Motor deficits are a hallmark of Down syndrome (DS), yet little is known about their exact cause. Despite the rich understanding of the neurobiology of DS, there is still a lack of targetable mechanisms for early intervention aimed at alleviating motor changes in people with DS. Therefore, we utilized a mouse model of DS known as Ts65Dn to characterize for the first time the effects of trisomy 21 on spinal cord (SC) development. A central molecular player in SC patterning and cell-type specification, Oligodendrocyte transcription factor 2 (Olig2), is located on human chromosome 21 (Hsa21) and is triplicated in both people with DS and in Ts65Dn mice. To observe the effects of the supernumerary Olig2, we used immunohistochemistry to visualize the OLIG2-derived cellular populations (i.e., motor neurons (MNs) and oligodendrocytes (OLs)), as well as adjacent and interacting cell populations (i.e., ventral spinal interneurons (INs)). We limited our analyses to two embryonic ages—embryonic days (E) 12.5 and 14.5. Our results indicate that there is no overall change in the numbers of OLs at either E12.5 or E14.5. However, there tend to be more OL-fated cells within the pMN domain, where they originate, and migrating cells tend to be clustered closer to the pMN domain at E12.5. IN populations show some changes in Ts65Dn mice at E12.5, with both total and abventricular PAX6+ cell numbers and abventricular NKX2.2+ cell numbers increased in Ts65Dn embryos compared to euploid mice. However, at E14.5 the number of NKX2.2+ cells is unchanged. No difference in the NKX6.1+ population was seen at either time-point. In contrast, there are significant changes in the MN population at both E12.5 and E14.5. Specifically, at E12.5, the total ISL1+ MN population is significantly increased and shows altered regional distribution in the ventral horn of Ts65Dn SCs. Conversely, the Ts65Dn spinal MN population is normalized to euploid levels at E14.5. Overall, our results suggest that neurogenesis, gliogenesis, and cell-type specification of OLIG2-lineage cells are altered in the developing SC of Ts65Dn mice. Thus, this work identifies a novel target for future therapeutic interventions aimed at ameliorating motor changes in DS.

Transplants of olfactory ensheathing cells (OECs) from olfactory bulbs have recently been shown to support regrowth and reinnervation of damaged spinal cord, which has led to improved functional recovery. Using complete transection in adult rat, the studies presented in this thesis examine the role of peripherally derived olfactory tissue in promoting axonal regeneration and functional recovery. Chapter One and Two provide the background to the area of spinal cord regeneration and the methods used in this thesis. Chapter Three shows that transplants of OECs from rat olfactory lamina propria (OLP) are able to support axon regrowth in the lesioned spinal cord. The BBB score was significantly higher in experimental rats (5.4???0.84) compared with control animals (1.9???0.33) (P&lt0.001). These dissociated OECs from OLP can promote axonal regrowth through the lesion. Histological assessment showed that: 1) axons labelled with Fluororuby grew into the injury site in OECs-transplanted rats, with occasional fibres extending into the rostral cord; 2) brainstem neurons in the raphe nucleus were retrogradely labeled with Fluororuby; and 3) serotonergic axons were detectable distal to the lesion in OECs-transplanted rats. No fibres grew into the injured region and no retrograde labeling or serotonergic fibres were seen in control animals. The role of regenerated serotonergic fibres in OECs-transplanted rats is discussed. Chapter Four demonstrates that solid pieces of OLP dissected from the nose can re-establish the continuity of the transected cord and supply the OECs that can migrate to the cord stumps to support the axon regeneration. Experimental rats which received OLP from olfactory mucosa showed significantly greater locomotive recovery (BBB scores: OLP, 5.0???1.9; control, 1.5???0.5, p&lt0.0001). In animals with OLP transplants, histological analysis indicated that nerve fibres, expressing neurofilament and serotonin were present at the transection site. Locomotive recovery of the hindlimbs occurred, similar to that seen after OECs transplantation. Retrograde labeling of medullary raphe neurons and gigantocellular reticular nucleus occurred following Fluororuby injection in the cord distal to the lesion, further supporting the supraspinal origin of the 5-HT innervation in the present studies. These results indicate that OLP is effective in promoting partial spinal cord repair. Chapter Five examines functional recovery of spinal reflex circuitry, ie., H-reflex excitability using paired stimuli, in OLP-transplanted rats compared with normal and respiratory lamina propria (RLP) transplanted animals. H-reflex amplitude of the conditioned response was significantly reduced in OLP transplanted rats compared to RLP transplanted animals (p&lt 0.05). Therefore, hindlimb reflex excitability can be modulated by OLP transplants after transection of the spinal cord in adult rats. Chapter Six examines whether functional recovery can occur if transplantation of OLP tissue is delayed by 1 month after the spinal cord transection. The BBB score was significantly higher in experimental rats (4.3???0.8 for OLP) compared with control animals (1.0???0.3, P&lt 0.001), but recovery was less than after acute transplantation. Asx before, histological assessment of OLP animals showed: a) serotonergic axons were present in the cord below the transection site; b) brainstem raphe nuclei was retrogradely labeled; c) bisbenzimide pre-labeled cells from OLP transplants migrated in host spinal cord. These changes were not seen in control animals. These results indicate that OLP has the ability to promote axonal regeneration in chronically injured cord of adult rats. Chapter Seven compares the results from these three types of intervention. In conclusion, these studies show that peripherally derived OECs or solid pieces of OLP can promote partial spinal cord repair in acute or chronic transection injuries. Such tissue might provide a potential source for autologous grafting in human paraplegia.

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.

Graduation date: 1994

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.