Spinal cord injury (SCI) causes life-long neurological impairment, with loss of sensory and motor function distal to the point of injury. There are approximately 300,000 patients living with SCI in the United States, and currently no effective treatment, reducing their quality of life. Amongst other things, proprioception, which has been determined essential for normal locomotion, can be lost with SCI. Epidural Electric Stimulation (EES), that is thought to excite large diameter afferent fibers (LDAF), has been found to improve recovery from spinal cord injury in conjunction with movement rehabilitation in animal models and humans. This represents an exciting new approach to help these patients. However, many open questions remain about how and why EES works. Chief among them are 1) which of the afferent fibers are necessary and sufficient to promote better recovery, and 2) what are the mechanisms of plasticity in the spinal cord that underly improvement. Here, we sought to address the first question by using viral and genetic tools to begin to target specific subsets of LDAF. First, we use a viral vector that preferably transduces only in the large diameter afferent fibers (LDAF) in the Dorsal Root Ganglia (DRG), and then specifically only the proprioceptors within the LDAF, by using a transgenic rat line that expresses Cre recombinase in Parvalbumin, a marker for proprioceptive neurons in the DRG. This approach consists of using the chemogenetic modulator of neuronal activity Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), which are activated by a putatively inert drug, clozapine-N-oxide (CNO), that crosses the blood brain barrier. While we were able to specifically target LDAF with excitatory DREADDs in L3-L5 DRGs in wild type rats, we were unsuccessful at specifically targeting proprioceptors by using the Pvalb-iCre rat line. Additionally, we studied the effect of exciting LDAF on rats with a 200KDyn SCI. CNO withdrawal on the week 7 stage of the recovery was associated with worse ladder performance than the previous and following weeks, as well as worse kinematic behavior of the same week on lower speeds in ankle movement. These results suggest that DREADDs activation is necessary for changes in movement at longer times post injury. It does not rule out that plasticity in neural circuitry has occurred but suggests that plasticity may rely on afferent activation. Finally, we sought to develop new methods to overcome skin motion artifact in rat kinematics by tattooing the knee area under the skin and recording infrared high-speed videos of moving rats which would correct joint calculations beyond just triangulation methods, as well as a novel MATLAB application that can accurately and reliably perform automated H-Reflex measurements, test the stimulating electrodes, and carry out typical instantaneous analyses, which in return allows for faster data collection with reduced human error, and subsequently result in higher research quality. / Bioengineering

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Summary: The effects of clonidine, a central alpha2 agonist, on changes in blood pressure caused by muscle afferent nerve (ergoreceptor) activation and baroreceptor manipulation were studied in cats. Prolonged isometric contractions (ergoreceptor activation) of the gastrocnemius and plantaris muscles increased mean arterial pressure by 53 mmHg. This pressor response was not altered by naloxone (0.5 μmol·litre-1) but was eliminated by clonidine (0.5-2.0 μg) when injected into the cerebral aqueduct. Brief occlusion of the carotid artery (15-30 s) caused mean arterial pressure to increase by 32-42 mmHg at rest. Neither naloxone nor clonidine altered the magnitude of the reflex pressor response to carotid occlusion. Similar increases in pressure were measured when occlusion was applied during fatiguing isometric contractions; thus baroreceptor induced increases in pressure were superimposed on the ergoreceptor induced blood pressure changes. Naloxone did not affect the changes in pressure caused by either reflex response. Clonidine continued to eliminate the pressor response to muscular contraction but did not affect the pressure increase when the carotid occlusion was applied during contractions. Electrical stimulation of the carotid sinus nerve caused blood pressure to decrease by 36 mmHg during rest and by 41 mmHg during fatiguing isometric contractions. Clonidine did not alter the depressor response to carotid sinus nerve stimulation. These data may indicate that separate pathways centrally mediate the changes in blood pressure caused by ergoreceptor and baroreceptor afferent activation. The integration of the ergoreceptor pathway may involve a catecholaminergic-opioidergic system but the present results do not suggest a similar interaction for the baroreceptor integration.

The overall focus of this research project was to develop an in vitro tissue-engineered system that accurately reproduced the physiology of the sensory elements of the stretch reflex arc as well as engineer the myelination of neurons in the systems. In order to achieve this goal we hypothesized that myelinating culture systems, intrafusal muscle fibers and the sensory circuit of the stretch reflex arc could be bioengineered using serum-free medium formulations, growth substrate interface design and microfabrication technology. The monosynaptic stretch reflex arc is formed by a direct synapse between motoneurons and sensory neurons and is one of the fundamental circuits involved in motor control. The circuit serves as a proprioceptive feedback system, relaying information about muscle length and stretch to the central nervous system (CNS). It is composed of four elements, which are split into two circuits. The efferent or motor circuit is composed of an [alpha]-motoneuron and the extrafusal skeletal muscle fibers it innervates, while the afferent or sensory circuit is composed of a Ia sensory neuron and a muscle spindle. Structurally, the two muscular units are aligned in parallel, which plays a critical role modulating the system's performance. Functionally, the circuit acts to maintain appropriate muscle length during activities as diverse as eye movement, respiration, locomotion, fine motor control and posture maintenance. Myelination of the axons of the neuronal system is a vertebrate adaptation that enables rapid conduction of action potentials without a commensurate increase in axon diameter. In vitro neuronal systems that reproduce these effects would provide a unique modality to study factors influencing sensory neuronal deficits, neuropathic pain, myelination and diseases associated with myelination. In this dissertation, results for defined in vitro culture conditions resulting in myelination of motoneurons by Schwann cells, pattern controlled myelination of sensory neurons, intrafusal fiber formation, patterned assembly of the mechanosensory complex and integration of the complex on bio-MEMS cantilever devices. Using these systems the stretch sensitive sodium channel BNaC1 and the structural protein PICK1 localized at the sensory neuron terminals associated with the intrafusal fibers was identified as well as the Ca2+ waves associated with sensory neuron electrical activity upon intrafusal fiber stretch on MEMS cantilevers. The knowledge gained through these multi-disciplinary approaches could lead to insights for spasticity inducing diseases like Parkinson's, demyelinating diseases and spinal cord injury repair. These engineered systems also have application in high-throughput drug discovery. Furthermore, the use of biomechanical systems could lead to improved fine motor control for tissue-engineered prosthetic devices.

This doctoral project presents the basis for the development and evaluation of the Eagles Wings Exercise Program. Eagles Wings was designed for Veterans with Post-traumatic Stress Disorder (PTSD). Evidence of the common trauma-focused treatments shows that their effectiveness is limited. The Eagles Wings program draws from theories and evidence of the therapeutic contribution of decreasing stress through physical exercise to promote relaxation, body awareness, and proper protective reflexes. Eagles Wings includes a protocol-based 8-week program of weekly group exercises along with a home exercise program. The program will be evaluated using formative and summative evaluations. A single-case design study will be conducted to identify changes in PTSD symptoms, psychosocial functioning, and resting heart rate. Study findings will be disseminated to stakeholders, including end-users (Veterans), family members, and decision-makers at the U.S. Department of Veterans Affairs. To keep the program accessible and at a minimal cost for participants, program funding will be secured from local sources such as nonprofit organizations that serve Veterans and crowdsourcing. The developer hopes that this program is found to be effective and is broadly used to enhance the well-being of veterans with PTSD across the United States.

Reflex inhibition of the quadriceps muscle group is a frequent and significant consequence of knee trauma, disease and surgical insult. The resultant quadriceps atrophy can be expected to delay rehabilitation and render the joint vulnerable to repeated injury resulting in capsular and synovial thickening, effusion and pain. A major purpose of this study was to examine the degree of quadriceps inhibition experienced by patients who undergo arthroscopic menisectomy. A secondary goal of this study was to investigate the efficacy of transcutaneous electrical nerve stimulation on the relief of reflex inhibition. Tests were performed on 12 patients prior to, and on day 1 and day 2 post surgery. True and placebo treatments of transcutaneous electrical nerve stimulation were administered on day 1 and day 2 post surgery. Measurements were made on the injured and normal limb with the knee fixed at 38G of flexion. Motor unit activation was determined by the twitch interpolation technique. Reduced motor unit activation was considered indicative of quadriceps reflex inhibition. Testing demonstrated that at all times the injured leg was weaker than the normal leg (p=.OOl). Following surgery, strength of the injured limb was significantly less than its pre operative score (p=.Ol). No significant recovery of strength was observed during the first two days following surgery. Injured legs were characterized by significantly lower motor unit activation at all times of testing Cp=.003). Following surgery, motor unit activation for the injured leg was significantly lower than its pre operative value (p=.Ol). By day 2 post surgery, motor unit activation had recovered Cp=.05) and was similar to the pre operative values for that leg. Transcutaneous electrical nerve stimulation had no effect on strength or motor unit activation. Recovery following arthroscopic surgery is characterized by an initial loss of strength and motor unit activation. By day 2, isometric strength remains depressed, however motor unit activation returns to pre surgery levels. / Thesis / Master of Science (MSc)

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