Antifouling Photograftable Zwitterionic Coatings on PDMS Substrates.

The foreign body response (FBR) to implantable materials can negatively impact performance of medical devices such as the cochlear implant. Engineering surfaces that resist the FBR could lead to enhanced functionality including potentially improving outcomes for cochlear implant recipients through reduction in fibrosis. In this work, we coat poly(dimethylsiloxane) (PDMS) surfaces with two zwitterionic polymers, poly(sulfobetaine methacrylate) (pSBMA) and poly(carboxybetaine methacrylate) (pCBMA), using a simultaneous photografting/photo-cross-linking process to produce a robust grafted zwitterionic hydrogel. reduce nonspecific protein adsorption, the first step of the FBR. The coating process uses benzophenone, a photografting agent and type II photoinitiator, to covalently link the cross-linked zwitterionic thin film to the PDMS surface. As the concentration of benzophenone on the surface increases, the adhesive strength of the zwitterionic thin films to PDMS surfaces increases as determined by shear adhesion. Additionally, with increased concentration of the adsorbed benzophenone, failure of the system changes from adhesive delamination to cohesive failure within the hydrogel, demonstrating that durable adhesive bonds are formed from the photografting process. Interestingly, antifouling properties of the zwitterionic polymers are preserved with significantly lower levels of nonspecific protein adsorption on zwitterion hydrogel-coated samples compared to uncoated controls. Fibroblast adhesion is also dramatically reduced on coated substrates. These results show that cross-linked pSBMA and pCBMA hydrogels can be readily photografted to PDMS substrates and show promise in potentially changing the fibrotic response to implanted biomaterials.

Photopolymerizable Zwitterionic Polymer Patterns Control Cell Adhesion and Guide Neural Growth.

Developing materials that reduce or eliminate fibrosis encapsulation of neural prosthetic implants could significantly enhance implant fidelity by improving the tissue/electrode array interface. Here, we report on the photografting and patterning of two zwitterionic materials, sulfobetaine methacrylate (SBMA) and carboxybetaine methacrylate (CBMA), for controlling the adhesion and directionality of cells relevant to neural prosthetics. CBMA and SBMA polymers were photopolymerized and grafted on glass surfaces then characterized by X-ray photoelectron spectroscopy, water contact angle, and protein adsorption. Micropatterned surfaces were fabricated with alternating zwitterionic and uncoated bands. Fibroblasts, cells prevalent in fibrotic tissue, almost exclusively migrate and grow on uncoated bands with little to no cells present on zwitterionic bands, especially for CBMA-coated surfaces. Astrocytes and Schwann cells showed similarly low levels of cell adhesion and morphology changes when cultured on zwitterionic surfaces. Additionally, Schwann cells and inner ear spiral ganglion neuron neurites aligned well to zwitterionic patterns.

Cortical glial fibrillary acidic protein-positive cells generate neurons after perinatal hypoxic injury.

Glial fibrillary acidic protein-positive (GFAP(+)) cells give rise to new neurons in the neurogenic niches; whether they are able to generate neurons in the cortical parenchyma is not known. Here, we use genetic fate mapping to examine the progeny of GFAP(+) cells after postnatal hypoxia, a model for the brain injury observed in premature children. After hypoxia, immature cortical astroglia underwent a shift toward neuronal fate and generated cortical excitatory neurons that appeared synaptically integrated into the circuitry. Fate-mapped cortical GFAP(+) cells derived ex vivo from hypoxic, but not normoxic, mice were able to form pluripotent, long-term self-renewing neurospheres. Similarly, exposure to low oxygen conditions in vitro induced stem-cell-like potential in immature cortical GFAP(+) cells. Our data support the conclusion that hypoxia promotes pluripotency in GFAP(+) cells in the cortical parenchyma. Such plasticity possibly explains the cognitive recovery found in some preterm children.

Photograftable Zwitterionic Coatings Prevent Staphylococcus aureus and Staphylococcus epidermidis Adhesion to PDMS Surfaces.

Due to its attractive mechanical properties and biocompatibility, poly(dimethyl)siloxane (PDMS) is widely used in the fabrication of biomedical materials. On the other hand, PDMS is also prone to adsorption of both proteins and bacteria, making PDMS implants susceptible to infection. Herein, we examine the use of durably cross-linked zwitterionic coatings for PDMS surfaces to mitigate bacterial adhesion. Using a single-step photografting technique, poly(sulfobetaine methacrylate) (pSBMA) and poly(carboxybetaine methacrylate) (pCBMA) thin films were covalently attached to PDMS substrates. The abilities of these coatings to resist the adhesion of Staphylococcus aureus and Staphylococcus epidermidis were tested in vitro under both wet and droplet conditions, as well as in subcutaneous and transcutaneous implantation models using Sprague-Dawley rats. Zwitterionic thin films effectively reduced bacterial adhesion in both in vitro and in vivo conditions. This was particularly true for pCBMA-coated materials, which exhibited significant reduction in bacterial adhesion and growth with respect to S. aureus and S. epidermidis for all in vitro conditions as well as the ability to resist bacterial growth on PDMS implants. The results of this study suggest that a simple and durable photografting process can be used to produce polymer thin films capable of preventing infection of implantable medical devices.

Fgfr1 is required for cortical regeneration and repair after perinatal hypoxia.

Chronic postnatal hypoxia causes an apparent loss of cortical neurons that is reversed during recovery (Fagel et al., 2006). The cellular and molecular mechanisms underlying this plasticity are not understood. Here, we show that chronic hypoxia from postnatal days 3 (P3) to 10 causes a 30% decrease in cortical neurons and a 24% decrease in cortical volume. T-brain-1 (Tbr1)(+) and SMI-32(+) excitatory neuron numbers were completely recovered 1 month after the insult, but the mice showed a residual deficit in Parvalbumin(+) and Calretinin(+) GABAergic interneurons. In contrast, hypoxic mice carrying a disrupted fibroblast growth factor receptor-1 (Fgfr1) gene in GFAP+ cells [Fgfr1 conditional knock-out (cKO)], demonstrated a persistent loss of excitatory cortical neurons and a worsening of the interneuron defect. Labeling proliferating progenitors at P17 revealed increased generation of cortical NeuN(+) and Tbr1(+) excitatory neurons in wild-type mice subjected to hypoxic insult, whereas Fgfr1 cKO failed to mount a cortical neurogenetic response. Hypoxic wild-type mice also demonstrated a twofold increase in cell proliferation in the subventricular zone (SVZ) at P17 and a threefold increase in neurogenesis in the olfactory bulb (OB) at P48, compared with normoxic mice. In contrast, Fgfr1 cKO mice had decreased SVZ cell proliferation and curtailed reactive neurogenesis in the OB. Thus, the activation of FGFR-1 in GFAP+ cells is required for neuronal recovery after neonatal hypoxic injury, which is attributable in part to enhanced cortical and OB neurogenesis. In contrast, there is incomplete recovery of inhibitory neurons after injury, which may account for persistent behavioral deficits.

Precursors with glial fibrillary acidic protein promoter activity transiently generate GABA interneurons in the postnatal cerebellum.

Neural stem or progenitor cells (NSC/NPCs) able to generate the different neuron and glial cell types of the cerebellum have been isolated in vitro, but their identity and location in the intact cerebellum are unclear. Here, we use inducible Cre recombination in GFAPCreER(T2) mice to irreversibly activate reporter gene expression at P2 (postnatal day 2), P5, and P12 in cells with GFAP (glial fibrillary acidic protein) promoter activity and analyze the fate of genetically tagged cells in vivo. We show that cells tagged at P2-P5 with beta-galactosidase or enhanced green fluorescent proteins reporter genes generate at least 30% of basket and stellate GABAergic interneurons in the molecular layer (ML) and that they lose their neurogenic potential by P12, after which they generate only glia. Tagged cells in the cerebellar white matter (WM) were initially GFAP/S100beta+ and expressed the NSC/NPCs proteins LeX, Musashi1, and Sox2 in vivo. One week after tagging, reporter+ cells in the WM upregulated the neuronal progenitor markers Mash1, Pax2, and Gad-67. These Pax2+ progenitors migrated throughout the cerebellar cortex, populating the ML and leaving the WM by P18. These data suggest that a pool of GFAP/S100beta+ glial cells located in the cerebellar WM generate a large fraction of cerebellar interneurons for the ML within the first postnatal 12 days of cerebellar development. This restricted critical period implies that powerful inhibitory factors may restrict their fate potential in vivo at later stages of development.

Quantifying Spiral Ganglion Neurite and Schwann Behavior on Micropatterned Polymer Substrates.

The first successful in vitro experiments on the cochlea were conducted in 1928 by Honor Fell (Fell, Arch Exp Zellforsch 7(1):69-81, 1928). Since then, techniques for culture of this tissue have been refined, and dissociated primary culture of the spiral ganglion has become a widely accepted in vitro model for studying nerve damage and regeneration in the cochlea. Additionally, patterned substrates have been developed that facilitate and direct neural outgrowth. A number of automated and semi-automated methods for quantifying this neurite outgrowth have been utilized in recent years (Zhang et al., J Neurosci Methods 160(1):149-162, 2007; Tapias et al., Neurobiol Dis 54:158-168, 2013). Here, we describe a method to study the effect of topographical cues on spiral ganglion neurite and Schwann cell alignment. We discuss our microfabrication process, characterization of pattern features, cell culture techniques for both spiral ganglion neurons and spiral ganglion Schwann cells. In addition, we describe protocols for reducing fibroblast count, immunocytochemistry, and methods for quantifying neurite and Schwann cell alignment.

Dual-gene, dual-cell type therapy against an excitotoxic insult by bolstering neuroenergetics.

Increasing evidence suggests that glutamate activates the generation of lactate from glucose in astrocytes; this lactate is shuttled to neurons that use it as a preferential energy source. We explore this multicellular "lactate shuttle" with a novel dual-cell, dual-gene therapy approach and determine the neuroprotective potential of enhancing this shuttle. Viral vector-driven overexpression of a glucose transporter in glia enhanced glucose uptake, lactate efflux, and the glial capacity to protect neurons from excitotoxicity. In parallel, overexpression of a lactate transporter in neurons enhanced lactate uptake and neuronal resistance to excitotoxicity. Finally, overexpression of both transgenes in the respective cell types provided more protection than either therapy alone, demonstrating that a dual-cell, dual-gene therapy approach gives greater neuroprotection than the conventional single-cell, single-gene strategy.

Marked differences in the efficacy of post-insult gene therapy with catalase versus glutathione peroxidase.

It is now recognized that the generation of reactive oxygen species (ROS) following necrotic neurological insults plays a central role in the subsequent neuron death. A key step in ROS detoxification is the conversion of hydrogen peroxide to water and oxygen by either catalase (CAT) or glutathione peroxidase (GPX). We have previously shown that overexpression of CAT or GPX protects cultured neurons against subsequent excitotoxic insults. Because of the unpredictability of most acute neurological insults, gene therapy will most often need to be carried out after rather than in anticipation of an insult. Thus, we have tested whether herpes virus amplicon vectors expressing CAT or GPX still protect cultured hippocampal neurons from oxygen/glucose deprivation if introduced following an insult. CAT-expressing vectors were protective even when introduced 8 h post-insult. In contrast, there was no post-insult time window in which GPX overexpression protected. While CAT requires no cofactor, GPX action requires glutathione as a cofactor. Thus, we speculated that the post-insult decline in glutathione compromises the protective potential of GPX. Supporting this, reversing the post-insult glutathione decline with glutathione supplementation was neuroprotective.

Neuroprotective effects of bcl-2 overexpression in hippocampal cultures: interactions with pathways of oxidative damage.

Overexpression of bcl-2protects neurons from numerous necrotic insults, both in vitro and in vivo. While the bulk of such protection is thought to arise from Bcl-2 blocking cytochrome c release from mitochondria, thereby blocking apoptosis, the protein can target other steps in apoptosis, and can protect against necrotic cell death as well. There is evidence that these additional actions may be antioxidant in nature, in that Bcl-2 has been reported to protect against generators of reactive oxygen species (ROS), to increase antioxidant defenses and to decrease levels of ROS and of oxidative damage. Despite this, there are also reports arguing against either the occurrence, or the importance of these antioxidant actions. We have examined these issues in neuron-enriched primary hippocampal cultures, with overexpression of bcl-2 driven by a herpes simplex virus amplicon: (i) Bcl-2 protected strongly against glutamate, whose toxicity is at least partially ROS-dependent. Such protection involved reduction in mitochondrially derived superoxide. Despite that, Bcl-2 had no effect on levels of lipid peroxidation, which is thought to be the primary locus of glutamate-induced oxidative damage; (ii) Bcl-2 was also mildly protective against the pro-oxidant adriamycin. However, it did so without reducing levels of superoxide, hydrogen peroxide or lipid peroxidation; (iii) Bcl-2 protected against permanent anoxia, an insult likely to involve little to no ROS generation. These findings suggest that Bcl-2 can have antioxidant actions that may nonetheless not be central to its protective effects, can protect against an ROS generator without targeting steps specific to oxidative biochemistry, and can protect in the absence of ROS generation. Thus, the antioxidant actions of Bcl-2 are neither necessary nor sufficient to explain its protective actions against these insults in hippocampal neurons.

Over-expression of antioxidant enzymes protects cultured hippocampal and cortical neurons from necrotic insults.

There is now considerable knowledge concerning neuron death following necrotic insults, and it is believed that the generation of reactive oxygen species (ROS) and oxidative damage play a pivotal role in the neuron death. Prompted by this, we have generated herpes simplex virus-1 amplicon vectors over-expressing the genes for the antioxidant enzymes catalase (CAT) or glutathione peroxidase (GPX), both of which catalyze the degradation of hydrogen peroxide. Over-expression of each of these genes in primary hippocampal or cortical cultures resulted in increased enzymatic activity of the cognate protein. Moreover, each enzyme potently decreased the neurotoxicity induced by kainic acid, glutamate, sodium cyanide and oxygen/glucose deprivation. Finally, these protective effects were accompanied by parallel decreases in hydrogen peroxide accumulation and the extent of lipid peroxidation. These studies not only underline the key role played by ROS in the neurotoxicity of necrotic insults, but also suggest potential gene therapy approaches.