Injuries to the central nervous system (CNS) have traumatic consequences such as irreparable disability due to the inability of the CNS to regenerate injured nerve fibres. The aim of the work presented here was to develop a scaffold which potentially provides guidance to axons in the injured spinal cord thus facilitating signal transduction. A poly-(lactic-co-glycolic acid) (PLGA, PLA:PGA ratio of 75:25) flat sheet membrane scaffold was created using phase inversion with N-methyl pyrrolidinone (NMP) as the solvent and water as the non-solvent for immersion precipitation. PLGA flat sheet membranes were exposed to surface treatments including aminolysis, peptide immobilisation and ozonation in order to achieve higher cell attachment of PC12 cells, a cell line which was cloned from a solid pheochromocytoma tumour of white rats, and used as a tool for measurement of regeneration. Cell attachment studies revealed no significant difference in cell attachment between modified and not-modified PLGA flat sheet membranes. However, the absence of foetal calf serum (FCS) resulted in fivefold higher cell attachment compared to medium supplemented with 10% FCS. A second scaffold was produced by electrospinning 10% (w/w) PLGA in a chloroform:methanol (CHCl3:MeOH) mixture in ratio of 3:1 resulting in a nanofibrous scaffold. Optimum settings for electrospinning were found to be 3 ml/h feeding rate, 15kV applied voltage and 11cm collector-to-needle distance. Random and aligned PLGA nanofibres were produced, with a fibre diameter of 530±140nm. PC12 cells attached and differentiated to the nanofibrous scaffold. When exposed to NGF these cells stopped dividing and extended neurites. On random fibres, neurite orientation was random, whereas on aligned fibres 63% of neurites grew with the fibre orientation ±15��ᵒ. After 7 days of exposure to NGF, cells had 1-4 neurites on random fibres, reaching a maximum length of 188μm, whereas on aligned fibres, cells had 1-2 neurites, reaching a maximum length of 400μm. PLGA nanofibres were also investigated as a delivery vehicle for bioactive molecules. For this, poly-L-lysine (PLL) was incorporated into electrospun PLGA nanofibres via emulsion electrospinning. PLGA-PLL nanofibres were significantly larger than PLGA nanofibres having a diameter of 830±190nm. In order to visualise the incorporation of PLL, FITC-PLL was electrospun und the resulting nanofibres fluoresced greed. Attachment of PC12s to PLGA-PLL nanofibres was not significantly different compared to PLGA nanofibres. Aligned PLGA-PLL nanofibres were shown to promote neurite outgrowth of PC12s with resulting neurites of up to twice the length compared to aligned PLGA nanofibres. The results suggest that PLGA nanofibres strongly influences neurite organisation, which is potentially useful for future therapeutic approaches. The work in this thesis has shown that electrospun PLGA nanofibre mats have the potential to be used as scaffolds for spinal cord repair addressing topographical guidance and delivery of bioactive molecules to the site of injury.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / From the need for a specific instrument to evaluate the quality of life of people with spinal cord injury in the Brazilian language, this study was conducted and had the following objectives: to translate and culturally adapt to Portuguese Ferrans and Powers Quality of Life Index Spinal Cord Injury Version â III and characterise the sample in terms of sociodemographic and clinical aspects. Methodological procedure, in which the authorization from the author was obtained. First, there was a stage of translating with the participation of six translators. Then, there was a cultural adaptation taking into account the opinion of five judges. The pretest sample consisted on 30 patients with spinal cord injury, which were selected from the database of the Center for Research and Consulting in Neurological Nursing, and which also met the criteria for inclusion. Data was collected from August to November of 2013 by the final version in Portuguese called Ãndice de LesÃo Medular e Qualidade De Vida â VersÃo III and a questionnaire with social, demographic and clinical data. The study was approved by the Ethics in Research Committee, under the number: 344.927/2013. An index with 74 items was obtained, it was divided into two parts (satisfaction / importance ), on which some adjustments were made for a better understanding. These adjustments resulted in: the addition of four new pronouns or articles to change the genre; nine exclusions of words or articles; two changes from a word to a term; four additional expressions for the purpose of illustrating; four adjusts in expressions; nine changes of only one word. Therefore, there was 24 modifications. It was observed that, in the criteria of semantics equivalence of the index, ortography was rated as âa very appropriate translationâ by a number greater than 87 % ; vocabulary and grammar, by 86%; idiomatic equivalence was rated by a number higher than 74%; experimental equivalence, higher than 78% and conceptual equivalence, by a number greater than 70%. Statistical analysis on semantic, idiomatic, experimental and conceptual evaluations by kappa showed a slight to moderate conformity between the pairs of analysis, adopting (p ≤ 0.05). It was concluded that this instrument, after being transculturally adapted, has proved to be appropriate under the semantic , idiomatic, experimental and conceptual views, as well as easy to use to evaluate the quality of life of people with spinal cord injury. The study enabled the expansion of scientific knowledge of nursing, especially for professionals in the neurological field, by making it possible to plan, intercede and evaluate the caring for the special needs of these patients. It is now possible to resort to a technology that takes into account a great number of subjective relationships that are not, generally, observed or disclosed. / A partir da necessidade de um instrumento especÃfico para avaliar a qualidade de vida de pessoas com lesÃo medular no idioma brasileiro, realizou-se este estudo que teve como objetivos: traduzir e adaptar culturalmente para a lÃngua portuguesa Ferrans and Powers Quality of Life Index Spinal Cord Injury Version â III e caracterizar a amostra quanto aos aspectos sociodemogrÃficos e clÃnicos. Estudo do tipo metodolÃgico, no qual obteve-se autorizaÃÃo da autora para utilizaÃÃo do instrumento. Inicialmente procedeu-se a etapa de traduÃÃo com a participaÃÃo de seis tradutores, em seguida a adaptaÃÃo cultural pela confrontaÃÃo de cinco juÃzes. A amostra do prÃ-teste constituiu-se de 30 pacientes com lesÃo medular selecionados a partir do banco de dados disponibilizados pelo NÃcleo de Pesquisa e ExtensÃo em Enfermagem NeurolÃgica, e que atenderam aos critÃrios de inclusÃo. A coleta de dados ocorreu nos meses de agosto a novembro/2013, por meio da utilizaÃÃo da versÃo final traduzida para o portuguÃs denominada de Ãndice de LesÃo Medular e Qualidade De Vida â VersÃo III e um questionÃrio com dados sociodemogrÃficos e clÃnicos. O estudo foi aprovado pelo Comità de Ãtica em Pesquisa, Parecer n 344.927/2013. Obteve-se um Ãndice com 74 itens, divididos em duas partes (satisfaÃÃo / importÃncia), com alguns ajustes para facilitar a compreensÃo, dos quais resultaram em quatro acrÃscimos de pronome ou artigo para variaÃÃo do gÃnero; nove retiradas de uma palavra ou artigo; duas mudanÃas de uma palavra para uma expressÃo; quatro expressÃes adicionais com o intuito de exemplificar; quatro ajustes em expressÃes; nove mudanÃas de apenas uma palavra. Desse modo, obteve-se ao todo 24 modificaÃÃes. Quanto aos critÃrios de equivalÃncia semÃntica do Ãndice observou-se que, em relaÃÃo a ortografia o percentual de itens avaliados como TraduÃÃo Muito Adequada foi superior a 87%, vocabulÃrio e gramÃtica a 86%. Na equivalÃncia idiomÃtica obteve-se valores superior a 74%, na equivalÃncia experimental superior a 78% e na equivalÃncia conceitual superior a 70%. A anÃlise estatÃstica para as avaliaÃÃes semÃntica, idiomÃtica, experimental e conceitual por meio do kappa apontou de ligeira a moderada a concordÃncia das anÃlises entre os pares, ao adotar (p ≤ 0,05). Conclui-se que este instrumento apÃs ser adaptado transculturalmente demonstrou ser adequado do ponto de vista semÃntico, idiomÃtico, experimental e conceitual, alÃm de fÃcil aplicaÃÃo para avaliar a qualidade de vida de pessoas com lesÃo medular. O estudo possibilitou a ampliaÃÃo do saber cientÃfico da enfermagem, em especial aos profissionais que atuam na Ãrea neurolÃgica, por possibilitar o planejamento, as intervenÃÃes e avaliaÃÃo de cuidados direcionados Ãs necessidades tÃo peculiares destes pacientes, podendo agora recorrer a uma tecnologia que mensura muitas relaÃÃes subjetivas e que nÃo sÃo observadas ou reveladas.

A forward dynamic model was developed to predict the performance of Spinal Cord Injured (SCI) individuals cycling an isokinetic ergometer using Neuromuscular Electrical Stimulation (NMES) to elicit contractions of the quadriceps, hamstring and gluteal muscles. Computer simulations were performed using three inter-connected models: a kinematic model of segmental linkages, a muscle model predicting forces in response to stimulation, and a kinetic model predicting ergometer pedal forces resulting from muscle stimulation. Specific model parameters for SCI individuals were determined through measurements from isometric and isokinetic contractions of the quadriceps muscles elicited using surface stimulation. The muscle model was fitted to data resulting from these isolated experiments in order to tailor the model's parameters to characteristics of muscles from SCI individuals. Isometric data from a range of knee angles were used to fit tendon slack lengths to the rectus femoris and vastus muscles. Adjustments to the quadriceps moment arm function were not able to improve the match between measured and modelled knee extension torques beyond those using moment arms taken from available literature. Similarly, literature values for constants from the muscle force - velocity relationship provided a satisfactory fit to the decline in torque with angular velocity, and parameter fitting did not improve this fit. Passive visco-elastic resistance remained constant for all velocities of extension except the highest (240 deg/s). Since knee angular velocities this high were not experienced during cycling, a visco-elastic dampener was not included within the present cycling model. The rise and fall in torque following NMES onset and cessation were used to fit constants to match the rate of change in torque. Constants for the rise in torque following NMES onset were significantly altered by changes in knee angle, with more extended angles taking longer for torque to rise. This effect was small, however, within the range of angles used during cycling, and consequently was not included within the cycling model. The decline in torque after NMES cessation was not affected by knee angle. A period of five minutes cyclical isometric activity of the quadriceps resulted in torque declining by more than 75% from rested levels. The activation time constants were largely unaffected by this fatigue, however, with only a small increase in the time for torque to decline, and no change in rise time or the delay between stimulation changes and resulting torque changes. The cycling model, therefore, did not incorporate any effect for changes in activation timing with fatigue. Performance of the full model was evaluated through measurements taken from SCI individuals cycling a constant velocity ergometer using NMES elicited contractions of the quadriceps, hamstring and gluteal muscles. Pedal transducers measured forces applied to the pedals for comparison between measured and modelled values. A five minute period of continuous cycling using just the quadriceps muscles produced similar results to those found for isolated knee extension. External power output dropped by 50% over the five-minute period, however there was no change in the pattern of torque production with fatigue. Cycling experiments were conducted using single muscle groups across a range of NMES firing angles. Experimental protocols were designed to seek the firing angles for each muscle that maximised power output by that group. Changes in power output in response to firing angle changes were not large, however, in comparison to the effects of cumulative fatigue and inconsistent power output between trials. This lead to large uncertainties in the determination of those firing angles that maximised power output by each muscle. Results suggest that NMES firing angles to maximise power output by the quadriceps muscles were relatively similar for each subject. For the hamstring muscles, however, substantial differences were observed in the range of firing angles that maximised power output. Results for the gluteal muscles were variable, with some subjects not applying any measurable torque to the cranks, even with maximal stimulation applied. The model produced a good match to experimental data for the quadriceps muscles, both in the shape of pedal force curves and the firing angles that maximised external power output. The individual variability in hamstring responses was not, however, predicted by the model. Modification of the relative size of the hamstrings' moment arms about the hip and knee substantially improved the match between measured and modelled data. Analysis of results suggests that individual variability in the relative size of these moment arms is a major cause of variation in individual's response to hamstring stimulation. There were apparent limitations in the model's ability to predict the shape of crank torques resulting from stimulation of the gluteus maximus muscle. It is suggested that further research be conducted to enable modelling of this muscle using a range of fibre lengths and moment arms.

Neural prostheses (NPs) are electronic stimulators that activate nerves to restore sensory or motor functions. Surface NPs are non-invasive and inexpensive, but are often poorly selective, activating non-targeted muscles and cutaneous sensory nerves that can cause pain or discomfort. Implanted NPs are highly selective, but invasive and costly. The stimulus router system (SRS) is a novel NP consisting of fully implanted leads that capture and route some of the current flowing between a pair of surface electrodes to the vicinity of a target nerve. One end of an SRS lead has a pick-up terminal that is implanted subcutaneously under the location of a surface electrode and the other end has a delivery terminal that is secured on or near the target nerve. The studies presented in this thesis address the development of the SRS from animal testing to its implementation as an upper extremity NP in a tetraplegic subject. Chapters 2 and 3 describe the SRSs basic properties, provide proof-of-principle of the system in animal studies and identify aspects that maximize its performance as a motor NP. The studies showed that selective and graded activation of deep-lying nerves can be achieved with the SRS over the full physiological range. Long term reliability of the system was demonstrated in chronic animal studies. The surface current needed to activate nerves with a SRS was found to depend on the proximity of the delivery terminal(s) to the target nerve, contact areas of the surface electrodes and implanted terminals, electrode configuration and the distances from the surface anode to the surface cathode and delivery terminal. Chapter 4 describes the first human proof-of-principle of the SRS during an intra-operative test. Finally, Chapter 5 describes the implementation of the SRS for restoration of hand function in a tetraplegic subject. Stimulation parameters and force elicited through the SRS, along with usage of the device were monitored up to 10 months after implantation. The system was found to be useful, reliable and robust. It is argued that the results of these studies indicate that the SRS provides the basis for a new family of NPs.

Involuntary electromyographic (EMG) activity has been recorded in the thenar (thumb) muscles of spinal cord injured (SCI) subjects for only short time periods (minutes), but it is unknown if this motor unit activity is ongoing. Longer duration EMG recordings can investigate the physiological significance of this neuromuscular activity. Analysis of these data is complex and time consuming. Since no software is currently capable of classifying 24 hours of data at a single motor unit level, the goal of this research was to devise an algorithm to automatically classify motor unit potentials over 24-hours. Twenty-four-hour, 2-channel thenar muscle EMG recordings were obtained from four different SCI subjects with cervical level injuries using a data logging device with custom software. The automatic motor unit classification algorithm used to classify the 24-hour recordings was a procedure consisting of four stages that included segmentation, clustering, and motor unit template uniting. All individual potentials were then classified and any superimposed potentials were resolved into their constituent classes. Finally, the algorithm found the firing patterns for each of the stable motor unit classes. The classification algorithm performance was compared to the analysis of a human operator and assessed in 2 ways: Tracking global classes over the 24 hours and correctly classifying individual motor unit potentials as to belonging to particular global classes. The algorithm was able to track an average of 13 global classes in four 24-hour recordings with a mean accuracy of 92 %. It was also able to classify individual potentials with a mean accuracy of 86% over four recordings, greater than the inter-rater reliability of two human operators (79%). The activities of the motor units tracked by the algorithm ranged from tonic firing to sporadic activity. The algorithm could analyze 24 hours of data in 2-3 weeks, while a human operator was estimated to take more than 2 years. In conclusion, the motor unit classification algorithm accomplished its goal of automatically tracking motor unit classes over a 24-hour recording with high accuracy. The 24-hour classification method developed here could be applied towards classifying long term recordings of other biological signals.

Adult mammalian central nervous system (CNS) neurons are unable to extend axons after injury, partially owing to the inhibitory myelin and chondroitin sulfate proteoglycans (CSPGs) present in the environment. A neuron's intrinsic state is also important for determining its regenerative potential. Peripheral nervous system (PNS) neurons, unlike their CNS counterparts, have increased ability to regrow their axons after injury, even in the presence of inhibitory molecules. With the goal of discovering novel regeneration associated genes, we have isolated the genes differentially expressed by PNS neurons. We then developed a high throughput neuronal transfection method to test whether these genes were sufficient to modify neurite growth in vitro. Using high content screening, we measured the ability of cerebellar neurons to initiate neurite outgrowth on inhibitory and permissive substrates. This combination of technologies (subtractive hybridization, microarray, high throughput electroporation and high content screening) allowed phenotypic examination of neurons after the overexpression of over a thousand genes. Additionally, kinases and phosphatases were assayed for their ability to modify neurite outgrowth in hippocampal neurons. Results from both of these large unbiased screens confirmed many of the existing candidates for neurite growth during development and regeneration. We also discovered many novel genes which promoted neurite outgrowth such as GPX3, EIF2B5, RBMX, CHKA, IRF6, and PKN2. To accurately interpret the large volume of data, new methods of analysis were performed. Finally, we developed novel techniques that took advantage of public databases to cluster genes and determine whether those clusters produced robust changes in neurite growth. In summary, we have provided a vast repository of functional data to study axon development and regeneration after injury as well as developing the tools needed to interpret that data.

Persons with spinal cord injury (SCI) suffer life-long consequences including paralysis, loss of involuntary bodily functions, and chronic pain. A subset of SCI patients develop neuropathic pain (NP), a chronic condition resulting from damage to the spinal cord. Hyperexcitability of spinal cord sensory neurons near damaged tissue is believed to underlie SCI-related NP. Although many therapies have been employed clinically to combat SCI-NP, few give satisfactory long-term relief. Transplantation of cells that release GABA, a molecule that inhibits neuronal activity, is being explored as an alternative to current SCI-NP therapies. My experiments made progress toward preclinical modeling of GABA cell therapy for SCI-NP. First, I sought to determine whether quisqualic acid (QUIS)-induced SCI altered responses to tonic pain stimuli or altered GABAergic neural circuitry in rats. Second, I sought to determine whether a combination of genetic and trophic manipulations could promote a GABAergic phenotype in rat embryonic neural precursor cells (NPCs) in an in vitro culture system. The results revealed that QUIS-SCI rats exhibit unusually prolonged nocifensive responses to hind paw formalin injections. There was no significant difference between QUIS-SCI and sham surgery rats in c-Fos immunolabeling of spinal cord sensory neurons after formalin-induced neuronal activity. However, immunohistochemistry revealed substantial decreases in staining for markers of GABA presynaptic vesicles in injured spinal cord tissue. NPCs were enriched for a neuronal phenotype by combining withdrawal of the growth factor FGF-2 from culture media and overexpression of the transcription factor MASH1 in transfected cells. Although glial marker expression was suppressed in NPCs by these manipulations, expression of neuronal markers none the less declined through time. MASH1-overexpressing NPCs exhibited greater clonal expansion and decreased stress-induced PDI expression after FGF-2 withdrawal as compared to naïve. In light of existing data, these results suggest that the QUIS-SCI model may be useful for testing the efficacy of GABAergic NPC transplantation to reduce neuropathic pain. MASH1 overexpression and FGF-2 withdrawal could serve as a first step toward enriching GABA in NPCs for transplantation. Although the mechanism for MASH1 cytoprotection remains unclear, MASH1 may enhance survival of NPCs grafted into the spinal cord. These experiments contributed to the preclinical basis for application of therapeutic GABAergic stem cell transplantation for NP in human SCI patients.

In individuals with spinal cord injury (SCI) who have disrupted communication between the brain and spinal cord, vibration may mimic functions formerly served by the lost or impaired supraspinal inputs resulting in more normal reflex modulation and improved walking function. Three experiments assessed the effects of vibration on spinal locomotor-generating circuitry, spinal reflex activity, and walking function. In Experiment 1, localized leg vibration was used to elicit air-stepping responses in the lower extremities. We compared responses of individuals with SCI to those of non-disabled (ND) individuals and assessed the influence of severity injury and locomotor training on the air-stepping response in individuals with SCI. Our results indicate that vibration of the tensor fascia latae elicited more consistent and robust responses than vibration of the quadriceps or hamstrings muscles. Individuals with SCI had less consistent and robust responses than ND individuals. In those with SCI, neither severity of injury nor locomotor training influenced the robustness or consistency of the response. In Experiment 2, we investigated the effect of whole-body vibration (WBV) on spasticity, as measured by spinal stretch reflex (SSR) excitability, in individuals with SCI. We also assessed differences in the influence of WBV among individuals who used antispastic medications and those who did not. Subjects were tested before and after participation in a 3 day/week, 12-session WBV intervention. There was a significant reduction in spasticity that persisted for several days following the WBV intervention. The amount by which spasticity was reduced was not different in those who used antispastic agents compared to those who did not use these agents. In Experiment 3, we assessed the effects of the 12-session WBV intervention on walking function. WBV was associated with significant increases in walking speed, cadence, step length of the stronger leg, and consistency of hip-knee intralimb coordination. Increases in cadence and stronger-leg step length correlated with improvements in walking speed. These results suggest that WBV may represent an approach to decreasing spasticity, and may be useful for individuals in whom spasticity interferes with function. Furthermore, vibration appears to have a beneficial effect on walking function, perhaps by influencing spinal locomotor-generating circuitry.

Spinal cord injury (SCI) often results in irreversible paralysis and widespread oligodendrocyte death and white matter damage. While the mechanisms underlying this phenomenon are poorly understood, previous studies from our laboratory indicate that inhibition of astroglial-NF-κB activation reduces white matter damage and improves functional recovery in a mouse model of SCI. Here we provide novel evidence demonstrating that astrocytes directly regulate oligodendrocyte fate after trauma by a glutamate-mediated AMPA receptor dependent mechanism. Following trauma, elevated expression of the SLC39a10 zinc transporter correlated with an increase in zinc uptake by astrocytes, thereby reducing extracellular zinc concentrations required for AMPA receptor inhibition. Stimulation of AMPA receptors on oligodendrocytes by glutamate induced oligodendrocyte toxicity through the activation of the NADPH oxidase enzyme within oligodendrocytes. Genetic and pharmacological inhibition of active NADPH oxidase was sufficient to attenuate oligodendrocyte death in vitro. Following SCI, NADPH oxidase inhibition reduced oligodendrocyte death by ~75%, suggesting that glutamate-mediated oligodendrocyte death is dependent on the activation of the NADPH oxidase enzyme within oligodendrocytes. Combined treatment of the NADPH oxidase inhibitor apocynin and the AMPA receptor inhibitor NBQX significantly improved hind limb locomotor behavior, reduced white matter damage and lesion volume, and significantly spared descending serotonergic fibers. These studies provide a novel mechanism of oligodendrocyte death and may lead to clinically relevant therapeutics after SCI.

It was hypothesized that superparamagnetic nanoparticles encapsulated in a silica shell with a fluorescent dye could be functionalized with axonal tracers and could be used for serial, non-invasive imaging with magnetic resonance imaging (MRI) for axon tract tracing. Nanoparticles functionalized with amine, octadecyl, silica, and biotinylated dextran amine were manufactured and characterized with MRI, scanning electron microscopy, and UV-visible, infrared, and fluorescence spectroscopy. Nanoparticle concentrations of 10 mM were not toxic to adult rat neural progenitor cells (NPCs) and labeled approximately 90% of cells. Nanoparticles were assessed for anterograde and retrograde tract tracing in adult rat models. With MRI and microscopy, the nanoparticles did not appear to trace axons but did provide an MRI signal for up to 3 weeks post implantation. While functionalized nanoparticles did not appear to trace axons, they are not toxic to NPCs and may be used as a MRI contrast agent in the neural axis.