Stability of Conducting Polymer-Coated Carbon Microfibers for Long-Term Electrical Stimulation of Injured Neural Tissue.

Electroactive microfiber-based scaffolds aid neural tissue repair. Carbon microfibers (CMFs) coated with the conducting polymer poly(3,4-ethylenedioxythiophene) doped with poly[(4-styrenesulfonic acid)-co-(maleic acid)] (PEDOT:PSS-co-MA) provide efficient support and guidance to regrowing axons across spinal cord lesions in rodents and pigs. We investigated the electrical and structural performance of PEDOT:PSS-co-MA-coated carbon MFs (PCMFs) for long-term, biphasic electrical stimulation (ES). Chronopotentiometry and electrochemical impedance spectroscopy (EIS) allowed the characterization of charge transfer in PCMFs during ES in vitro, and morphological changes were assessed by scanning electron microscopy (SEM). PCMFs that were 4 mm long withstood two-million-biphasic pulses without reaching cytotoxic voltages, with a 6 mm length producing optimal results. Although EIS and SEM unveiled some polymer deterioration in the 6 mm PCMFs, no significant changes in voltage excursions appeared. For the preliminary testing of the electrical performance of PCMFs in vivo, we used 12 mm long, 20-microfiber assemblies interconnected by metallic microwires. PCMFs-assemblies were implanted in two spinal cord-injured pigs and submitted to ES for 10 days. A cobalt-alloy interconnected assembly showed safe voltages for about 1.5 million-pulses and was electrically functional at 1-month post-implantation, suggesting its suitability for sub-chronic ES, as likely required for spinal cord repair. However, improving polymer adhesion to the carbon substrate is still needed to use PCMFs for prolonged ES.

Composite Fibrin/Carbon Microfiber Implants for Bridging Spinal Cord Injury: A Translational Approach in Pigs.

Biomaterials may enhance neural repair after spinal cord injury (SCI) and testing their functionality in large animals is essential to achieve successful clinical translation. This work developed a porcine contusion/compression SCI model to investigate the consequences of myelotomy and implantation of fibrin gel containing biofunctionalized carbon microfibers (MFs). Fourteen pigs were distributed in SCI, SCI/myelotomy, and SCI/myelotomy/implant groups. An automated device was used for SCI. A dorsal myelotomy was performed on the lesion site at 1 day post-injury for removing cloths and devitalized tissue. Bundles of MFs coated with a conducting polymer and cell adhesion molecules were embedded in fibrin gel and used to bridge the spinal cord cavity. Reproducible lesions of about 1 cm in length were obtained. Myelotomy and lesion debridement caused no further neural damage compared to SCI alone but had little positive effect on neural regrowth. The MFs/fibrin gel implant facilitated axonal sprouting, elongation, and alignment within the lesion. However, the implant also increased lesion volume and was ineffective in preventing fibrosis, thus precluding functional neural regeneration. Our results indicate that myelotomy and lesion debridement can be advantageously used for implanting MF-based scaffolds. However, the implants need refinement and pharmaceuticals will be necessary to limit scarring.

Composite Fibrin and Carbon Microfibre Implant to Modulate Postraumatic Inflammation after Spinal Cord Injury.

Poor functional recovery after spinal cord injury (SCI) drives the development of novel strategies to manage this devastating condition. We recently showed promising immunomodulatory and pro-regenerative actions of bio-functionalized carbon microfibres (MFs) implanted in a rodent model of SCI. In order to maximize tissue repair while easing MF implantation, we produced a composite implant based on the embedding of several MFs within a fibrin hydrogel. We used intravital imaging of fluorescent reporter mice at the early stages and spinal sections of the same animals 3 months later to characterize the neuroinflammatory response to the implant and its impact on axonal regeneration. Whereas fibrin alone was inert in the first week, its enzymatic degradation drove the chronic activation of microglial cells and axonal degeneration within 3 months. However, the presence of MFs inside the fibrin hydrogel slowed down fibrin degradation and boosted the early recruitment of immune cells. Noteworthy, there was an enhanced contribution of monocyte-derived dendritic cells (moDCs), preceding a faster transition toward an anti-inflammatory environment with increased axonal regeneration over 3 months. The inclusion of MF here ensured the long-term biocompatibility of fibrin hydrogels, which would otherwise preclude successful spinal cord regeneration.

The Cortical Motor System in the Domestic Pig: Origin and Termination of the Corticospinal Tract and Cortico-Brainstem Projections.

The anatomy of the cortical motor system and its relationship to motor repertoire in artiodactyls is for the most part unknown. We studied the origin and termination of the corticospinal tract (CST) and cortico-brainstem projections in domestic pigs. Pyramidal neurons were retrogradely labeled by injecting aminostilbamidine in the spinal segment C1. After identifying the dual origin of the porcine CST in the primary motor cortex (M1) and premotor cortex (PM), the axons descending from those regions to the spinal cord and brainstem were anterogradely labeled by unilateral injections of dextran alexa-594 in M1 and dextran alexa-488 in PM. Numerous corticospinal projections from M1 and PM were detected up to T6 spinal segment and showed a similar pattern of decussation and distribution in the white matter funiculi and the gray matter laminae. They terminated mostly on dendrites of the lateral intermediate laminae and the internal basilar nucleus, and some innervated the ventromedial laminae, but were essentially absent in lateral laminae IX. Corticofugal axons terminated predominantly ipsilaterally in the midbrain and bilaterally in the medulla oblongata. Most corticorubral projections arose from M1, whereas the mesencephalic reticular formation, superior colliculus, lateral reticular nucleus, gigantocellular reticular nucleus, and raphe received abundant axonal contacts from both M1 and PM. Our data suggest that the porcine cortical motor system has some common features with that of primates and humans and may control posture and movement through parallel motor descending pathways. However, less cortical regions project to the spinal cord in pigs, and the CST neither seems to reach the lumbar enlargement nor to have a significant direct innervation of cervical, foreleg motoneurons.

Neuropathological and Motor Impairments after Incomplete Cervical Spinal Cord Injury in Pigs.

Humans, primates, and rodents with cervical spinal cord injury (SCI) show permanent sensorimotor dysfunction of the upper/forelimb as consequence of axonal damage and local neuronal death. This work aimed at characterizing a model of cervical SCI in domestic pigs in which hemisection with excision of 1 cm of spinal cord was performed to reproduce the loss of neural tissue observed in human neuropathology. Posture and motor control were assessed over 3 months by scales and kinematics of treadmill locomotion. Histological measurements included lesion length, atrophy of the adjacent spinal cord segments, and neuronal death. In some animals, the retrograde neural tracer aminostilbamidine was injected in segments caudal to the lesion to visualize propriospinal projection neurons. Neuronal loss extended for 4-6 mm from the lesion borders and was more severe in the ipsilateral, caudal spinal cord stump. Axonal Wallerian degeneration was observed caudally and rostrally, associated with marked atrophy of the white matter in the spinal cord segments adjacent to the lesion. The pigs showed chronic monoplegia or severe monoparesis of the foreleg ipsilateral to the lesion, whereas the trunk and the other legs had postural and motor impairments that substantially improved during the first month post-lesion. Adaptations of the walking cycle such as those reported for rats and humans ameliorated the negative impact of focal neurological deficits on locomotor performance. These results provide a baseline of behavior and histology in a porcine model of cervical spinal cord hemisection that can be used for translational research in SCI therapeutics.

Electro-responsive films containing voltage responsive gated mesoporous silica nanoparticles grafted onto PEDOT-based conducting polymer.

The characteristics and electromechanical properties of conductive polymers together to their biocompatibility have boosted their application as a suitable tool in regenerative medicine and tissue engineering. However, conducting polymers as drug release materials are far from being ideal. A possibility to overcome this drawback is to combine conducting polymers with on-command delivery particles with inherent high-loading capacity. In this scenario, we report here the preparation of conduction polymers containing gated mesoporous silica nanoparticles (MSN) loaded with a cargo that is delivered on command by electro-chemical stimuli increasing the potential use of conducting polymers as controlled delivery systems. MSNs are loaded with Rhodamine B (Rh B), anchored to the conductive polymer poly(3,4-ethylenedioxythiophene) (PEDOT) doped with poly[(4-styrenesulfonic acid)-co-(maleic acid)], functionalized with a bipyridinium derivative and pores are capped with heparin (P3) by electrostatic interactions. P3 releases the entrapped cargo after the application of -640 mV voltage versus the saturated calomel electrode (SCE). Pore opening in the nanoparticles and dye delivery is ascribed to both (i) the reduction of the grafted bipyridinium derivative and (ii) the polarization of the conducting polymer electrode to negative potentials that induce detachment of positively charged heparin from the surface of the nanoparticles. Biocompatibility and cargo release studies were carried out in HeLa cells cultures.

Enhanced spinal cord microstimulation using conducting polymer-coated carbon microfibers.

Intraspinal microstimulation (ISMS) may help to restore motor functions after spinal cord injury. ISMS caudal to the lesion activates motoneurons and evokes selective movements with graded force in rats and other mammals. We investigated the safety and effectiveness of conducting polymer (CP)-coated carbon microfibers (CMFs) for ISMS. 7-µm-diameter CMFs coated with poly(3,4-ethylenedioxythiophene) doped with poly[(4-styrenesulfonic acid)-co-(maleic acid)] (PEDOT:PSS-co-MA) were used to apply current-controlled biphasic electric pulses at the cervical spinal cord (C7) of anesthetized rats. Electrode performance and motoneuron activation, as readout by voltage transients, cyclic voltammetry, electrochemical impedance spectroscopy, electromyography (EMG) and foreleg kinematics, were investigated as a function of microfiber length (50 µm vs. 250 µm) and presence of polymer coating. The microfibers were very effective in activating specific spinal motoneurons, with the lowest stimulus thresholds varying between -28 µA and -46 µA in the cathodic phase. EMG and kinematic thresholds decreased when the microfiber tip approached the targeted motor nucleus (triceps brachii, t.b.) from the dorsal spinal cord surface. ISMS with polymer-coated CMFs produced higher electrical activity in the t.b. fascicles compared to bare CMFs. PEDOT:PSS-co-MA coating of 250-µm CMFs avoided the generation of unsafe overvoltages for biphasic pulses up to -80/+40 µA in vivo, although the positive effect of the conducting polymer was lost after the application of a few thousands of electric pulses. Thus, CP-coated CMFs may provide an effective and minimally invasive electrode for ISMS; however, polymer optimization is still required to improve its electrical stability and safety for long-term use. Statement of significance Intraspinal microstimulation may restore motor functions after spinal cord injury. In the present study we demonstrate that carbon microfibers (CMFs) coated with the conducting polymer PEDOT:PSS-co-MA can be advantageously used for this purpose. These microfibers allow for both effective and temporarily safe electrical activation of spinal motor circuits with high spatial resolution. The presence of the polymer enhances the effectiveness of the electrical stimuli to recruit spinal motoneurons. Thus, conducting polymer-coated CMFs have potential for the development of advanced neuroprosthetic devices, although further improvements are needed regarding their electrochemical and mechanical stability.

Favorable Biological Responses of Neural Cells and Tissue Interacting with Graphene Oxide Microfibers.

Neural tissue engineering approaches show increasing promise for the treatment of neural diseases including spinal cord injury, for which an efficient therapy is still missing. Encouraged by both positive findings on the interaction of carbon nanomaterials such as graphene with neural components and the necessity of more efficient guidance structures for neural repair, we herein study the potential of reduced graphene oxide (rGO) microfibers as substrates for neural growth in the injured central neural tissue. Compact, bendable, and conductive fibers are obtained. When coated with neural adhesive molecules (poly-l-lysine and N-cadherin), these microfibers behave as supportive substrates of highly interconnected cultures composed of neurons and glial cells for up to 21 days. Synaptic contacts close to rGO are identified. Interestingly, the colonization by meningeal fibroblasts is dramatically hindered by N-cadherin coating. Finally, in vivo studies reveal the feasible implantation of these rGO microfibers as a guidance platform in the injured rat spinal cord, without evident signs of subacute local toxicity. These positive findings boost further investigation at longer implantation times to prove the utility of these substrates as components of advanced therapies for enhancing repair in the damaged central neural tissue including the injured spinal cord.

Biofunctionalized PEDOT-coated microfibers for the treatment of spinal cord injury.

Poly(3, 4-ethylenedioxythiophene)-coated carbon microfibers (PEDOT-MFs) hold promise for developing advanced neuroprostheses and neural repair devices. We investigated the chronic cellular responses to PEDOT-MFs implanted into the uninjured and the transected rat spinal cord, and compared the effects of polymer surface biofunctionalization with covalently attached polylysine (PLL) or a multimolecular complex of PLL, heparin, basic fibroblast growth factor (bFGF), and fibronectin. An alginate gel was used to facilitate microfiber implantation and reduce connective tissue scarring after spinal cord injury (SCI). PLL/heparin/bFGF/fibronectin-functionalized PEDOT-MFs showed excellent integration within the uninjured and injured spinal cord, frequently establishing contact with neuronal somas, axons, dendrites and glial cells, accompanied by very little or absent scarring response. On the contrary, non-functionalized and PLL-functionalized microfibers provoked inflammation and fibrosis with loss of neural elements in the surrounding tissue. Within the lesion, the PEDOT-MFs by themselves facilitated longitudinal alignment of migratory cells and growing axons, and their modification with PLL/heparin/bFGF/fibronectin promoted tissue healing, enhancing blood vessel formation and axonal regeneration without increasing inflammation. These results support the incorporation of biofunctionalized electroconducting microfibers in neuro-electronic interfaces and lesion-bridging systems for the treatment of SCI.

Glial progenitor cell migration promotes CNS axon growth on functionalized electroconducting microfibers.

Electroactive systems that promote directional axonal growth and migration of glial progenitor cells (GPC) are needed for the treatment of neurological injuries. We report the functionalization of electroconducting microfibers with multiple biomolecules that synergistically stimulate the proliferation and migration of GPC, which in turn induce axonal elongation from embryonic cerebral cortex neurons. PEDOT doped with poly[(4-styrenesulfonic acid)-co-(maleic acid)] was synthesized on carbon microfibers and used for covalent attachment of molecules to the electroactive surface. The molecular complexes that promoted GPC proliferation and migration, followed by axonal extension, were composed of polylysine, heparin, basic fibroblast growth factor (bFGF), and matricellular proteins; the combination of bFGF with vitronectin or fibronectin being indispensable for sustained glial and axonal growth. The rate of glial-induced axonal elongation was about threefold that of axons growing directly on microfibers functionalized with polylysine alone. Electrical stimuli applied through the microfibers released bFGF and fibronectin from the polymer surface, consequently reducing GPC proliferation and promoting their differentiation into astrocytes, without causing cell detachment or toxicity. These results suggest that functionalized electroactive microfibers may provide a multifunctional tool for controlling neuron-glia interactions and enhancing neural repair. STATEMENT OF SIGNIFICANCE: We report a multiple surface functionalization strategy for electroconducting microfibers (MFs), in order to promote proliferation and guided migration of glial precursor cells (GPC) and consequently create a permissive substrate for elongation of central nervous system (CNS) axons. GPC divided and migrated extensively on the functionalized MFs, leading to fast elongation of embryonic cerebral cortex axons. The application of electric pulses thorough the MFs controlled glial cell division and differentiation. The functionalized MFs provide an advanced tool for neural tissue engineering and for controlling neuron-glial interactions. CNS axonal growth associated to migratory glial precursors, together with the possibility of directing glial differentiation by electrical stimuli applied through the MFs, open a new research avenue to explore for CNS repair.

Biofunctionalized Conducting Polymer/Carbon Microfiber Electrodes for Ultrasensitive Neural Recordings.

Carbon microfibers (MFs) coated with conducting polymers may provide a solution for long-term recording of activity from individual or small groups of neurons. Attaching cell adhesion molecules to the electro-sensitive surface might further improve electrode-neuron contact, thus enhancing signal stability and fidelity. We fabricated biofunctionalized microelectrodes consisting of 7-µm diameter carbon MFs coated with poly(3,4-ethylenedioxythiophene) doped with poly[(4-styrenesulfonic acid)-co-(maleic acid)] ( PEDOT: PSS-co-MA), and linked N-Cadherin to the polymer surface. These electrodes were tested for recording artificially generated electric potentials, as well as multiunit activity (MUA), sharp wave-ripple complexes (SWRs), and field excitatory postsynaptic potentials (fEPSPs) in rat hippocampal slices. The effects of electrode length and functionalization were compared. PEDOT: PSS-co-MA coating improved electric current detection and reduced the electrical noise but had no significant effect on the amplitude of recorded biopotentials. Surface biofunctionalization lowered the electric current flow, and further reduced the electrical noise. Additionally, it increased the amplitude of the recorded MUA, finally doubling the signal-to-noise ratio achieved with bare carbon MFs. Biofunctionalization benefits were apparent only for potentials from cells putatively adjacent to the microelectrode. Analysis of fEPSPs excluded adverse effects of functionalized electrodes in basal synaptic transmission. These results demonstrate the possibility of enhancing the amplitude and signal-to-noise ratio of neural recordings by coating the microelectrodes with conducting polymers modified with neural cell adhesion molecules, and support the use of biofunctionalized MFs in advanced neuroprosthetic devices.

Influence of hypotension and nerve root section on the ability to mobilize the spinal cord during spine surgery. An experimental study in a pig model.

BACKGROUND CONTEXT: The correction of severe spinal deformities by an isolated posterior approach often involves cord manipulation together with hypotensive anesthesia. To date, the efficiency of methods to increase the tolerance of the cord to displacement and the influence of hypotension on this tolerance is yet to be assessed. PURPOSE: The objective of this study was to determine the limits of cord displacement before the disappearance of neurophysiologic signals. The influence of the type of force applied, the section of the roots, and the induced hypotension on the cord's tolerance to displacement was also assessed. STUDY DESIGN: Experimental study using a domestic pig model. OUTCOME MEASURES: Successive records of cord-to-cord motor evoked potentials were obtained during displacement maneuvers. Displacing forces were released immediately after the absence of neurophysiologic signals. METHODS: Surgical procedures were performed under conventional general anesthesia. The spinal cord and nerve roots from T6 to T10 levels were exposed by excision of the posterior elements, allowing for free cord movement. Three groups were established according to the method of spinal cord displacement: the separation (Group 1, n=5), the root stump pull (Group 2, n=5), and the torsion groups (Group 3, n=5). An electromechanical external device was used to apply the displacing forces. The three displacement tests were repeated after sectioning the adjacent nerve roots. The experiments were first carried out under normotension and afterward under induced hypotension. RESULTS: In Group 1, evoked potential disappeared with a displacement of 10.1±1.6 mm with unharmed roots and 15.3±4.7 mm after the sectioning of four adjacent roots (p<.01). After induced hypotension, potentials were lost at 4.0±1.2 mm (p<.01). In Group 2, the absence of potentials occurred at 20.0±4.3 mm and increased to 23.5±2.1 mm (p<.05) after cutting the two contralateral roots. Under hypotensive conditions, the loss of neurophysiologic signals was detected at 5.3±1.2 mm (p<.01). In Group 3, the cord allowed torsion of 95.3±.2° that increased to 112.4±7.1° if the contralateral roots were cut. Under hypotension, the loss of potentials was found at 20±6.2° (p<.01). CONCLUSIONS: In this experimental model, it was possible to displace the thoracic spinal cord by a distance superior to the spinal cord width without suffering neurophysiologic changes. The limits of cord displacement increase when the adjacent nerve roots are sacrificed. Induced hypotension had a dramatic effect on the tolerance of the spinal cord for displacement. This work has an important clinical significance because induced hypotension during specific spine surgery procedures requiring spinal cord manipulation in humans may increase the risk of neurologic spinal cord injury.

Chondroitin sulphate-based 3D scaffolds containing MWCNTs for nervous tissue repair.

Nervous tissue lesions are an important social concern due to their increasing prevalence and their high sanitary costs. Their treatment still remains a challenge because of the reduced ability of nervous tissue to regenerate, its intrinsic structural and functional complexity and the rapid formation of fibroglial scars inhibiting neural repair. Herein, we show that 3D porous scaffolds made of chondroitin sulphate (CS), a major regulatory component of the nervous tissue, and multi-walled carbon nanotubes (MWCNTs) are selective substrates for the formation of a viable and neuron-enriched network with a transitory low glial content. Scaffolds have been fabricated by using the ice segregation-induced self-assembly technique and cultured with embryonic neural progenitor cells. Cell adhesion, morphology, viability, neuron/glial differentiation, calcium signaling dynamics, and mitochondrial activity have been studied over time on the scaffolds and compared to appropriate 2D control substrates. Our results indicate the formation of viable cultures enriched in neuron cells for up to 20 days, with ability to display calcium transients and active mitochondria, even in the absence of poly-D-lysine coating. A synergistic neural-permissive signaling from both the scaffold structure and its components (i.e., MWCNTs and CS) is suggested as the major responsible factor for these findings. We anticipate that these scaffolds may serve nerve regeneration if implanted in the acute phase after injury, as it is during the first stages of graft implantation when the most critical sequence of phenomena takes place to drive either nervous regeneration or fibroglial scar formation. The temporary glial inhibition found may be, indeed, beneficial for promoting the formation of neuron-enriched circuits at early phases while guaranteeing posterior glial integration to support longer-term neuron survival and activity.

N-Cadherin- and L1-functionalised conducting polymers for synergistic stimulation and guidance of neural cell growth.

Conducting polymers are promising materials for advanced neuroprostheses and neural repair devices. However, these challenging technologies demand stable presentation of multiple biomolecules on the polymer surface and fabrication of scaffolds suitable for implantation. We electrosynthesised poly(3,4-ethylenedioxythiophene) doped with poly[(4-styrenesulfonic acid)-co-(maleic acid)] (PEDOT:PSS-co-MA) on gold-coated surfaces or carbon microfibres, functionalised the polymer by covalent immobilisation of anti-IgG antibodies and subsequent binding of N-Cadherin and L1 recombinant proteins, and used these materials as substrates for culturing cerebral cortex neurons. N-Cadherin and L1 were much more effective than polylysine in promoting axonal elongation and collateralisation on the polymer. However, N-Cadherin also induced cell migration and dendritic extension and branching, whereas L1 inhibited dendrites. Dual functionalisation with N-Cadherin and L1 produced synergistic effects on neuronal growth that could not be achieved with either of the proteins when used alone. PEDOT:PSS-co-MA electrosynthesised on carbon microfibres showed good electrochemical properties and, when biofunctionalised with N-Cadherin or L1, stimulated very long and guided axonal elongation. Finally, electrochemical impedance spectroscopy, cyclic voltammetry and chronoamperometry showed that the good electrical properties of PEDOT:PSS-co-MA were not degraded by covalent peptide attachment, indicating that this polymer is suitable for multiple biofunctionalisation of electroactive surfaces in neuroprosthetic and lesion-bridging applications.

Dynamic motor compensations with permanent, focal loss of forelimb force after cervical spinal cord injury.

Incomplete cervical lesion is the most common type of human spinal cord injury (SCI) and causes permanent paresis of arm muscles, a phenomenon still incompletely understood in physiopathological and neuroanatomical terms. We performed spinal cord hemisection in adult rats at the caudal part of the segment C6, just rostral to the bulk of triceps brachii motoneurons, and analyzed the forces and kinematics of locomotion up to 4 months postlesion to determine the nature of motor function loss and recovery. A dramatic (50%), immediate and permanent loss of extensor force occurred in the forelimb but not in the hind limb of the injured side, accompanied by elbow and wrist kinematic impairments and early adaptations of whole-body movements that initially compensated the balance but changed continuously over the follow-up period to allow effective locomotion. Overuse of both contralateral legs and ipsilateral hind leg was evidenced since 5 days postlesion. Ipsilateral foreleg deficits resulted mainly from interruption of axons that innervate the spinal cord segments caudal to the lesion, because chronic loss (about 35%) of synapses was detected at C7 while only 14% of triceps braquii motoneurons died, as assessed by synaptophysin immunohistochemistry and retrograde neural tracing, respectively. We also found a large pool of propriospinal neurons projecting from C2-C5 to C7 in normal rats, with topographical features similar to the propriospinal premotoneuronal system of cats and primates. Thus, concurrent axotomy at C6 of brain descending axons and cervical propriospinal axons likely hampered spontaneous recovery of the focal neurological impairments.

Spinal cord direct current stimulation: finite element analysis of the electric field and current density.

Applied low-intensity direct current (DC) stimulates and directs axonal growth in models of spinal cord injury (SCI) and may have therapeutic value in humans. Using higher electric strengths will probably increase the beneficial effects, but this faces the risk of tissue damage by electricity or toxic reactions at the electrode-tissue interface. To inform the optimisation of DC-based therapeutics, we developed a finite element model (FEM) of the human cervical spine and calculated the electric fields (EFs) and current densities produced by electrodes of different size, geometry and location. The presence of SCI was also considered. Three disc electrodes placed outside the spine produced low-intensity, uneven EFs, whereas the EFs generated by the same electrodes located epidurally were about three times more intense. Changes in electrical conductivity after SCI had little effect on the EF magnitudes. Uniformly distributed EFs were obtained with five disc electrodes placed around the dura mater, but not with a paddle-type electrode placed in the dorsal epidural space. Replacing the five disc electrodes by a single, large band electrode yielded EFs > 5 mV/mm with relatively low current density (2.5 µA/mm(2)) applied. With further optimisation, epidural, single-band electrodes might enhance the effectiveness of spinal cord DC stimulation.

Bioelectrochemical control of neural cell development on conducting polymers.

Electrically conducting polymers hold promise for developing advanced neuroprostheses, bionic systems and neural repair devices. Among them, poly(3, 4-ethylenedioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS) exhibits superior physicochemical properties but biocompatibility issues have limited its use. We describe combinations of electrochemical and molecule self-assembling methods to consistently control neural cell development on PEDOT:PSS while maintaining very low interfacial impedance. Electro-adsorbed polylysine enabled long-term neuronal survival and growth on the nanostructured polymer. Neurite extension was strongly inhibited by an additional layer of PSS or heparin, which in turn could be either removed electrically or further coated with spermine to activate cell growth. Binding basic fibroblast growth factor (bFGF) to the heparin layer inhibited neurons but promoted proliferation and migration of precursor cells. This methodology may orchestrate neural cell behavior on electroactive polymers, thus improving cell/electrode communication in prosthetic devices and providing a platform for tissue repair strategies.

Compartmentalization in the triceps brachii motoneuron nucleus and its relation to muscle architecture.

Each fascicle of the triceps brachii (TB) can be independently recruited during movement execution. To investigate the anatomical basis of this selective control and to gain insight into its functional role in adult rats, we carried out a triple retrograde tracing of the motoneurons (MNs) innervating each TB head, and performed muscle ATPase histochemistry and histology to correlate the size of the MN pools with the number and type of muscle fibers innervated. No double-labeled MNs were found, demonstrating that each TB head is innervated by a completely independent MN subnucleus. Absolute cell counts determined that the long fascicle had the largest MN subnucleus, followed by the medial and the lateral fascicles. MNs of the three fascicles intermingled extensively in the rostral part of the spinal motor column, while the caudal part of the column comprised mostly MNs innervating the long fascicle. Muscle histology and average innervation ratios estimated from absolute MN counts showed that the medial head was predominantly formed by small type I fibers and motor units (69 fibers/MN). In contrast, the lateral fascicle comprised a great quantity of large type IIb fibers and motor units (179 fibers/MN), whereas the long head consisted of a more balanced mixture of fiber types and motor units (99 fibers/MN). Taking into account the mechanical and physiological heterogeneity of the TB, our findings suggest that each fascicle may be considered an independent muscle with specific functional roles.

Titanium oxide as substrate for neural cell growth.

Titanium oxide has antiinflammatory activity and tunable electrochemical behavior that make it an attractive material for the fabrication of implantable devices. The most stable composition is TiO2 and occurs mainly in three polymorphs, namely, anatase, rutile, and brookite, which differ in its crystallochemical properties. Here, we report the preparation of rutile surfaces that permit good adherence and axonal growth of cultured rat cerebral cortex neurons. Rutile disks were obtained by sinterization of TiO2 powders of commercial origin or precipitated from hydrolysis of Ti(IV)-isopropoxide. Commercial powders sintered at 1300-1600 degrees C produced rutile surfaces with abnormal grain growth, probably because of impurities of the powders. Neurons cultured on those surfaces survived in variable numbers and showed fewer neurites than on control materials. On the other hand, rutile sintered from precipitated powders had less contaminants and more homogenous grain growth. By adjusting the thermal treatment it was possible to obtain surfaces performing well as substrate for neuron survival for at least 10 days. Some surfaces permitted normal axonal elongation, whereas dendrite growth was generally impaired. These findings support the potential use of titanium oxide in neuroprostheses and other devices demanding materials with enhanced properties in terms of biocompatibility and axon growth promotion.

Locomotor deficits and adaptive mechanisms after thoracic spinal cord contusion in the adult rat.

The rat is widely used for modeling human spinal cord injury (SCI) and paraplegia. However, quadruped animals adapt trunk, forelimb and hindlimb movements to compensate for deficits, improving their behavioral scores and complicating the interpretation of spontaneous and treatment-induced function recovery. The kinematics of locomotion was studied in rats, both normal and after SCI (T9 contusion), and variables indicative of hindlimb function were related to brain-spinal cord connections (BSCC) spared during lesioning. Normal animals showed forward velocities characteristic of fast walking. The hind paw was placed approximately three centimeters in front of the hip at the initial contact. Hip height decreased during the first third of hindlimb stance and increased later. Mild and moderate spinal cord contusions destroyed the gray matter and adjacent axons but spared the ventrolateral tracts to various degrees. Injured animals placed the hindpaw in a more caudal position than normal and showed reduced forward velocity and hip height. Knee extension was also impaired, and both hindlimb and forelimb steps were adapted to compensate for the deficits. BSCC was estimated by averaging the transverse area of white matter at the lesion epicenter and the percentage of brain neurons labeled after peroxidase injection into L2 and L3. Recovery of hindlimb motor function was proportional to the amount of BSCC. On average, injured animals retained 18% of BSSC and recovered 23% of hindlimb function. These findings show that kinematic analysis is a reliable tool for assessing locomotor deficits and compensations and suggest limited spontaneous motor plasticity after SCI.