Experimental Study Using Multiple Strains of Prion Disease in Cattle Reveals an Inverse Relationship between Incubation Time and Misfolded Prion Accumulation, Neuroinflammation, and Autophagy.

Proteinopathies result from aberrant folding and accumulation of specific proteins. Currently, there is a lack of knowledge about the factors that influence disease progression, making this a key challenge for the development of therapies for proteinopathies. Because of the similarities between transmissible spongiform encephalopathies (TSEs) and other protein misfolding diseases, TSEs can be used to understand other proteinopathies. Bovine spongiform encephalopathy (BSE) is a TSE that occurs in cattle and can be subdivided into three strains: classic BSE and atypical BSEs (H and L types) that have shorter incubation periods. The NACHT, LRR, and PYD domains-containing protein 3 inflammasome is a critical component of the innate immune system that leads to release of IL-1ß. Macroautophagy is an intracellular mechanism that plays an essential role in protein clearance. In this study, the retina was used as a model to investigate the relationship between disease incubation period, prion protein accumulation, neuroinflammation, and changes in macroautophagy. We demonstrate that atypical BSEs present with increased prion protein accumulation, neuroinflammation, and decreased autophagy. This work suggests a relationship between disease time course, neuroinflammation, and the autophagic stress response, and may help identify novel therapeutic biomarkers that can delay or prevent the progression of proteinopathies.

Accelerated accumulation of retinal α-synuclein (pSer129) and tau, neuroinflammation, and autophagic dysregulation in a seeded mouse model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by accumulation of misfolded α-synuclein within the central nervous system (CNS). Visual problems in PD patients are common, although retinal pathology associated with PD is not well understood. The purpose of this study was to investigate retinal pathology in a transgenic mouse model (TgM83) expressing the human A53T α-synuclein mutation and assess the effect of α-synuclein "seeding" on the development of retinal pathology. Two-month-old TgM83 mice were intracerebrally inoculated with brain homogenate from old (12-18 months) TgM83 mice. Retinas were then analyzed at 5 months of age. We analyzed retinas from 5-month-old and 8-month-old uninoculated healthy TgM83 mice, and old (12-18 months) mice that were euthanized following the development of clinical signs. Retinas of B6C3H mice (genetic background of the TgM83 mouse) served as control. We used immunohistochemistry and western blot analysis to detect accumulation of α-synuclein, pTauThr231, inflammation, changes in macroautophagy, and cell death. Raman spectroscopy was used to test the potential to differentiate between retinal tissues of healthy mice and diseased mice. This work demonstrates retinal changes associated with the A53T mutation. Retinas of non-inoculated TgM83 mice had accumulation of α-synuclein, "pre-tangle" tau, activation of retinal glial cells, and photoreceptor cell loss by 8 months of age. The development of these changes is accelerated by inoculation with brain homogenate from clinically ill TgM83 mice. Compared to non-inoculated 5-month-old TgM83 mice, retinas of inoculated 5-month-old mice had increased accumulation of α-synuclein (pSer129) and pTauThr231 proteins, upregulated microglial activation, and dysregulated macroautophagy. Raman spectroscopic analysis was able to discriminate between healthy and diseased mice. This study describes retinal pathology resulting from the A53T mutation. We show that seeding with brain homogenates from old TgM83 mice accelerates retinal pathology. We demonstrate that Raman spectroscopy can be used to accurately identify a diseased retina based on its biochemical profile, and that α-synuclein accumulation may contribute to accumulation of pTauThr231 proteins, neuroinflammation, metabolic dysregulation, and photoreceptor cell death. Our work provides insight into retinal changes associated with Parkinson's disease, and may contribute to a better understanding of visual symptoms experienced by patients.

Lasting Retinal Injury in a Mouse Model of Blast-Induced Trauma.

Traumatic brain injury due to blast exposure is currently the most prevalent of war injuries. Although secondary ocular blast injuries due to flying debris are more common, primary ocular blast exposure resulting from blast wave pressure has been reported among survivors of explosions, but with limited understanding of the resulting retinal pathologies. Using a compressed air-driven shock tube system, adult male and female C57BL/6 mice were exposed to blast wave pressure of 300 kPa (43.5 psi) per day for 3 successive days, and euthanized 30 days after injury. We assessed retinal tissues using immunofluorescence for glial fibrillary acidic protein, microglia-specific proteins Iba1 and CD68, and phosphorylated tau (AT-270 pThr181 and AT-180 pThr231). Primary blast wave pressure resulted in activation of Müller glia, loss of photoreceptor cells, and an increase in phosphorylated tau in retinal neurons and glia. We found that 300-kPa blasts yielded no detectable cognitive or motor deficits, and no neurochemical or biochemical evidence of injury in the striatum or prefrontal cortex, respectively. These changes were detected 30 days after blast exposure, suggesting the possibility of long-lasting retinal injury and neuronal inflammation after primary blast exposure.

Adult Stem Cell-Based Strategies for Peripheral Nerve Regeneration.

Peripheral nerve injuries (PNI) occur as the result of sudden trauma and can lead to life-long disability, reduced quality of life, and heavy economic and social burdens. Although the peripheral nervous system (PNS) has the intrinsic capacity to regenerate and regrow axons to a certain extent, current treatments frequently show incomplete recovery with poor functional outcomes, particularly for large PNI. Many surgical procedures are available to halt the propagation of nerve damage, and the choice of a procedure depends on the extent of the injury. In particular, recovery from large PNI gaps is difficult to achieve without any therapeutic intervention or some form of tissue/cell-based therapy. Autologous nerve grafting, considered the "gold standard" is often implemented for treatment of gap formation type PNI. Although these surgical procedures provide many benefits, there are still considerable limitations associated with such procedures as donor site morbidity, neuroma formation, fascicle mismatch, and scarring. To overcome such restrictions, researchers have explored various avenues to improve post-surgical outcomes. The most commonly studied methods include: cell transplantation, growth factor delivery to stimulate regenerating axons and implanting nerve guidance conduits containing replacement cells at the site of injury. Replacement cells which offer maximum benefits for the treatment of PNI, are Schwann cells (SCs), which are the peripheral glial cells and in part responsible for clearing out debris from the site of injury. Additionally, they release growth factors to stimulate myelination and axonal regeneration. Both primary SCs and genetically modified SCs enhance nerve regeneration in animal models; however, there is no good source for extracting SCs and the only method to obtain SCs is by sacrificing a healthy nerve. To overcome such challenges, various cell types have been investigated and reported to enhance nerve regeneration.In this review, we have focused on cell-based strategies aimed to enhance peripheral nerve regeneration, in particular the use of mesenchymal stem cells (MSCs). Mesenchymal stem cells are preferred due to benefits such as autologous transplantation, routine isolation procedures, and paracrine and immunomodulatory properties. Mesenchymal stem cells have been transplanted at the site of injury either directly in their native form (undifferentiated) or in a SC-like form (transdifferentiated) and have been shown to significantly enhance nerve regeneration. In addition to transdifferentiated MSCs, some studies have also transplanted ex-vivo genetically modified MSCs that hypersecrete growth factors to improve neuroregeneration.

Polycaprolactone Microfibrous Scaffolds to Navigate Neural Stem Cells.

Fibrous scaffolds have shown promise in tissue engineering due to their ability to improve cell alignment and migration. In this paper, poly(ε-caprolactone) (PCL) fibers are fabricated in different sizes using a microfluidic platform. By using this approach, we demonstrated considerable flexibility in ability to control the size of the fibers. It was shown that the average diameter of the fibers was obtained in the range of 2.6-36.5 µm by selecting the PCL solution flow rate from 1 to 5 µL min-1 and the sheath flow rate from 20 to 400 µL min-1 in the microfluidic channel. The microfibers were used to create 3D microenvironments in order to investigate growth and differentiation of adult hippocampal stem/progenitor cells (AHPCs) in vitro. The results indicated that the 3D topography of the PCL substrates, along with chemical (extracellular matrix) guidance cues supported the adhesion, survival, and differentiation of the AHPCs. Additionally, it was found that the cell deviation angle for 44-66% of cells on different types of fibers was less than 10°. This reveals the functionality of PCL fibrous scaffolds for cell alignment important in applications such as reconnecting serious nerve injuries and guiding the direction of axon growth as well as regenerating blood vessels, tendons, and muscle tissue.

Enabling nanomaterial, nanofabrication and cellular technologies for nanoneuromedicines.

Nanoparticulate delivery systems represent an area of particular promise for nanoneuromedicines. They possess significant potential for desperately needed therapies designed to combat a range of disorders associated with aging. As such, the field was selected as the focus for the 2014 meeting of the American Society for Nanomedicine. Regenerative, protective, immune modulatory, anti-microbial and anti-inflammatory products, or imaging agents are readily encapsulated in or conjugated to nanoparticles and as such facilitate the delivery of drug payloads to specific action sites across the blood-brain barrier. Diagnostic imaging serves to precisely monitor disease onset and progression while neural stem cell replacement can regenerate damaged tissue through control of stem cell fates. These, taken together, can improve disease burden and limit systemic toxicities. Such enabling technologies serve to protect the nervous system against a broad range of degenerative, traumatic, metabolic, infectious and immune disorders. From the clinical editor: Nanoneuromedicine is a branch of nanomedicine that specifically looks at the nervous system. In the clinical setting, a fundamental hurdle in nervous system disorders is due to an inherent inability of nerve cells to regenerate after damage. Nanotechnology can offer new approaches to overcome these challenges. This review describes recent developments in nanomedicine delivery systems that would affect stem cell repair and regeneration in the nervous system.

Correlation of Hemorrhage Near Developing Opossum Skull With Pulsed Ultrasound Exposure Parameters.

OBJECTIVES: High-intensity focused ultrasound (HIFU) has been used noninvasively for therapeutic applications. Before HIFU can be used therapeutically on a human fetus, the bioeffects related to HIFU must be studied, and the mechanism causing the bioeffects should be understood. Previous studies have shown that HIFU, when targeted on fetal rat and mice bones. resulted in hemorrhage. However, the mechanism responsible has not been identified. In this study, we looked at ultrasound parameters related to hemorrhage in an effort to better understand the mechanism. METHODS: Brazilian opossum pups (7-8 postnatal days) were exposed to a 1.1-MHz f/1 spherically focused transducer (6.3 cm focal length). Four treatment groups of n = 14 and a control group of n = 14 were exposed to rarefactional pressures of 3.6 to 6 MPa with spatial-peak temporal average intensity values of 5.4 to 10.8 W/cm(2). The pulse repetition frequency was varied from 500 to 1000 Hz with exposure durations of 1 to 4 minutes. RESULTS: Four groups with sample sizes of 14 had hemorrhage percentages of 43%, 36%, 29%, and 36%, respectively. Hemorrhage occurrence and size were found to correlate strongly with the nonlinear product of energy density and number of pulses, with correlation values of 0.92 and 0.97, respectively. CONCLUSIONS: The dependence of hemorrhage on energy density and the number of pulses suggests that the hemorrhage may be due to high-stress, low-cycle mechanical fatigue damage. Hence, for therapeutic applications, the product of energy density and number of pulses should not exceed a certain predetermined limit.

Assessment of the Behaviors of an In Vitro Brain Model On-Chip under Shockwave Impacts.

Herein we report the assessment of the effects of shockwave (SW) impacts on adult rat hippocampal progenitor cell (AHPC) neurospheres (NSs), which are used as in vitro brain models, for enhancing our understanding of the mechanisms of traumatic brain injury (TBI). The assessment has been achieved by using culture dishes and a new microchip. The microchip allows the chemicals released from the brain models cultured inside the cell culture chamber under SW impacts to diffuse to the nanosensors in adjacent sensor chambers through built-in diffusion barriers, which are used to prevent the cells from entering the sensor chambers, thereby mitigating the biofouling issues of the sensor surface. Experiments showed the negative impact of the SW on the viability, proliferation, and differentiation of the cells within the NSs. A qPCR gene expression analysis was performed and appeared to confirm some of the immunocytochemistry (ICC) results. Finally, we demonstrated that the microchip can be used to monitor lactate dehydrogenase (LDH) released from the AHPC-NSs subjected to SW impacts. As expected, LDH levels changed when AHPC-NSs were injured by SW impacts, verifying this chip can be used for assessing the degrees of injuries to AHPC-NSs by monitoring LDH levels. Taken together, these results suggest the feasibility of using the chip to better understand the interactions between SW impacts and in vitro brain models, paving the way for potentially establishing in vitro TBI models on a chip.

An integrated microfluidic chip for studying the effects of neurotransmitters on neurospheroids.

To improve our understanding of how the central nervous system functions in health and disease, we report the development of an integrated chip for studying the effects of the neurotransmitters dopamine and serotonin on adult rat hippocampal progenitor cell (AHPC) neurospheroids. This chip allows dopamine or serotonin located in one chamber to diffuse to AHPC neurospheroids cultured in an adjacent chamber through a built-in diffusion barrier created by an array of intentionally misaligned micropillars. The gaps among the micropillars are filled with porous poly(ethylene glycol) (PEG) gel to tune the permeability of the diffusion barrier. An electrochemical sensor is also integrated within the chamber where the neurospheroids can be cultured, thereby allowing monitoring of the concentrations of dopamine or serotonin. Experiments show that concentrations of the neurotransmitters inside the neurospheroid chamber can be increased over a period of several hours to over 10 days by controlling the compositions of the PEG gel inside the diffusion barrier. The AHPC neurospheroids cultured in the chip remain highly viable following dopamine or serotonin treatment. Cell proliferation and neuronal differentiation have also been observed following treatment, revealing that the AHPC neurospheroids are a valuable in vitro brain model for neurogenesis research. Finally, we show that by tuning the permeability of diffusion barrier, we can block transfer of Escherichia coli cells across the diffusion barrier, while allowing dopamine or serotonin to pass through. These results suggest the feasibility of using the chip to better understand the interactions between microbiota and brain via the gut-brain axis.

Characterization of structure and function of the mouse retina using pattern electroretinography, pupil light reflex, and optical coherence tomography.

OBJECTIVE: To perform in vivo analysis of retinal functional and structural parameters in healthy mouse eyes. ANIMAL STUDIED: Adult C57BL/6 male mice (n = 37). PROCEDURES: Retinal function was evaluated using pattern electroretinography (pERG) and the chromatic pupil light reflex (cPLR). Structural properties of the retina and nerve fiber layer (NFL) were evaluated using spectral-domain optical coherence tomography (SD-OCT). RESULTS: The average pERG amplitudes were found to be 11.2 ± 0.7 µV (P50-N95, mean ± SEM), with an implicit time for P50-N95 interval of 90.4 ± 5.4 ms. Total retinal thickness was 229.5 ± 1.7 µm (mean ± SEM) in the area centralis region. The thickness of the retinal nerve fiber layer (mean ± SEM) using a circular peripapillary retinal scan centered on the optic nerve was 46.7 ± 0.9 µm (temporal), 46.1 ± 0.9 µm (superior), 45.8 ± 0.9 µm (nasal), and 48.4 ± 1 µm (inferior). The baseline pupil diameter was 2.1 ± 0.05 mm in darkness, and 1.1 ± 0.05 and 0.56 ± 0.03 mm after stimulation with red (630 nm, luminance 200 kcd/m(2)) or blue (480 nm, luminance 200 kcd/m(2)) light illumination, respectively. CONCLUSIONS: Pattern electroretinography, cPLR and SD-OCT analysis are reproducible techniques, which can provide important information about retinal and optic nerve function and structure in mice.

Astrocyte-derived interleukin-6 promotes specific neuronal differentiation of neural progenitor cells from adult hippocampus.

The purpose of this study was to investigate the ability of astrocyte-derived factors to influence neural progenitor cell differentiation. We previously demonstrated that rat adult hippocampal progenitor cells (AHPCs) immunoreactive for the neuronal marker class III beta-tubulin (TUJ1) were significantly increased in the presence of astrocyte-derived soluble factors under noncontact coculture conditions. Using whole-cell patch-clamp analysis, we observed that the cocultured AHPCs displayed two prominent voltage-gated conductances, tetraethyl ammonium (TEA)-sensitive outward currents and fast transient inward currents. The outward and inward current densities of the cocultured AHPCs were approximately 2.5-fold and 1.7-fold greater, respectively, than those of cells cultured alone. These results suggest that astrocyte-derived soluble factors induce neuronal commitment of AHPCs. To investigate further the activity of a candidate neurogenic factor on AHPC differentiation, we cultured AHPCs in the presence or absence of purified rat recombinant interleukin-6 (IL-6). We also confirmed that the astrocytes used in this study produced IL-6 by ELISA and RT-qPCR. When AHPCs were cultured with IL-6 for 6-7 days, the TUJ1-immunoreactive AHPCs and the average length of TUJ1-immunoreactive neurites were significantly increased compared with the cells cultured without IL-6. Moreover, IL-6 increased the inward current density to an extent comparable to that of coculture with astrocytes, with no significant differences in the outward current density, apparent resting potential, or cell capacitance. These results suggest that astrocyte-derived IL-6 may facilitate AHPC neuronal differentiation. Our findings have important implications for understanding injury-induced neurogenesis and developing cell-based therapeutic strategies using neural progenitors.

Differentiation and behavior of human neural progenitors on micropatterned substrates and in the developing retina.

In this study we investigated the differentiation of human neural progenitor cells (hNPCs) in vitro to evaluate their differentiation potential and in vivo to explore their viability and behavior following transplantation. Progenitors were maintained as neurospheres in media containing basic fibroblast growth factor and epidermal growth factor. Micropatterned polystyrene substrates were fabricated and coated with ECL (entactin, collagen, and laminin) to provide physical and chemical guidance during the differentiation of the hNPCs. The hNPCs growing on the micropatterned substrates showed no differences in proliferation or differentiation potential compared with those hNPCs growing on the nonpatterned substrates. However, hNPCs cultured on the micropatterned substrates were aligned in the direction of the micropattern compared with those cells growing on the nonpatterned substrates. Furthermore, hNPC migration was directed in alignment with the micropatterned substrates. Transplantation of the hNPCs into the developing retina was used to evaluate their behavior in vivo. Cells displayed extensive survival, differentiation, and morphological integration following xenotransplant into the retina, even in the absence of immunosuppression. Taken together, our results show that these multipotent hNPCs are a neurogenic progenitor population that can be maintained in culture for extended periods. Although the micropatterned substrates have no major effect on the proliferation or differentiation of the hNPCs, they clearly promoted alignment and directed neurite outgrowth along the pattern as well as directing migration of the cells. These approaches may provide important strategies to guide the growth and differentiation of NPCs in vitro and in vivo.

In situ probing of cholesterol in astrocytes at the single-cell level using laser desorption ionization mass spectrometric imaging with colloidal silver.

Mass spectrometric imaging has been utilized to localize individual astrocytes and to obtain cholesterol populations at the single-cell level in laser desorption ionization (LDI) with colloidal silver. The silver ion adduct of membrane-bound cholesterol was monitored to detect individual cells. Good correlation between mass spectrometric and optical images at different cell densities indicates the ability to perform single-cell studies of cholesterol abundance. The feasibility of quantification is confirmed by the agreement between the LDI-MS ion signals and the results from a traditional enzymatic fluorometric assay. We propose that this approach could be an effective tool to study chemical populations at the cellular level.

Exogenous modulation of intrinsic optic nerve neuroprotective activity.

BACKGROUND: To characterize the molecular and functional status of the rat retina and optic nerve after acute elevation of intraocular pressure (IOP). METHODS: Retinal ischemia was induced in rats by increasing the IOP (110 mmHg/60 minutes). Microarray analysis, quantitative RT-PCR (qRT-PCR) and immunohistochemistry were used to characterize retinal tissue. PLGA microspheres containing neurotrophic factors (BDNF, GDNF, or CNTF) or empty microspheres were injected into the vitreous of operated animals 1 day after elevation of IOP. Pupil light reflex (PLR) parameters and electroretinograms (ERG) were monitored at multiple time points during the 60-day postoperative recovery period. RESULTS: Molecular analysis showed a significant intrinsic up-regulation of CNTF at 10 and 25 days after induction of the acute ocular hypertension (p = 0.0067). Molecular tissue analysis of GDNF and its receptors (GDNFR1, GDNFR2), and BDNF and its receptor (trkB) showed no change in expression. Animals that received CNTF microspheres had no significant functional recovery compared to animals which received blank microspheres (p > 0.05). Animals that received GDNF or BDNF microspheres showed significant PLR recovery (p < 0.05 and p < 0.001 respectively) compared to non-treated animals. CONCLUSIONS: Continuous release of neurotrophic growth factors (NGFs) significantly protects optic nerve function in the experimental model of retinal ischemia observed by PLR analysis.

Recovery of Encapsulated Adult Neural Progenitor Cells from Microfluidic-Spun Hydrogel Fibers Enhances Proliferation and Neuronal Differentiation.

Because of the limitations imposed by traditional two-dimensional (2D) cultures, biomaterials have become a major focus in neural and tissue engineering to study cell behavior in vitro. 2D systems fail to account for interactions between cells and the surrounding environment; these cell-matrix interactions are important to guide cell differentiation and influence cell behavior such as adhesion and migration. Biomaterials provide a unique approach to help mimic the native microenvironment in vivo. In this study, a novel microfluidic technique is used to encapsulate adult rat hippocampal stem/progenitor cells (AHPCs) within alginate-based fibrous hydrogels. To our knowledge, this is the first study to encapsulate AHPCs within a fibrous hydrogel. Alginate-based hydrogels were cultured for 4 days in vitro and recovered to investigate the effects of a 3D environment on the stem cell fate. Post recovery, cells were cultured for an additional 24 or 72 h in vitro before fixing cells to determine if proliferation and neuronal differentiation were impacted after encapsulation. The results indicate that the 3D environment created within a hydrogel is one factor promoting AHPC proliferation and neuronal differentiation (19.1 and 13.5%, respectively); however, this effect is acute. By 72 h post recovery, cells had similar levels of proliferation and neuronal differentiation (10.3 and 8.3%, respectively) compared to the control conditions. Fibrous hydrogels may better mimic the natural micro-environment present in vivo and be used to encapsulate AHPCs, enhancing cell proliferation and selective differentiation. Understanding cell behavior within 3D scaffolds may lead to the development of directed therapies for central nervous system repair and rescue.

Brain-derived neurotrophic factor released from engineered mesenchymal stem cells attenuates glutamate- and hydrogen peroxide-mediated death of staurosporine-differentiated RGC-5 cells.

The purpose of this study was to determine the viability of cell-based delivery of brain-derived neurotrophic factor (BDNF) from genetically modified mesenchymal stem cells (MSCs) for neuroprotection of RGC-5 cells. RGC-5 cells were differentiated with the protein kinase inhibitor staurosporine (SS) and exposed to the cellular stressors glutamate or H2O2. As a neuroprotective strategy, these cells were then co-cultured across a membrane insert with mesenchymal stem cells (MSCs) engineered with a lentiviral vector for production of BDNF (BDNF-MSCs). As a positive control, recombinant human BDNF (rhBDNF) was added to stressed RGC-5 cells. After SS-differentiation RGC-5s developed neuronal-like morphologies, and a significant increase in the proportion of RGC-5s immunoreactive for TuJ-1 and Brn3a was observed. Differentiated RGC-5s also had prominent TrkB staining, demonstrating expression of the high-affinity BDNF receptor. Treatment of SS-differentiated RGC-5s with glutamate or H2O2, produced significant cell death (56.0 +/- 7.02 and 48.90 +/- 4.58% of control cells, respectively) compared to carrier-solution treated cells. BDNF-delivery from MSCs preserved more RGC-5 cells after treatment with glutamate (80.0 +/- 5.40% cells remaining) than control GFP expressing MSCs (GFP-MSCs, 57.29 +/- 1.89%, p < 0.01). BDNF-MSCs also protected more RGC-5s after treatment with H2O2 (65.6 +/- 3.47%) than GFP-MSCs (46.0 +/- 4.20%, p < 0.01). We have shown survival of differentiated RGC-5s is reduced by the cellular stressors glutamate and H2O2. Additionally, our results demonstrate that genetically modified BDNF-producing MSCs can enhance survival of stressed RGC-5 cells and therefore, may be effective vehicles to deliver BDNF to retinal ganglion cells affected by disease.

Genetic Manipulation and Selection of Mouse Mesenchymal Stem Cells for Delivery of Therapeutic Factors In Vivo.

Bone marrow-derived mesenchymal stem cells (MSCs) hold great potential as an ex vivo cellular system for delivery of therapeutic proteins to the diseased or damaged central nervous system (CNS). This adult stem cell population has considerable translational potential for autologous transplantation due to lack of ethical concerns, accessibility, multipotent nature, and plasticity. Here we describe a methodology and outline a strategy using lentiviral vectors for producing lines of MSCs hypersecreting neurotrophic growth factors (e.g., brain-derived neurotrophic factor (BDNF) and/or glial cell line-derived neurotrophic factor (GDNF)) together with a reporter protein such as green fluorescent protein (GFP) that may be used for in vitro and in vivo neuroprotection bioassays. This approach provides exciting opportunities for basic research and proof-of-concept studies.

Isolation and culture of primary embryonic zebrafish neural tissue.

BACKGROUND: Primary cell culture is a valuable tool to utilize in parallel with in vivo studies in order to maximize our understanding of the mechanisms surrounding neurogenesis and central nervous system (CNS) regeneration and plasticity. The zebrafish is an important model for biomedical research and primary neural cells are readily obtainable from their embryonic stages viatissue dissociation. Further, transgenic reporter lines with cell type-specific expression allows for observation of distinct cell populations within the dissociated tissue. NEW METHOD: Here, we define an efficient method for ex vivo quantification and characterization of neuronal and glial tissue dissociated from embryonic zebrafish. RESULTS: Zebrafish brain dissociated cells have been documented to survive in culture for at least 9 days in vitro (div). Anti-HuC/D and anti-Acetylated Tubulin antibodies were used to identify neurons in culture; at 3 div approximately 48% of cells were HuC/D positive and 85% expressed serotonin, suggesting our protocol can efficiently isolate neurons from whole embryonic zebrafish brains. Live time-lapse imaging was also carried out to analyze cell migration in vitro. COMPARISON WITH EXISTING METHODS: Primary cultures of zebrafish neural cells typically have low rates of survivability in vitro. We have developed a culture system that has long term cell viability, enabling direct analysis of cell-cell and cell-extracellular matrix interactions. CONCLUSIONS: These results demonstrate a practical method for isolating, dissociating and culturing of embryonic zebrafish neural tissue. This approach could further be utilized to better understand zebrafish regeneration in vitro.

3D Microfibrous Scaffolds Selectively Promotes Proliferation and Glial Differentiation of Adult Neural Stem Cells: A Platform to Tune Cellular Behavior in Neural Tissue Engineering.

Biomaterials are essential for the development of innovative biomedical and therapeutic applications. Biomaterials-based scaffolds can influence directed cell differentiation to improve cell-based strategies. Using a novel microfluidics approach, poly (ε-caprolactone) (PCL), is used to fabricate microfibers with varying diameters (3-40 µm) and topographies (straight and wavy). Multipotent adult rat hippocampal stem/progenitor cells (AHPCs) are cultured on 3D aligned PCL microfibrous scaffolds to investigate their ability to differentiate into neurons, astrocytes, and oligodendrocytes. The results indicate that the PCL microfibers significantly enhance proliferation of the AHPCs compared to control, 2D planar substrates. While the AHPCs maintained their multipotent differentiation capacity when cultured on the PCL scaffolds, there is a significant and dramatic increase in immunolabeling for astrocyte and oligodendrocyte differentiation when compared with growth on planar surfaces. Our results show a 3.5-fold increase in proliferation and 23.4-fold increase in astrocyte differentiation for cells on microfibers. Transplantation of neural stem/progenitor cells within a PCL microfiber scaffold may provide important biological and topographic cues that facilitate the survival, selective differentiation, and integration of transplanted cells to improve therapeutic strategies.

Photo-Cross-Linked Poly(ethylene glycol) Diacrylate Hydrogels: Spherical Microparticles to Bow Tie-Shaped Microfibers.

Bow tie-shaped fibers and spherical microparticles with controlled dimensions and shapes were fabricated with poly(ethylene glycol) diacrylate hydrogel utilizing hydrodynamic shear principles and a photopolymerization strategy under a microfluidic regime. Decreasing the flow rate ratio between the core and sheath fluids from 25 (50:2) to 1.25 (100:80) resulted in increasing the particles size and reducing the production rate by 357 and 86%, respectively. The width of the fibers increased by a factor of 1.4 when the flow rate ratio was reduced from 2.5 to 1 due to the decrease of the shear force at the fluid/fluid interface. The stress at break and Young's modulus of the fibers were enhanced by 32 and 63%, respectively, when the sheath-to-core flow rate ratio decreased from 100:40 to 100:80. The fiber fabrication was simulated using the finite element method, and the numerical and experimental results were in agreement. Adult hippocampal stem/progenitor cells and bone-marrow-derived multipotent mesenchymal stromal cells were seeded onto the fibrous scaffolds in vitro, and cellular adhesion, proliferation, and differentiation were investigated. Microgrooves on the fibers' surface were shown to positively affect cell adhesion when compared to flat fibers and planar controls.