of the amount of transgene-expressing cells and the migratory capacity of these cells 3 and 8 weeks posttransplantation. Quantification was made of the number of β-galactosidase- and GFP-expressing cells with a semiautomatized stereological cell counting system. With the same system, delineation of the distribution area of the grafted cells was also performed. At 3 weeks, 68% of the grafted eyes contained marker-expressing cells, whereas at 8 weeks only 35% of the eyes contained such cells. Counting of marker-expressing cells demonstrated a lower number of transgene-expressing cells at 3 weeks compared with 8 weeks post-transplantation. The distribution pattern of marker gene-expressing cells revealed cells occupying up to 21% at 3 weeks and up to 68% at 8 weeks of the entire host retina postgrafting. The precursor cells survived well in the adult retina although the most striking feature of the RN33B cell line was its extraordinary migratory capacity. This capability could be useful if precursor cells are used to deliver necessary genes or gene products that need to be distributed over a large diseased area.

The potential use of in vitro-expanded precursor cells or cell lines in brain repair includes transplantation of such cells for cell replacement purposes and the activation of host cells to provide 'self-repair'. Recently, it has been reported that the immortalized brain-derived cell line RN33B (derived from the embryonic rat medullary raphe) survive, integrate and differentiate after subretinal grafting to normal adult rats. Here, it is demonstrated that grafts of these cells survive for at least 6 weeks after implantation into postnatal days 21 and 35 retinas of normal and Royal College of Surgeons rats, a model of retinal degeneration. Implanted cells integrate into the retinal pigment epithelium and the inner retinal layers, and the anterior part of the optic nerve of both normal and Royal College of Surgeons rats. The RN33B cells migrate within the retina, occupying the whole retina from one eccentricity to the other. A significant number of the grafted cells differentiate into glial cells, as shown by the double labelling of the reporter genes LacZ or green fluorescent protein, with several glial markers, including oligodendrocytic markers. Many implanted cells in the host retina were in a proliferative stage judging from proliferative cell nuclear antigen and SV40 large T-antigen immunohistochemistry. Interestingly, there was a promotion of photoreceptor survival, extending over more than 2/3 of the superior hemisphere, in Royal College of Surgeons rats transplanted at postnatal day 21, but not at postnatal day 35. In addition, grafted cells were found in the surviving photoreceptor layer in these rats.

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.

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.