The use of a dual PEDOT and RGD-functionalized alginate hydrogel coating to provide sustained drug delivery and improved cochlear implant function.

Cochlear implants provide hearing by electrically stimulating the auditory nerve. Implant function can be hindered by device design variables, including electrode size and electrode-to-nerve distance, and cochlear environment variables, including the degeneration of the auditory nerve following hair cell loss. We have developed a dual-component cochlear implant coating to improve both the electrical function of the implant and the biological stability of the inner ear, thereby facilitating the long-term perception of sound through a cochlear implant. This coating is a combination of an arginine-glycine-aspartic acid (RGD)-functionalized alginate hydrogel and the conducting polymer poly(3, 4-ethylenedioxythiophene) (PEDOT). Both in vitro and in vivo assays on the effects of these electrode coatings demonstrated improvements in device performance. We found that the coating reduced electrode impedance, improved charge delivery, and locally released significant levels of a trophic factor into cochlear fluids. This coating is non-cytotoxic, clinically relevant, and has the potential to significantly improve the cochlear implant user's experience.

In vitro and in vivo evaluation of PEDOT microelectrodes for neural stimulation and recording.

Cortical neural prostheses require chronically implanted small-area microelectrode arrays that simultaneously record and stimulate neural activity. It is necessary to develop new materials with low interface impedance and large charge transfer capacity for this application and we explore the use of conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) for the same. We subjected PEDOT coated electrodes to voltage cycling between -0.6 and 0.8 V, 24 h continuous biphasic stimulation at 3 mC/cm² and accelerated aging for four weeks. Characterization was performed using cyclic voltammetry, electrochemical impedance spectroscopy, and voltage transient measurements. We found that PEDOT coated electrodes showed a charge injection limit 15 times higher than Platinum Iridium (PtIr) electrodes and electroplated Iridium Oxide (IrOx) electrodes when using constant current stimulation at zero voltage bias. In vivo chronic testing of microelectrode arrays implanted in rat cortex revealed that PEDOT coated electrodes show higher signal-to-noise recordings and superior charge injection compared to PtIr electrodes.

Poly(3,4-ethylenedioxythiophene) (PEDOT) polymer coatings facilitate smaller neural recording electrodes.

We investigated using poly(3,4-ethylenedioxythiophene) (PEDOT) to lower the impedance of small, gold recording electrodes with initial impedances outside of the effective recording range. Smaller electrode sites enable more densely packed arrays, increasing the number of input and output channels to and from the brain. Moreover, smaller electrode sizes promote smaller probe designs; decreasing the dimensions of the implanted probe has been demonstrated to decrease the inherent immune response, a known contributor to the failure of long-term implants. As expected, chronically implanted control electrodes were unable to record well-isolated unit activity, primarily as a result of a dramatically increased noise floor. Conversely, electrodes coated with PEDOT consistently recorded high-quality neural activity, and exhibited a much lower noise floor than controls. These results demonstrate that PEDOT coatings enable electrode designs 15 µm in diameter.

In vivo electrical conductivity across critical nerve gaps using poly(3,4-ethylenedioxythiophene)-coated neural interfaces.

BACKGROUND: Bionic limbs require sensitive, durable, and physiologically relevant bidirectional control interfaces. Modern central nervous system interfacing is high risk, low fidelity, and failure prone. Peripheral nervous system interfaces will mitigate this risk and increase fidelity by greatly simplifying signal interpretation and delivery. This study evaluates in vivo relevance of a hybrid peripheral nervous system interface consisting of biological acellular muscle scaffolds made electrically conductive using poly(3,4-ethylenedioxythiophene). METHODS: Peripheral nervous system interfaces were tested in vivo using the rat hind-limb conduction-gap model for motor (peroneal) and sensory (sural) nerves. Experimental groups included acellular muscle, iron(III) chloride-treated acellular muscle, and poly(3,4-ethylenedioxythiophene) polymerized on acellular muscle, each compared with intact nerve, autogenous nerve graft, and empty (nonreconstructed) nerve gap controls (n=5 for each). Interface lengths tested included 0, 5, 10, and 20 mm. Immediately following implantation, the interface underwent electrophysiologic characterization in vivo using nerve conduction studies, compound muscle action potentials, and antidromic sensory nerve action potentials. RESULTS: Both efferent and afferent electrophysiology demonstrates acellular muscle-poly(3,4-ethylenedioxythiophene) interfaces conduct physiologic action potentials across nerve conduction gaps of at least 20 mm with amplitude and latency not differing from intact nerve or nerve grafts, with the exception of increased velocity in the acellular muscle-poly(3,4-ethylenedioxythiophene) interfaces. CONCLUSIONS: Nonmetallic, biosynthetic acellular muscle-poly(3,4-ethylenedioxythiophene) peripheral nervous system interfaces both sense and stimulate physiologically relevant efferent and afferent action potentials in vivo. This demonstrates their relevance not only as a nerve-electronic coupling device capable of reaching the long-sought goal of closed-loop neural control of a prosthetic limb, but also in a multitude of other bioelectrical applications.

Layered carbon nanotube-polyelectrolyte electrodes outperform traditional neural interface materials.

The safety, function, and longevity of implantable neuroprosthetic and cardiostimulating electrodes depend heavily on the electrical properties of the electrode-tissue interface, which in many cases requires substantial improvement. While different variations of carbon nanotube materials have been shown to be suitable for neural excitation, it is critical to evaluate them versus other materials used for bioelectrical interfacing, which have not been done in any study performed so far despite strong interest to this area. In this study, we carried out this evaluation and found that composite multiwalled carbon nanotube-polyelectrolyte (MWNT-PE) multilayer electrodes substantially outperform in one way or the other state-of-the-art neural interface materials available today, namely activated electrochemically deposited iridium oxide (IrOx) and poly(3,4-ethylenedioxythiophene) (PEDOT). Our findings provide the concrete experimental proof to the much discussed possibility that carbon nanotube composites can serve as excellent new material for neural interfacing with a strong possibility to lead to a new generation of implantable electrodes.

Poly(3,4-ethylenedioxythiophene) as a Micro-Neural Interface Material for Electrostimulation.

Chronic microstimulation-based devices are being investigated to treat conditions such as blindness, deafness, pain, paralysis, and epilepsy. Small-area electrodes are desired to achieve high selectivity. However, a major trade-off with electrode miniaturization is an increase in impedance and charge density requirements. Thus, the development of novel materials with lower interfacial impedance and enhanced charge storage capacity is essential for the development of micro-neural interface-based neuroprostheses. In this report, we study the use of conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) as a neural interface material for microstimulation of small-area iridium electrodes on silicon-substrate arrays. Characterized by electrochemical impedance spectroscopy, electrodeposition of PEDOT results in lower interfacial impedance at physiologically relevant frequencies, with the 1 kHz impedance magnitude being 23.3 +/- 0.7 kOmega, compared to 113.6 +/- 3.5 kOmega for iridium oxide (IrOx) on 177 mum(2) sites. Further, PEDOT exhibits enhanced charge storage capacity at 75.6 +/- 5.4 mC/cm(2) compared to 28.8 +/- 0.3 mC/cm(2) for IrOx, characterized by cyclic voltammetry (50 mV/s). These improvements at the electrode interface were corroborated by observation of the voltage excursions that result from constant current pulsing. The PEDOT coatings provide both a lower amplitude voltage and a more ohmic representation of the applied current compared to IrOx. During repetitive pulsing, PEDOT-coated electrodes show stable performance and little change in electrical properties, even at relatively high current densities which cause IrOx instability. These findings support the potential of PEDOT coatings as a micro-neural interface material for electrostimulation.

Electrochemical polymerization of conducting polymers in living neural tissue.

A number of biomedical devices require extended electrical communication with surrounding tissue. Significant improvements in device performance would be achieved if it were possible to maintain communication with target cells despite the reactive, insulating scar tissue that forms at the device-tissue interface. Here, we report that the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) can be polymerized directly within living neural tissue resulting in an electrically conductive network that is integrated within the tissue. Nano and microscale PEDOT filaments extend out from electrode sites, presumably forming within extracellular spaces. The cloud of PEDOT filaments penetrates out into the tissue far enough that it should be possible to bypass fibrous scar tissue and contact surrounding healthy neurons. These electrically functional, diffuse conducting polymer networks grown directly within tissue signify a new paradigm for creating soft, low impedance implantable electrodes.

Polymerization of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) around living neural cells.

In this paper, we describe interactions between neural cells and the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) toward development of electrically conductive biomaterials intended for direct, functional contact with electrically active tissues such as the nervous system, heart, and skeletal muscle. We introduce a process for polymerizing PEDOT around living cells and describe a neural cell-templated conducting polymer coating for microelectrodes and a hybrid conducting polymer-live neural cell electrode. We found that neural cells could be exposed to working concentrations (0.01 m) of the EDOT monomer for as long as 72 h while maintaining 80% cell viability. PEDOT could be electrochemically deposited around neurons cultured on electrodes using 0.5-1 microA/mm(2) galvanostatic current. PEDOT polymerized on the electrode and surrounded the cells, covering cell processes. The polymerization was impeded in regions where cells were well adhered to the substrate. The cells could be removed from the PEDOT matrix to generate a neural cell-templated biomimetic conductive substrate with cell-shaped features that were cell attracting. Live cells embedded within the conductive polymer matrix remained viable for at least 120 h following polymerization. Dying cells primarily underwent apoptotic cell death. PEDOT, PEDOT+live neurons, and neuron-templated PEDOT coatings on electrodes significantly enhanced the electrical properties as compared to the bare electrode as indicated by decreased electrical impedance of 1-1.5 orders of magnitude at 0.01-1 kHz and significantly increased charge transfer capacity. PEDOT coatings showed a decrease of the phase angle of the impedance from roughly 80 degrees for the bare electrode to 5-35 degrees at frequencies >0.1 kHz. Equivalent circuit modeling indicated that PEDOT-coated electrodes were best described by R(C(RT)) circuit. We found that an RC parallel circuit must be added to the model for PEDOT+live neuron and neuron-templated PEDOT coatings.

Toward a self-deploying shape memory polymer neuronal electrode.

The widespread application of neuronal probes for chronic recording of brain activity and functional stimulation has been slow to develop partially due to long-term biocompatibility problems with existing metallic and ceramic probes and the tissue damage caused during probe insertion. Stiff probes are easily inserted into soft brain tissue but cause astrocytic scars that become insulating sheaths between electrodes and neurons. In this communication, we explore the feasibility of a new approach to the composition and implantation of chronic electrode arrays. We demonstrate that softer polymer-based probes can be inserted into the olfactory bulb of a mouse and that slow insertion of the probes reduces astrocytic scarring. We further present the development of a micromachined shape memory polymer probe, which provides a vehicle to self-deploy an electrode at suitably slow rates and which can provide sufficient force to penetrate the brain. The deployment rate and composition of shape memory polymer probes can be tailored by polymer chemistry and actuator design. We conclude that it is feasible to fabricate shape memory polymer-based electrodes that would slowly self-implant compliant conductors into the brain, and both decrease initial trauma resulting from implantation and enhance long-term biocompatibility for long-term neuronal measurement and stimulation.

Inhibition of Rac GTPase triggers a c-Jun- and Bim-dependent mitochondrial apoptotic cascade in cerebellar granule neurons.

Rho GTPases are key transducers of integrin/extracellular matrix and growth factor signaling. Although integrin-mediated adhesion and trophic support suppress neuronal apoptosis, the role of Rho GTPases in neuronal survival is unclear. Here, we have identified Rac as a critical pro-survival GTPase in cerebellar granule neurons (CGNs) and elucidated a death pathway triggered by its inactivation. GTP-loading of Rac1 was maintained in CGNs by integrin-mediated (RGD-dependent) cell attachment and trophic support. Clostridium difficile toxin B (ToxB), a specific Rho family inhibitor, induced a selective caspase-mediated degradation of Rac1 without affecting RhoA or Cdc42 protein levels. Both ToxB and dominant-negative N17Rac1 elicited CGN apoptosis, characterized by cytochrome c release and activation of caspase-9 and -3, whereas dominant-negative N19RhoA or N17Cdc42 did not cause significant cell death. ToxB stimulated mitochondrial translocation and conformational activation of Bax, c-Jun activation, and induction of the BH3-only protein Bim. Similarly, c-Jun activation and Bim induction were observed with N17Rac1. A c-jun N-terminal protein kinase (JNK)/p38 inhibitor, SB203580, and a JNK-specific inhibitor, SP600125, significantly decreased ToxB-induced Bim expression and blunted each subsequent step of the apoptotic cascade. These results indicate that Rac acts downstream of integrins and growth factors to promote neuronal survival by repressing c-Jun/Bim-mediated mitochondrial apoptosis.

Minocycline delays disease onset and mortality in reovirus encephalitis.

Minocycline is neuroprotective in many experimental models of neurodegenerative diseases and central nervous system (CNS) injury but has not previously been tested in a model of viral encephalitis. Experimental infection of neonatal mice with neurotropic reoviruses is a classic model for studying the pathogenesis of viral encephalitis. Intracerebral inoculation of serotype 3 reovirus strain Dearing (T3D) in neonatal mice results in lethal encephalitis caused by neuronal apoptosis throughout the CNS. Minocycline significantly delayed death in mice to 11.6 +/- 0.9 days post-infection vs. 8.6 +/- 0.7 days post-infection in controls (P < 0.01). Virus-induced CNS injury, apoptosis, viral titer and antigen expression were significantly decreased in the brains of minocycline-treated mice on 6 and 8 days post-infection compared to controls. Virus-induced injury and viral titer in minocycline-treated infected mice at 11 days post-infection were similar to those seen in untreated T3D-infected mice at 8 days post-infection. Little microglial or astrocytic invasion of brain regions with viral injury was found at any time-point in untreated or minocycline-treated mice, suggesting that in this model system the neuroprotective effect exerted by minocycline is more likely due to its anti-apoptotic properties rather than its capacity to inhibit microglial activation and limit gliosis. These findings, similar to those reported for neurodegenerative diseases, indicate that minocycline does not prevent development of fatal reovirus encephalitis but delays disease onset and progression, suggesting that minocycline treatment may provide a useful adjunctive therapy in viral CNS infections.

Nonstructural protein sigma1s is a determinant of reovirus virulence and influences the kinetics and severity of apoptosis induction in the heart and central nervous system.

The mechanisms by which viruses kill susceptible cells in target organs and ultimately produce disease in the infected host remain poorly understood. Dependent upon the site of inoculation and strain of virus, experimental infection of neonatal mice with reoviruses can induce fatal encephalitis or myocarditis. Reovirus-induced apoptosis is a major mechanism of tissue injury, leading to disease development in both the brain and heart. In cultured cells, differences in the capacity of reovirus strains to induce apoptosis are determined by the S1 gene segment, which also plays a major role as a determinant of viral pathogenesis in both the heart and the central nervous system (CNS) in vivo. The S1 gene is bicistronic, encoding both the viral attachment protein sigma-1 and the nonstructural protein sigma-1-small (sigma1s). Although sigma1s is dispensable for viral replication in vitro, we wished to investigate the expression of sigma1s in the infected heart and brain and its potential role in reovirus pathogenesis in vivo. Two-day-old mice were inoculated intramuscularly or intracerebrally with either sigma1s(-) or sigma1s(+) reovirus strains. While viral replication in target organs did not differ between sigma1s(-) and sigma1s(+) viral strains, virus-induced caspase-3 activation and resultant histological tissue injury in both the heart and brain were significantly reduced in sigma1s(-) reovirus-infected animals. These results demonstrate that sigma1s is a determinant of the magnitude and extent of reovirus-induced apoptosis in both the heart and CNS and thereby contributes to reovirus pathogenesis and virulence.

JNK regulates the release of proapoptotic mitochondrial factors in reovirus-infected cells.

Reovirus-induced apoptosis is associated with activation of the proapoptotic mitogen-activated protein kinase c-Jun N-terminal kinase (JNK) and the JNK-associated transcription factor c-Jun. Here we show that reovirus-induced apoptosis and activation of caspase 3 are inhibited in cells deficient in MEK kinase 1, an upstream activator of JNK in reovirus-infected cells. Inhibition of JNK activity following reovirus infection delays the release of proapoptotic mitochondrial factors and the subsequent onset of apoptosis. In contrast, reovirus-induced apoptosis is not blocked by infection with adenovirus expressing dominant-negative c-Jun, and c-Jun activation does not correlate with apoptosis in reovirus-infected cells. This is the first report demonstrating that JNK is associated with regulation of mitochondrial pathways of apoptosis following viral infection.

Regional differences in viral growth and central nervous system injury correlate with apoptosis.

Infection of neonatal mice with reovirus T3 Dearing (T3D), the prototypic neurotropic reovirus, causes fatal encephalitis associated with neuronal injury and virus-induced apoptosis throughout the brain. T3D variant K (VarK) is an antigenic variant that has a nearly 1 million-fold reduction in neurovirulence following intracerebral (i.c.) inoculation compared to T3D and a restricted pattern of central nervous system injury with damage limited to the hippocampus, sparing other brain regions. We wished to determine whether the restricted pattern of VarK-induced injury was due to a reduced capacity to replicate in or injure cortical, as opposed to hippocampal, tissue. We found that following i.c. inoculation, VarK grew to similar titers as T3D in the hippocampus but had significantly lower titers in the cortex. Both viruses grew to identical titers and infected the same percentage of cells in mouse primary hippocampal cultures (MHC). In mouse primary cortical cultures (MCC) both the number of infected cells and the viral yield per infected cell were significantly lower for VarK than T3D. VarK-induced apoptosis was limited to the hippocampus in vivo, and in vitro both viruses induced apoptosis equally in MHC but VarK induced significantly less apoptosis than T3D in MCC. Growth of T3D in MCC was reduced to levels comparable to those of VarK following treatment of MCC with caspase inhibitors. Conversely, induction of apoptosis in VarK-infected MCC with fatty acid synthase-activating antibody significantly enhanced viral yield. These results suggest that the decreased neurovirulence of VarK may be due to its failure to efficiently induce apoptosis in cortical neurons.

Does Toll-like receptor 3 play a biological role in virus infections?

The Toll-like receptor (TLR) family functions to recognize conserved microbial and viral structures with the purpose of activating signal pathways to instigate immune responses against infections by these organisms. For example, in vitro studies reveal that the TLR3 ligand is a double-stranded RNA (dsRNA), a product of viral infections. From this observation, it has been proposed that TLR3 is likely an important first signal for virus infections. We approached this issue by investigating the role of TLR3 in four different infectious viral models (lymphocytic choriomeningitis virus (LCMV), vesicular stomatitis virus (VSV), murine cytomegalovirus (MCMV), and reovirus) and in TLR3 genetically deficient ((-/-)) mice. Our results indicate that TLR3 is not universally required for the generation of effective antiviral responses because the absence of TLR3 does not alter either viral pathogenesis or impair host's generation of adaptive antiviral responses to these viruses.

Progressive multifocal leukoencephalopathy and apoptosis of infected oligodendrocytes in the central nervous system of patients with and without AIDS.

CONTEXT: Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system (CNS) caused by JC virus (JCV) that occurs in immunocompromised patients. Demyelination of the CNS is a consequence of virus-induced killing of oligodendrocytes, although the exact mechanism of cell death is unknown. OBJECTIVE: To examine archival autopsy and surgical pathologic specimens from 8 patients with PML, including 6 patients with human immunodeficiency virus (HIV)-associated PML and 2 patients with non-HIV-associated PML, for evidence of apoptosis. DESIGN: Apoptotic cells were identified by TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end in situ labeling) or immunohistochemical detection of activated caspase 3. The JCV-infected cells were identified by in situ hybridization for viral transcripts or immunohistochemical analysis for JCV T antigen. RESULTS: Apoptosis of JCV-infected oligodendrocyte apoptosis was a prominent feature in all cases of both HIV- and non-HIV-associated PML. There were no differences between number or distribution of apoptotic cells identified by TUNEL or immunohistochemical analysis for activated caspase 3. Bizarre astrocytes were occasionally positive for JCV but were not apoptotic. Neurons, astrocytes, macrophages, and oligodendrocytes remote from lesions were neither apoptotic nor JCV infected. CONCLUSIONS: Our study demonstrates that apoptosis occurs in oligodendrocytes associated with demyelinated lesions of patients with both HIV-associated and non-HIV-associated PML. There were no differences in degree, location, or type of infected or apoptotic cells between patients with HIV-associated and non-HIV-associated PML. The extent of apoptosis did not correlate with the presence or intensity of host inflammatory response. Accumulation of viral particles in nuclei of infected cells made it difficult to identify morphologic changes in the nucleus typically associated with apoptosis.

Central nervous system apoptosis in human herpes simplex virus and cytomegalovirus encephalitis.

Central nervous system (CNS) specimens from 10 immunocompetent patients with herpes simplex encephalitis (HSE) and 3 infants with congenital cytomegalovirus (CMV) encephalitis were analyzed to determine whether apoptosis is a feature of CNS injury in these patients. Apoptotic neurons and glia were detected in significant numbers in acute HSE and CMV encephalitis. Occurring predominantly in areas of productive viral infection, apoptosis appeared to result from direct viral injury to neurons and was not dependent on inflammatory T cell responses. In contrast to patients with acute cases, patients with late sequelae of HSE or CMV had no detectable virus and minimal neuronal or glial apoptosis, regardless of the degree of inflammation. This is the first demonstration of apoptotic neuronal death in humans with HSE. These results suggest that neuronal apoptosis is an important contributing factor to acute CNS injury and may serve as a novel therapeutic target in these patients.

Reovirus-induced neuronal apoptosis is mediated by caspase 3 and is associated with the activation of death receptors.

Reovirus infection of the central nervous system (CNS) is an important experimental system for understanding the pathogenesis of neurotropic viral infection. Infection of neonatal mice with T3 reoviruses causes lethal encephalitis in which injury results from virus-induced apoptosis. We now show that this apoptosis in vivo is associated with activation of caspase 3, and use neuroblastoma and primary neuronal cultures to identify the cellular pathways involved. Reovirus-induced apoptosis in neuronal cultures is initiated by activation of the tumor necrosis factor (TNF) receptor superfamily death receptors and is inhibited by treatment with soluble death receptors (DRs). The DR-associated initiator caspase, caspase 8, is activated following infection, this activation is inhibited by a cell-permeable peptide inhibitor (IETD-CHO). In contrast to our previous findings in non-neuronal cell lines, reovirus-induced neuronal apoptosis is not accompanied by significant release of cytochrome c from the mitochondria or with caspase 9 activation following infection. This suggests that in neuronal cells, unlike their non-neuronal counterparts, the mitochondria-mediated apoptotic pathway associated with cytochrome c release and caspase 9 activation does not play a significant role in augmenting reovirus-induced apoptosis. Consistent with these results, peptide caspase inhibitors show a hierarchy of efficacy in inhibiting reovirus-induced apoptosis, with inhibitors of caspase 3 > caspase 8 >>> caspase 9. These studies provide a comprehensive profile of the pattern of virus-induced apoptotic pathway activation in neuronal culture.