Knockdown of cortical transthyretin expression around implanted neural prosthetic devices using intraventricular siRNA injection in the brain.

Neural prosthetic devices are showing increasing clinical use for the treatment of a variety of neurological disorders. However the functions on these devices are often limited due to an inability to effectively and chronically interface with neural tissue. The insertion of devices has been shown to result in significant cellular and vascular trauma surrounding the insertion site. In particular, the up-regulation of genes involved in neuronal degeneration are believed to contribute to the loss of neuronal tissue. RNA interference is a novel technique for the development of antisense therapeutics for the post-transcriptional silencing of specific genes. In order to demonstrate the feasibility of RNA interference for gene-specific silencing in vivo, a short interfering RNA targeting transthyretin, was infused prior to unilateral device insertion. Injection of siRNA was found to significantly reduce the expression of transthyretin mRNA when expression was assessed at 1 week following device insertion. Concomitant decreases in transthyretin protein levels were also observed. These data demonstrate the feasibility of using RNA interference to modulate the initial reactive cellular responses that occur in the brain following insertion of neural prosthetic devices.

Optical monitoring of neural networks evoked by focal electrical stimulation on microelectrode arrays using FM dyes.

Patch-clamping or microelectrode arrays (MEA) are conventional methods to monitor the electrical activity in biological neural networks in vitro. Despite the effectiveness of these techniques, there are disadvantages including the limited number of electrodes and the predetermined location of electrodes in MEAs. In particular, these drawbacks raise a difficulty in monitoring a number of neurons outnumbering the electrodes. Here, we propose an optical technique to determine the effective range of focal electrical stimulation using FM dyes in neural networks grown on planar-type MEAs. After 3 weeks in culture, electrical stimulation was delivered to neural networks via an underlying electrode in the presence of FM dyes. The stimulation induced the internalization of the dye into the neurons around the stimulating electrodes. Fluorescent images of dye distribution successfully showed the effects of focal stimulation. A range of stimulus amplitudes and frequencies were examined to collect fluorescence images. FM-dye uptake after electrical stimulation resulted in the labeling of cells up to approximately 300 microm away from the stimulating electrode. Fluorescence intensity increased proportionally to stimulation amplitude. Tetrodotoxin was shown to inhibit the labeling of neurons except those located immediately adjacent (within 40 microm) from the stimulating electrode. In the presence of AMPA and NMDA receptors antagonists, the FM-dye labeling appeared within 80 microm from the electrode, indicating directly evoked neural networks via blocking of glutamatergic synaptic transmission. These results showed that FM dyes can be a useful tool for monitoring activity-dependent synaptic events and determining the effect of focal stimulation in cultured neural networks.

Effects of glial cells on electrode impedance recorded from neuralprosthetic devices in vitro.

Neural prosthetic devices hold the potential to be used in the treatment of a variety of neurological disorders. However, their long-term clinical success is currently limited by the ability to achieve stable interfaces between devices and the CNS. Immunohistochemical analysis has shown that cellular responses occur in tissue surrounding implanted devices. These cellular responses have been correlated with the impedance measured from device electrodes, leading to the hypothesis that a possible mechanism resulting in inconsistent device performance is the formation of an electrically insulating glial sheath at the implantation site. However, little is known about what cellular and tissue changes affect impedance values and thus contribute to the decreases in electrode performance. We have designed an in vitro system in which cell conditions can be varied within an artificial tissue matrix surrounding a neural prosthetic device. In this study, high-density cultures of glial cells were analyzed by immunohistochemical methods and impedance spectroscopy. Astrocytes and microglia were cultured at various ratios within the matrix surrounding the probes, and were observed over a period of 2 weeks. Cell seeding conditions and confocal images were compared to impedance data to enable the effects of glial cell type on electrode impedance to be determined.

Release of nerve growth factor from HEMA hydrogel-coated substrates and its effect on the differentiation of neural cells.

Local pharmacological intervention may be needed to ensure the long-term performance of neural prosthetic devices because of insertion-related neuron loss and reactive cell responses that form compact sheaths, leading to decreased device performance. We propose that local delivery of neurotrophins would enhance neuron survival and promote neuron sprouting toward device electrodes, thus providing improved electrode-neuron communication and device performance for recording and stimulating CNS activity. In this study, three different types of poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels were developed and assessed for storage capacity and release rates of the neurotrophin, nerve growth factor (NGF). Additionally, a method was developed for routine coating of microfabricated neuroprosthetic devices with the different pHEMA hydrogels. Biological responses to hydrogel-delivered NGF from the devices were measured using primary cell cultures of dorsal root ganglion (DRG) neurons. Neuron process growth was used to assess biological responses to released NGF. When targeted media concentrations were the same, responses to bath-applied NGF and NGF released from pHEMA hydrogels were not significantly different. When NGF was released from lysine-conjugated pHEMA hydrogels, a significant increase in process growth was observed. Our studies demonstrate that pHEMA coatings can be used on neural devices consistent with the needs for local neurotrophin delivery in the brain.

Biomedical Technologies for in vitro Screening and Controlled Delivery of Neuroactive Compounds.

Cell culture models can provide information pertaining to the effective dose, toxiciology, and kinetics, for a variety of neuroactive compounds. However, many in vitro models fail to adequately predict how such compounds will perform in a living organism. At the systems level, interactions between organs can dramatically affect the properties of a compound by alteration of its biological activity or by elimination of it from the body. At the tissue level, interaction between cell types can alter the transport properties of a particular compound, or can buffer its effects on target cells by uptake, processing, or changes in chemical signaling between cells. In any given tissue, cells exist in a three-dimensional environment bounded on all sides by other cells and components of the extracellular matrix, providing kinetics that are dramatically different from the kinetics in traditional two-dimensional cell culture systems. Cell culture analogs are currently being developed to better model the complex transport and processing that occur prior to drug uptake in the CNS, and to predict blood-brain barrier permeability. These approaches utilize microfluidics, hydrogel matrices, and a variety of cell types (including lung epithelial cells, hepatocytes, adipocytes, glial cells, and neurons) to more accurately model drug transport and biological activity. Similar strategies are also being used to control both the spatial and temporal release of therapeutic compounds for targeted treatment of CNS disease.

Cryopreservation of transfected primary dorsal root ganglia neurons.

Primary dorsal root ganglia (DRG) neurons are often used to investigate the relative strength of various guidance cues to promote re-growth in vitro. Current methods of neuron isolation are laborious and disposal of excess dissected cells is inefficient. Traditional immunostaining techniques are inadequate to visualize real-time neurite outgrowth in co-culture. Cryopreservation, in combination with transfection techniques, may provide a viable solution to both under-utilized tissue and insufficient methods of visualization. This study aims to qualitatively and quantitatively demonstrate successful cryopreservation of primary transfected and non-transfected DRG neurons. Fluorescent micrographs were used to assess morphology after 24h in culture and suggest similarities between freshly isolated neurons and neurons which have been transfected and/or cryopreserved. Quantitative measurements of neuron outgrowth (specifically, primary neurites, branch points and total neurite length) indicate that neuron outgrowth is not altered by cryopreservation. Transfected neurons have stunted outgrowth at 24h.

Modulation of cultured neural networks using neurotrophin release from hydrogel-coated microelectrode arrays.

Polyacrylamide and poly(ethylene glycol) diacrylate hydrogels were synthesized and characterized for use as drug release and substrates for neuron cell culture. Protein release kinetics was determined by incorporating bovine serum albumin (BSA) into hydrogels during polymerization. To determine if hydrogel incorporation and release affect bioactivity, alkaline phosphatase was incorporated into hydrogels and a released enzyme activity determined using the fluorescence-based ELF-97 assay. Hydrogels were then used to deliver a brain-derived neurotrophic factor (BDNF) from hydrogels polymerized over planar microelectrode arrays (MEAs). Primary hippocampal neurons were cultured on both control and neurotrophin-containing hydrogel-coated MEAs. The effect of released BDNF on neurite length and process arborization was investigated using automated image analysis. An increased spontaneous activity as a response to the released BDNF was recorded from the neurons cultured on the top of hydrogel layers. These results demonstrate that proteins of biological interest can be incorporated into hydrogels to modulate development and function of cultured neural networks. These results also set the stage for development of hydrogel-coated neural prosthetic devices for local delivery of various biologically active molecules.

Applications of hydrogels for neural cell engineering.

Hydrogels, three-dimensional networks of hydrophilic polymers, are currently being investigated for use in a variety of tissue-engineering applications. Hydrogel materials generally exhibit a number of properties including permeability to oxygen and nutrients, which make these materials attractive for use in biological applications. However, in order for such polymers to be successfully integrated into biological systems, these constructs must be engineered so as to mimic native biological interfaces. In this paper we discuss the use of acrylate-based polymers and their derivatives, for use in biomedical applications, including drug delivery, tissue engineering, cell encapsulation, and as templates for directed cell growth, attachment and proliferation.

Electrical stimulation-induced cell clustering in cultured neural networks.

Planar microelectrode arrays (MEAs) are widely used to record electrical activity from neural networks. However, only a small number of functional recording sites frequently show electrical activity. One contributing factor may be that neurons in vitro receive insufficient synaptic input to develop into fully functional networks. In this study, electrical stimulation was applied to neurons mimicking synaptic input. Various stimulation paradigms were examined. Stimulation amplitude and frequency were tailored to prevent cell death. Two effects of stimulation were observed when 3-week-old cultures were stimulated: (1) clusters of neural cells were observed adjacent to stimulating electrodes and (2) an increase in spontaneous neuronal activity was recorded at stimulating electrodes. Immunocytochemical analysis indicates that stimulation may cause both new neuron process growth as well as astrocyte activation. These data indicate that electrical stimulation can be used as a tool to modify neural networks at specific electrode sites and promote electrical activity.

Directed cell growth on protein-functionalized hydrogel surfaces.

Biochemical surface modification has been used to direct cell attachment and growth on a biocompatible gel surface. Acrylamide-based hydrogels were photo-polymerized in the presence of an acroyl-streptavidin monomer to create planar, functionalized surfaces capable of binding biotin-labelled proteins. Soft protein lithography (microcontact printing) of proteins was used to transfer the biotinylated extracellular matrix proteins, fibronectin and laminin, and the laminin peptide biotin-IKVAV, onto modified surfaces. As a biological assay, we plated LRM55 astroglioma and primary rat hippocampal neurons on patterned hydrogels. We found both cell types to selectively adhere to areas patterned with biotin-conjugated proteins. Fluorescence and bright-field modes of microscopy were used to assess cell attachment and cell morphology on modified surfaces. LRM55 cells were found to attach to protein-stamped regions of the hydrogel only. Neurons exhibited significant neurite extension after 72h in vitro, and remained viable on protein-stamped areas for more than 4 weeks. Patterned neurons developed functionally active synapses, as measured by uptake of the dye FM1-43FX. Results from this study suggest that hydrogel surfaces can be patterned with multiple proteins to direct cell growth and attachment.

Low-density neuronal networks cultured using patterned poly-l-lysine on microelectrode arrays.

Synaptic activity recorded from low-density networks of cultured rat hippocampal neurons was monitored using microelectrode arrays (MEAs). Neuronal networks were patterned with poly-l-lysine (PLL) using microcontact printing (microCP). Polydimethysiloxane (PDMS) stamps were fabricated with relief structures resulting in patterns of 2 microm-wide lines for directing process growth and 20 microm-diameter circles for cell soma attachment. These circles were aligned to electrode sites. Different densities of neurons were plated in order to assess the minimal neuron density required for development of an active network. Spontaneous activity was observed at 10-14 days in networks using neuron densities as low as 200 cells/mm(2). Immunocytochemistry demonstrated the distribution of dendrites along the lines and the location of foci of the presynaptic protein, synaptophysin, on neuron somas and dendrites. Scanning electron microscopy demonstrated that single fluorescent tracks contained multiple processes. Evoked responses of selected portions of the networks were produced by stimulation of specific electrode sites. In addition, the neuronal excitability of the network was increased by the bath application of high K(+) (10-12 mM). Application of DNQX, an AMPA antagonist, blocked all spontaneous activity, suggesting that the activity is excitatory and mediated through glutamate receptors.

Functionalized hydrogel surfaces for the patterning of multiple biomolecules.

Patterning of multiple proteins and enzymes onto biocompatible surfaces can provide multiple signals to control cell attachment and growth. Acrylamide-based hydrogels were photo-polymerized in the presence of streptavidin-acrylamide, resulting in planar gel surfaces functionalized with the streptavidin protein. This surface was capable of binding biotin-labeled biomolecules. The proteins fibronectin and laminin, the enzyme alkaline phosphatase, and the photo-protein R-phycoerythrin were patterned using soft lithographic techniques. Polydimethylsiloxane stamps were used to transfer biotinylated proteins onto streptavidin-conjugated hydrogel surfaces. Stamped biomolecules were spatially resolved to feature sizes of 10 mum. Fluorescence measurements were used to assess protein transfer and enzyme functionality on modified surfaces. Our results demonstrate that hydrogel surfaces can be patterned with multiple proteins and enzymes, with retention of biological and catalytic activity. These surfaces are biocompatible and provide cues for cell attachment and growth. (c) 2006 Wiley Periodicals, Inc. J Biomed Mater Res 2007.

Differential expression of N-methyl-D-aspartate receptor NR2 isoforms in Alzheimer's disease.

We have previously shown that the expression of NMDA receptor NR1 subunit mRNA splice variants in Alzheimer's disease (AD) brain varies according to regional susceptibility to pathological damage. Here we investigated the expression of the modulatory NR2 subunits of the NMDA receptor using quantitative RT-PCR to assay all NR2 isoforms. Significantly lower expression of NR2A and NR2B transcripts was found in susceptible regions of AD brain, whereas expression of NR2C and NR2D transcripts did not differ from that in controls. Western blot analysis confirmed a lower expression of the NR2A and NR2B isoforms at the protein level. The results suggest that NR2 subunit composition may modulate NMDA receptor-mediated excitotoxicity. NMDA receptor dysfunction might give rise to the regionally selective pattern of neuronal loss that is characteristic of AD.

Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia, accounting for 60-70% of cases in subjects over 65 years of age. Several postulates have been put forward that relate AD neuropathology to intellectual and functional impairment. These range from free-radical-induced damage, through cholinergic dysfunction, to beta-amyloid-induced toxicity. However, therapeutic strategies aimed at improving the cognitive symptoms of patients via choline supplementation, cholinergic stimulation or beta-amyloid vaccination, have largely failed. A growing body of evidence suggests that perturbations in systems using the excitatory amino acid L-glutamate (L-Glu) may underlie the pathogenic mechanisms of (e.g.) hypoxia-ischemia, epilepsy, and chronic neurodegenerative disorders such as Huntington's disease and AD. Almost all neurons in the CNS carry the N-methyl-D-aspartate (NMDA) subtype of ionotropic L-glutamate receptors, which can mediate post-synaptic Ca2+ influx. Excitotoxicity resulting from excessive activation of NMDA receptors may enhance the localized vulnerability of neurons in a manner consistent with AD neuropathology, as a consequence of an altered regional distribution of NMDA receptor subtypes. This review discusses mechanisms for the involvement of the NMDA receptor complex and its interaction with polyamines in the pathogenesis of AD. NMDA receptor antagonists have potential for the therapeutic amelioration of AD.

Selective loss of NMDA receptor NR1 subunit isoforms in Alzheimer's disease.

Previous work had shown that the ratio of NMDA receptor NR1 subunit mRNA transcripts containing an N-terminal splice cassette to those that do not is markedly lower in regions of the Alzheimer's disease (AD) brain that are susceptible to pathological damage, compared with spared regions in the same cases or homotropic regions in controls. To elucidate the origins of this difference in proportionate expression, we measured the absolute levels of each of the eight NR1 transcripts by quantitative internally standardized RT-PCR assay. Expression of transcripts with the cassette was strongly attenuated in susceptible regions of Alzheimer's brain, whereas expression of non-cassette transcripts differed little from that in controls. The expression of other NR1 splice variants was not associated with pathology relevant to disease status, although some combinations of splice cassettes were well maintained in AD cases. The population profile of NR1 transcripts in occipital cortex differed from the profiles in other brain regions studied. Western analysis confirmed that the expression of protein isoforms containing the N-terminal peptide was very low in susceptible areas of the Alzheimer's brain. Cells that express NR1 subunits with the N-terminal cassette may be selectively vulnerable to toxicity in AD.

Biochemical and molecular studies using human autopsy brain tissue.

The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT-PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable post-mortem. However, their abundance and integrity can exhibit marked intra- and intercase variability, making comparisons between case-groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante-mortem status, agonal state and post-mortem and post-autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante- and post-mortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.

Quantitation of alternatively spliced NMDA receptor NR1 isoform mRNA transcripts in human brain by competitive RT-PCR.

The N-methyl-D-aspartate (NMDA)-selective subtype of ionotropic glutamate receptor is of importance in neuronal differentiation and synapse consolidation, activity-dependent forms of synaptic plasticity, and excitatory amino acid-mediated neuronal toxicity [Neurosci. Res. Program Bull. 19 (1981) 1; Lab. Invest. 68 (1993) 372]. NMDA receptors exist in vivo as tetrameric or pentameric complexes comprising proteins from two families of homologous subunits, designated NR1 and NR2(A-D) [Biochem. Biophys. Res. Commun. 185 (1992) 826]. The gene coding for the human NR1 subunit (hNR1) is composed of 21 exons, three of which (4, 20 and 21) can be differentially spliced to generate a total of eight distinct subunit variants. We detail here a competitive RT-PCR (cRT-PCR) protocol to quantify endogenous levels of hNR1 splice variants in autopsied human brain. Quantitation of each hNR1 splice variant is performed using standard curve methodology in which a known amount of synthetic ribonucleic acid competitor (internal standard) is co-amplified against total RNA. This method can be used for the quantitation of hNR1 mRNA levels in response to acute or chronic disease states, in particular in the glutamatergic-associated neuronal loss observed in Alzheimer's disease [J. Neurochem. 78 (2001) 175]. Furthermore, alterations in hNR1 mRNA expression may be reflected at the translational level, resulting in functional changes in the NMDA receptor.

Quantitation of NMDA receptor NR2 mRNA transcripts in human brain by competitive RT-PCR.

The NMDA-selective ionotropic receptor constitutes one of the three principal classes of L-glutamate receptors within the mammalian brain. It plays key roles in neuronal differentiation and synapse consolidation, activity-dependent forms of synaptic plasticity, and excitatory amino acid-mediated neuronal toxicity [Lab. Invest., 68 (1993) 372-387]. NMDA receptors exist as multimeric complexes comprising proteins from two families, NR1 and NR2(A-D) [J. Biol. Chem., 271 (1996) 15669-15674]. Studies on recombinant receptors have revealed that while homomeric NR2 receptors are non-functional, co-expression of an NR1 with an NR2 subunit modulates the efficacy of the resulting channel [Nature, 357 (1992) 70-74]. The RT-PCR assay we describe here was developed to allow quantitation of all hNR2 transcripts in a single-tube PCR assay. Each hNR2 isoform is quantified on the basis of standard curves in which a known amount of synthetic ribonucleic acid competitor is co-amplified against total RNA. The protocol has been applied to the quantitation of hNR2 mRNA levels in autopsy brain. Used in conjunction with a method for the quantitation of hNR1 transcripts [Brain Res. Protoc., in press], a complete analysis of NMDA receptor mRNA expression can be obtained.