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People with diabetic peripheral neuropathy and limited joint mobility syndrome (LJMS) can experience increased forefoot peak plantar pressures (PPPs). Elevated PPPs are a risk factor for diabetic neuropathic foot ulceration. Diabetic peripheral neuropathy can also affect balance and increase the risk of falling. This thesis investigated the efficacy and acceptability of foot and ankle mobilisations combined with home stretches in people living at risk of neuropathic diabetic foot ulceration and falling. A systematic review and meta-analysis examined the effects of foot and ankle physical therapy modalities for the management of foot and ankle joint mobility, PPPs, and balance. The review highlighted that combined exercise intervention programmes integrating stretches increased total ankle ROM by 1.76 degrees (95% CI 0.78-2.7) and reduced PPPs by 32kPa (95%CI -42.42 - -19.21). The review highlighted that no studies to date have looked at combined foot and ankle mobilisations and stretches on foot and ankle mobility, PPPs, and balance. This informed a proof-of-concept randomised controlled trial (RCT). In my proof-of-concept RCT, people with diabetic peripheral neuropathy (n=61) were randomly assigned to an intervention (n=31) or control group (n=30). The intervention consisted of a 6-week programme of ankle and 1st metatarsophalangeal (MTP) joint mobilisations and home stretches. The control group received usual podiatry care. Outcome measures were taken at baseline (T0); 6-weeks post intervention (T1) and at 18-weeks follow up (T2) by a blinded assessor. The primary outcome was dynamic ankle dorsiflexion range of motion (ROM) at T1 using three-dimensional (3D) motion analysis. Secondary outcomes included static 1st MTP and ankle joint ROM, stride length, dynamic peak plantar pressure, forefoot-to-rearfoot pressure ratio, perceived walking ability and balance with measures of postural sway and functional reach. Results were reported according to CONSORT guidance. At T2 there was no difference between both groups in ankle dorsiflexion in stance phase (primary outcome measure) or any other dynamic measurements including plantar pressure and balance. Compared to the control group, the intervention group showed differences in static ankle dorsiflexion range (left 1.52cm and 2.9cms, right 1.62cm and 2.7cm) at 6 (T1) and 18 weeks (T2) respectively p<0.01). Between group differences were also seen in left hallux dorsiflexion (2.75°, p<0.05) at T1 and in right hallux dorsiflexion ROM (4.9°, p<0.01) at T2 follow up. Further, functional reach showed a difference in the intervention group (T1=3.13cm p<0.05 and T2=3.9cm p<0.01). A qualitative study was embedded in the RCT using semi-structured interviews with thematic analysis to explore the experiences of participants (n=16) in receiving foot and ankle mobilisations with home stretches to gain an insight into barriers and facilitators influencing adherence to treatment. Perceived improvement in foot and ankle function and building good rapport with the physiotherapist were factors that positively influenced participant exercise adherence and overall acceptability to the intervention. A focus group with the treating therapists (n=3) revealed concerns about the intervention because the study used a higher dosage of mobilisations than that usually seen in clinical practice. It also underlined whether it is appropriate to use outcomes that capture biomechanical data instead of functional outcome measures that are meaningful to patients. In conclusion, this is the first study to investigate whether mobilisations combined with stretches can reverse the effects of diabetic glycosylation on foot and ankle mobility. The findings from the RCT found no changes in the primary outcome. However, differences between groups in static ankle and hallux ROM, and functional reach test could be associated with some participants reporting functional improvement in their foot and ankle. Future work should focus on assessing the effects of mobilisation in a group with LJMS that affects ankle range while walking. Further, implementation issues such as who might be best suited to deliver the intervention within a healthcare setting needs to be considered prior to undertaking a multi-centre RCT.

Neuropathic pain is a frequent and disabling complication of diabetic polyneuropathy (DPN). However, it is not known why some patients develop painful DPN (pDPN) while others do not suffer pain. Although DPN is a primary disorder of the peripheral nerves it is increasingly recognised that processes in the central nervous system, including the spinal cord, are involved in generating neuropathic pain in DPN. One potential pain generating mechanism is spinal disinhibition whereby a loss of spinal inhibition leads to increased ascending nociceptive drive due to amplification of, or a failure to suppress, incoming signals from the periphery. Investigations in diabetic rodents implicate spinal disinhibition as a major mechanism of pain generation. A biomarker of spinal disinhibition in rodents is impaired rate dependent depression of the H-reflex (HRDD). The H-reflex is easily measured in humans and preliminary studies in patients have shown that HRDD is also impaired in pDPN associated with type 1 diabetes. This thesis aims to further investigate the role of HRDD as a biomarker of spinal disinhibition in pDPN in a larger sample size, including patients with type 2 diabetes, by deeply phenotyping neuropathy and pain using quantitative sensory testing, questionnaires, conditioned pain modulation and tests of neuropathy severity. The work establishes that HRDD is impaired in a larger cohort of patients with pDPN, including those with type 2 diabetes. It also shows that HRDD is enhanced in patients with painless DPN. By using receiver operator characteristic analysis the optimal HRDD parameters which delineate patients with pDPN from those without pain are defined. To aid transferability of H-reflex testing into the clinic a simple clinical scale, the Neuropathy Disability Score, is defined as a predictor of H-reflex amplitude. Finally, the work shows that increasing impairment abnormal HRDD, and by inference spinal disinhibition, is associated with a distinct pain phenotype characterised by a relative gain of function that would be expected to enhance ascending mechanical and thermal nociceptive drive despite sensory loss.

Hereditary peripheral neuropathy, also known as Charcot-Marie-Tooth disease (CMT) and related disorders, are a group of genetic disorders causing length-dependant neuropathy, resulting in motor and/or sensory loss progressing from the lower extremities toward the spine. With a population prevalence of 1 in 2,500, CMT is the most common inherited neuromuscular disorder and among the most common of all Mendelian diseases. Despite advances in the understanding of CMT genomics, a significant number of CMT patients remain genetically undiagnosed, and disease-modifying therapeutics are not available for the vast majority of CMT patients. In this thesis I present four topics addressing related areas of unmet need within the CMT field. My aim is to identify biological features relevant to a broad range of subtypes and experimental models, bridging the division between clinical and basic science. Firstly, I perform a systemic review of therapeutic studies of inherited peripheral neuropathies, with a focus specifically on gene and variant-level, rather than disease-level, treatment outcomes. Therapeutics for neuromuscular diseases are often gene- or mutation-specific, and I identify several variant-specific therapies and therapeutic approaches for inherited neuropathies, as well as variant-specific outcomes for gene-specific therapies, to be integrated into an online genetic diagnostic platform. Within the clinical trials I identify lack of responsive CMT biomarkers as a major limiting factor for therapeutic trials. Therefore, I next seek to identify novel biomarkers, by applying proteomics in a cohort of CMT patients and mouse models to identify neural cell adhesion molecular 1 (NCAM1) and growth differentiation factor 15 (GDF15) as serum biomarkers of CMT. NCAM1 may potentially function as a disease-progression biomarker, whereas GDF15 is indicated to be a very accurate diagnostic biomarker. Additionally, I find that broad elevation of serum complement proteins across CMT subtypes, which, while less promising as biomarkers, may indicate that dysregulation of this system. Thirdly, whilst the pathophysiology of some individual CMT genotypes is well understood, in many cases there is not currently a clear shared mechanism linking basic CMT-associated gene families to neuropathy pathogenesis. In particular, it is unclear why dominant missense mutations in several tRNA synthetases lead to axonal neuropathy, whilst other variants in these genes cause very variable human disease. Therefore, I develop an induced neural progenitor cell (iNPC) model derived from skin fibroblasts of patients with these genetic defects and perform transcriptomic and proteomic analyses of dysregulated pathways. Finally, to address the particularly large proportion of undiagnosed patients with distal hereditary motor neuropathy, I test the possibility of using functional transcriptomic and proteomic datasets to identify causative variants of axonal neuropathies, and develop a machine learning model to identify neuropathy-causative variants within large datasets of whole exome or genome sequencing. Although CMT is highly heterogenous at a genetic level, I show across these topics that there are several molecular signatures common across many subtypes. The identification of two non-subtype-specific biomarkers will aid the development of CMT therapeutics. Dysregulation of cell adhesion molecules is identified as a major feature in both patient sera and patient-derived iNPC models, while the machine learning model I develop demonstrates that protein and RNA enrichment in the axon is a key feature of CMT.

The research in this thesis has examined the use of texture and shape analysis to characterise Magnetic Resonance (MR) images of peripheral nerves in order to provide a potential quantitative tool for better diagnosis and treatments. Texture and shape can be considered as inherent properties of all surfaces and have the potential to provide sensitive information which cannot be quantitatively perceived by human vision. Texture analysis has been successfully used in image classification of aerial and satellite imagery and the diagnosis and prognosis of several types of cancer. However, to date, it has never been used in investigating peripheral nerve damage. In this thesis, we study the application of texture and shape analysis to the peripheral nerves in the upper extremities of patients suffering from Whiplash Associated Disorders (WAD). Specifically, quantitative texture analysis was performed on MR images of the carpal tunnel which contains the median nerve. The median nerve was studied to identify differences in textural patterns. Texture methods such as: first order features; co-occurrence matrices; run-length matrices and autocorrelation function were applied and their performance was assessed. Texture analysis was also performed to investigate nerve damage in the MR images of the brachial plexus, both in controls and patients. Further, spatial domain shape metrics were used to quantify and study the morphological differences of the median nerve in controls and patients. This highlighted that some significant differences exist between groups and thus could potentially be reliably used in combination with clinical scale metrics to identify possible nerve damage. As MR images contain noise, locating the median nerve accurately to perform image analysis is very important. Therefore, we further investigated the application of an enhanced correlation filtering method that could be trained on images of the median nerve and then applied to detect the median nerve in test images. The Optimal Trade-off Maximum Average Correlation Height (OT-MACH) filter includes the expected distortions in the target in the construction of the filter reference function. The OT-MACH filter was tuned in a bandpass to maximize the correlation peak and thereby successfully locate the position of the median nerve in the carpal tunnel. This study has successfully demonstrated that texture and shape analysis can be used to investigate possible peripheral nerve damage. Further research is required using larger datasets to establish a quantitative image analysis tool to support clinical decision making and thereby improve patient care and treatment outcome.

Corneal Confocal Microscopy (CCM) is a non-invasive imaging technique to quantify small nerve fibre structure in patients with diabetic somatic and autonomic neuropathy and increasingly other metabolic, hereditary, toxic and inflammatory peripheral neuropathies. This thesis establishes that CCM is indeed a powerful imaging technique which can identify early small fibre degeneration and regeneration in relation to the clinical phenotype of subjects with obesity, impaired glucose tolerance and Type1/2 diabetes. We demonstrate a precise relationship between small fibre neuropathy and erectile dysfunction in subjects with Type 1 diabetes. We also demonstrate the utility of CCM in demonstrating relative protection from small fibre damage in Type 1 patients with extreme duration diabetes (medallists) at baseline and over 3 years and repair in patients undergoing simultaneous pancreas and kidney transplantation. This thesis provides further evidence for the utility of CCM as a marker of early small fibre neuropathy by demonstrating nerve damage in subjects with morbid obesity with and without diabetes and explore the mechanisms underlying nerve damage at baseline and repair following bariatric surgery. We also show that CCM can track dynamic changes in small fibre degeneration and regeneration in subjects with impaired glucose tolerance in relation to change in glucose tolerance status and following continuous subcutaneous insulin infusion in subjects with Type 1 diabetes.

Painful diabetic neuropathy (PDN) is one microvascular complication of diabetes mellitus (DM) and the focus of this thesis. PDN is a neuropathic pain condition characterised by severe burning pain in the feet and sometimes hands. It has significant impacts on peoples’ mobility, sleep quality and overall quality of life. The personal and societal burden associated with DM and PDN is predicated to rise as prevalence rates increase. Pharmacological management of PDN is often less than optimal, and people are left with few strategies to cope. Multidisciplinary pain management programmes (PMPs) use physical activity and psychological coping strategies to help people live better with persistent pain, yet people with PDN are rarely referred. It is unknown whether these strategies would be appropriate to help people live with PDN. This thesis aimed to: 1) locate and appraise all literature relating to physical activity and psychological coping strategies in PDN; 2) interview people with PDN and explore how PDN impacted on their lives; 3) explore the perspectives of patients and clinicians on the relevance of PMP approaches; and 4) explore patients’ treatment priorities and whether these might be addressed by PMP strategies. To address these aims, firstly a systematic literature review was conducted. The review identified a paucity of studies investigating physical activity or psychological coping strategies for PDN. Two interview studies were conducted, and data were analysed using thematic analysis (TA). A study with patients (n=23) found the impacts of PDN were wide ranging, people had experimented with many coping strategies unsuccessfully and there was some scepticism that PMP strategies were relevant to PDN, though few participants had direct experience of them. The second study interviewed specialist diabetes and pain clinicians and representatives from primary care (n=19). Clinicians relied primarily on medication strategies and did not have alternatives when these failed. Diabetes clinicians highlighted that people with PDN were medically complex patients and were at risk of tissue damage from too much physical activity. Pain clinicians felt PMP strategies could be adapted to suit the population with PDN. Informed by the patient interview study, an Internet survey was developed to explore the management priorities of people with PDN (n=63 respondents). Sleep disturbance was the top priority in all subgroups analysed. There were six impacts most frequently prioritised by respondents, which did not include pain. Potential clinical management strategies for these impacts have been described, and suggestions made for future research. This thesis has shown a scarcity of existing evidence for non-pharmacological strategies in the management of PDN. PMP strategies were not necessarily viewed as appropriate by patient participants. The impacts prioritized by people with PDN could however be matched to management strategies from other conditions where persistent pain is common. There is no a priori reason why these strategies could not be trialled with PDN. Managing the impacts of PDN on peoples’ lives remains a complex process.

Spinal muscular atrophy (SMA) and Charcot-Marie-Tooth disease type 2D (CMT2D)/distal SMA type V (dSMAV) are two incurable neuromuscular disorders that predominantly manifest during childhood and adolescence. Both conditions are caused by mutations in widely and constitutively expressed genes that encode proteins with essential housekeeping functions, yet display specific lower motor neuron pathology. SMA results from recessive inactivating mutations in the survival motor neuron 1 (SMN1) gene, while CMT2D/dSMAV manifests due to dominant point mutations in the glycyl-tRNA synthetase (GlyRS) gene, GARS. Using a number of different model systems, ranging from Caenorhabditis elegans to the mouse, this thesis aimed to identify potential novel therapeutic compounds for SMA, and to increase our understanding of the mechanisms underlying both diseases. I characterised a novel C. elegans allele, which possesses a point mutation in the worm SMN1 orthologue, smn-1, and showed its potential for large-scale screening by highlighting 4-aminopyridine in a screen for compounds able to improve the mutant motility defect. Previously, the gene encoding three isoforms of chondrolectin (Chodl) was shown to be alternatively spliced in the spinal cord of SMA mice before disease onset. I performed functional analyses of the three isoforms in neuronal cells with experimentally reduced Smn levels, and determined that the dysregulation of Chodl likely reflects a combination of compensatory mechanism and contributor to pathology, rather than mis-splicing. Finally, working with two Gars mutant mice and a new Drosophila model, I have implicated semaphorin-plexin pathways and axonal guidance in the GlyRS toxic gain-of-function disease mechanism of CMT2D/dSMAV.

The goal of this thesis was to examine the processing of clinical pain in two patient groups with well defined primary pathologies, i.e. neuropathic pain patients and patients with rheumatoid arthritis (RA). It was hypothesized that chronic pain is associated with plastic changes in pain processing brain structures that can be detected using functional magnetic resonance imaging (FMRI). The first study, presented in Chapter 3, demonstrates that the neural representation of experimental heat pain is different in neuropathic pain patients than in age- and gender-matched healthy control subjects, although the pain stimulus was applied outside clinically affected areas. Increased activation was found in amygdala and anterior insula in the patient group and was accompanied by increased state anxiety and depression scores. Anterior insula is the focus of Chapter 4 in which it is demonstrated that clinical pain processing is located significantly more anteriorly in the insula than experimental pain processing, in close proximity to neural correlates of highly negative emotions and the conscious perception of bodily sensations. This offers a potential explanation for the shift of clinical pain processing. In Chapter 5, clinical pain is contrasted with experimental pain in the same patient population, i.e. patients with RA. In addition to comparing clinical and experimental pain processing, it was investigated if emotional and cognitive determinates of the pain experience, specifically depression and catastrophizing, exert different influences on the two types of pain. It is shown that clinical pain, but not experimental pain, is likely to be driven partially by depressive symptoms whereas catastrophizing is associated with the same neural activation pattern in both conditions. The cerebral representation of allodynic pain in neuropathic pain patients is presented Chapter 6. Chapters 6 and 7 demonstrate that the FMRI signal encodes the perceived intensity of clinical allodynic pain across subjects and that it reflects longitudinal variations of the perceived intensity within subjects. This thesis illustrates that FMRI can reveal subtle differences in the processing of clinical and experimental pain, despite brain activation patterns being similar on the whole. It also indicates that FMRI can be used to elucidate the origin of these differences, for instance by studying the influence of emotional and cognitive variables. This suggests that neuroimaging methods, in particular FMRI, have the potential to dissect clinical pain into its constituent parts, including central sensitization, brainstem facilitation and amplification by psychological factors. Such knowledge could potentially be exploited to target treatment selectively at different components of clinical pain and to monitor longitudinal changes of these components separately.

Conventional approaches to the study of neurotoxicants focus exclusively on the direct action of neurotoxicants on the nervous system. In fact, pharmacokinetic and metabolic factors can greatly modify the effect of neurotoxicants. This thesis presents the investigations of the pharmacokinetic and metabolic factors in chemical-induced neurotoxicities, p-Bromophenylacetylurea (BPAU) and m-dinitrobenzene (m-DNB) were selected as model neurotoxicants. Wistar (LACP) and F344 rats, and the analytical techniques of HPLC, MS and NMR were used in the present studies. This study has demonstrated that metabolism and pharmacokinetic behaviour of BPAU and m-DNB significantly modified their neurotoxicity. Three metabolites of BPAU, 3-hydroxy-5-(4-bromophenyl)-1,3 -diazapentane-2,4-dione (M1), 4-4-bromo-phenyl)-3-oxapyrrolidine-2,5-dione (M2) and 3-methyl-5-(4-bromophenyl)-1,3-diaza- pentane-2,4-dione (M3), were identified and characterised in this study. Phenylmethanesulfonyl fluoride (PMSF) which can intensify BPAU-induced neuropathy signiflcandy delayed BPAU elimination, increased M1 levels in tissues and decreased M2 concentration in serum This metabolic interaction and neurotoxic synergy between the two compounds represent a distinct model in the mechanistic studies of the promotion of delayed neuropathy. The present study also showed that 1-year old rats which are more susceptible to BPAU-induced neuropathy produced more M1 and less M2 than 6-week old rats. In vivo and in vitro studies in this thesis also demonstrated that m-DNB can be metabolised in brain. The major metabolite was 3-nitroaniline (3-NA). An activated intermediate, 3-nitrosonitrobenzene (3-NNB) was detected in the cultured astrocytes and endothelial cells. This suggests that metabolic activation may play an important role in m-DNB-induced neurotoxicity. The infusion-lesion model revealed that once a concentration threshold for m-DNB is exceeded, the time which m-DNB is maintained above that threshold is a more important factor in producing brain lesions than a higher transient concentration. This study has demonstrated that pharmacokinetic approaches have a great potential in understanding chemical-induced neurotoxicity.

Numerous patients who have uncontrolled diabetes have developed complications associated with diabetic neuropathy. The apparent knowledge deficit and noncompliance have led to the development of clinical concerns related to improving patient outcomes. This scholarly project assesses the effects of dietary, weight, and exercise management on improving diabetic neuropathy symptoms. The debilitating effects of diabetes continue to affect numerous people each day. Uncontrolled diabetes can create innumerable complications, including diabetic neuropathy. Appropriate nutrition associated with accountability, monitoring, and proper education can alleviate the progression of uncontrolled diabetes. Assessment of the improvement of diabetic neuropathy with dietary and exercise management is examined among a group of twelve participants selected randomly.

Environmental exposure to organophosphate (OP) pesticides, such as malathion, is a risk factor for neuropathy and neurodegeneration. Toxic levels of OPs irreversibly inhibit acetylcholinesterase (AchE) activity, leading to acute paralysis and even death as the neurotransmitter acetylcholine accumulates at cholinergic synapses. In addition, there is compelling evidence that repeated low-level “occupational-like” exposure to OPs is associated with somatosensory defects but the cellular mechanisms for this effect are unclear. We show sensory neuron cell size in the dorsal root ganglia is significantly reduced in rats exposed to occupational level malathion. However, co-administration of a reversible AchE inhibitor, galantamine, prevented this effect.

Artificial Light at Night Disrupts Pain Behavior and Cerebrovascular Structure in Mice Jacob R. Bumgarner Circadian rhythms are intrinsic biological processes that fluctuate in function with a period of approximately 24 hours. These rhythms are precisely synchronized to the 24- hour day of the Earth by external rhythmic signaling cues. Solar light-dark cycles are the most potent environmental signaling cue for terrestrial organisms to align internal rhythms with the external day. Proper alignment and synchrony of internal circadian rhythms with external environmental rhythms are essential for health and optimal biological function. The modern human environment on Earth is no longer conducive to properly aligned circadian rhythms. Following the industrial revolution, artificial lighting and an ever-growing 24-hour global economy have shifted humans away from natural environments suited for rhythmic behavior and physiology. Humans, and much of the natural environment, are routinely exposed to circadian rhythm disruptors. The most pervasive disruptor of circadian rhythms is artificial light at night (ALAN). A growing 80% of humans on Earth are exposed to ALAN beyond natural nighttime environmental lighting levels. ALAN exposure is associated with numerous negative consequences on behavior and physiology, including neuroinflammation, cardiovascular disease, and altered immune function. This dissertation examines two previously uninvestigated effects of ALAN exposure on physiology and behavior in mice. In Part 1, I investigated the effects of ALAN exposure on pain behavior in mice. I observed that ALAN exposure had detrimental effects on rodent pain behavior in contexts of both health and models of human disease. ALAN exposure heightened responsiveness to noxious cold stimuli and innocuous mechanical touch. Differences in these effects were noted based on sex and disease state. I conclude this section with a report on the mechanisms by which ALAN exposure altered pain behavior. In Part 2, I investigated the effects of ALAN exposure on cerebrovascular structure in mice. To conduct these investigations, I first developed VesselVio, an open-source application for the analysis and visualization of vasculature datasets. Using this application and additional analytical frameworks, I examined the effects of short-term ALAN exposure on hippocampal vasculature in mice. ALAN exposure reduced hippocampal vascular density in mice, with notable regional sex differences. I also observed that ALAN exposure altered hippocampal vascular network connectivity and structure, with persistent regional sex differences. The data in this dissertation contribute to the ever-growing field of circadian rhythm biology focused on studying circadian rhythm disruption. These data highlight the continuing need to mitigate the pervasiveness of ALAN in human and natural environments. Most importantly, the results presented in this dissertation emphasize the need to consider ALAN as a mitigating factor for the treatment of both cardiovascular disease and pain.

Chronic Inflammatory Demyelinating Polyneuropathy (CIDP) is considered a rare autoimmune disorder which makes it difficult to accurately diagnose and creates a lot of opportunity for active research. With its wide array of presentations and even similarities to other neurological disorders, improvement must be made in the field of diagnostic methods in order to offer concise and effective treatments. As with many other neurological disorders, CIDP patients are at a higher risk for falls. The dual effect of gait impairment due to neuropathy and general effects of older age create a dangerous combination and increase the risk for falls. By increasing the accuracy with which physicians and health professionals predict falls in patients, they can effectively prevent serious injury and improve quality of life. Consequently, in order to predict the risk of falls, and therefore prevent severe injury, the ability to accurately access the specific qualities of the patient’s gait is critical. Without the ability to precisely identify patients’ particular gait impairment presentation it is very difficult to establish their risk for falls. The current methods of diagnoses focuses mainly on PROs (patient reported outcomes) which are often gathered through patient questionnaires. Previous research has shown that such methods are simply not detailed and patient specific enough to offer a complete picture of a patient’s condition. We believe ProtoKinetics Movement Analysis Software (PKMAS) is an objective, examiner independent measure of patients’ gait, and offers a method of quantifying patients’ functional gait outcomes in a way that is superior to the current standard of care procedures. Therefore, in this study we aim to reveal the shortcomings of current standard of care procedures in the diagnosis and treatment of CIDP, while also demonstrating the superior value of PKMAS in providing a detailed patient disease profile for CIDP afflicted individuals. Specifically, we demonstrate PKMAS’ increased ability to predict fall risk in CIDP patients, as compared with currently used methods. In this study PKMAS data was collected under two conditions: Dual Task and PWS (preferred walking speed). During each condition the patient was asked to walk across the Gait Map/Zeno Walkway as the ProtoKinetics Software collected detailed information about the patients’ gait. For PWS, patients were asked to walk across the map at a speed they were most comfortable at as PKMAS data was collected. For Dual Task, patients are asked to walk at their preferred walking speed while simultaneously doing a simple cognitive task, for instance, counting backwards from a hundred. This second condition is particularly important. The point of such a task is to mimic real-life walking. When we walk on a daily basis we are usually thinking or doing something else simultaneously, even if we may not be consciously aware of this. As such, comparing the results for PWS and Dual Task for patients can shine light onto their real-life gait experience. In order to do so, we measured the percent change in abilities between PWS and Dual Task walking. A greater change signifies greater gait impairment, and a change of greater than 15% places the patient at risk for falls. Among the PROs, INCAT is the one most often referred to in neurological standard of care and as such we focused on this particular questionnaire separately as well. To do so, t-tests were completed to demonstrate the lack of validity in scoring, by looking at the PKMAS data as compared between two INCAT scores. In this study we seek to demonstrate the superiority of ProtoKinetics Movement Analysis Software (PKMAS) over the current standard of care for CIDP patients. Specifically, in accurately evaluating patients’ gait and future fall risk. The ability to do so is vitally important for elderly patients who already suffer from decreased gait stability and the additional impact of CIDP can accentuate that risk.

Peripheral neuropathy is a severe and debilitating adverse effect of anti-cancer therapies, including chemotherapy and immunotherapy. It is caused by neurotoxicity and neuroinflammation in the peripheral nervous system, involving the dorsal root ganglion (DRG), and is characterised by neuropathic symptoms. Currently, treatment options for peripheral neuropathy are limited, and therefore a better understanding of the underlying mechanisms is needed. In the first study, we investigated the effects of chemotherapy on neurite outgrowth using DRG explant cultures. We demonstrated for the first time that DRGs derived from chemotherapy (paclitaxel and oxaliplatin) treated mice exhibited reduced ex vivo neurite outgrowth at day 30 and a return to baseline levels at day 90. This study established the ex vivo DRG explant model as an important tool to investigate axonal outgrowth in neuropathic pain. Recently, chemotherapy in combination with immunotherapy, and particularly immune checkpoint inhibitors (ICIs), has become more widely used. Therefore, in the second study, we investigated the effects of paclitaxel and the ICI anti PD-1 on peripheral neuropathy. We demonstrated that both paclitaxel and anti PD-1 reduced ex vivo neurite outgrowth of DRG explants and induced macrophage or T cell infiltration into the DRG and microglia activation in the spinal cord. However, anti PD-1 did not exacerbate mechanical sensitivity, neurodegeneration or neuroinflammation when given in combination with paclitaxel, suggesting that using this co-therapy may be a safe cancer treatment avenue. Since we observed changes in spinal cord microglia in chemotherapy treated mice, in the third study we investigated the contribution of neuron-glia interactions to microglia changes. We showed that paclitaxel induced calcium release in DRG sensory neurons, which may alter upstream signalling and affect microglia that interact with DRG central terminals. Using microfluidic chambers, we found that microglia that interacted with paclitaxel treated DRG axons displayed a reactive state phenotype with decreased velocity, cell area and perimeter, and increased sphericity. This may contribute to neuropathic pain development and maintenance. Taken together, our results reveal some of the effects of chemotherapy and immunotherapy on the nervous system and demonstrate the involvement of neuroinflammation and neuroimmune crosstalk in the development of chemotherapy-induced peripheral neuropathy.

The meninges have long been accepted as purely structural, protecting the central nervous system (CNS). Similarly, the choroid plexus (CP) was thought to only produce cerebrospinal fluid. Advances have dispelled these limited views, and the meninges and CP are now known as critical sites of immune activity, housing various immune cells, including B and T cells, monocytes/macrophages, and dendritic cells, and are increasingly implicated in neuroinflammation. The broad aim of this thesis is to characterise the immune profile of the meninges, CP, and secondary lymphoid tissue in animal models of CNS and peripheral neuropathy. New evidence in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, suggests inflammation of the meninges and CP changes are crucial for disease induction, warranting further investigation. Firstly, we demonstrated significant changes in the meninges and lymphoid tissue in EAE. Adoptive transfer of immunosuppressive activated regulatory T cells (Tregs) resulted in their migration to the pia and CP, and drainage from the CNS back into lymph nodes 2-4 days following delivery. Treg treatment at the peak of EAE showed mild changes in meningeal/lymphatic immunity. Secondly, to study meningeal involvement in peripheral neuropathy, where literature is severely lacking, we utilised an animal model of peripheral nerve injury, sciatic nerve chronic constriction injury (CCI), and 2 animal models of chemotherapy-induced peripheral neuropathy (CIPN). The immune profile of the meninges, CP, and lymphoid tissue was compared across male and female mice in acute stages of CCI and in a chronic model. We found the immune profile of the meninges, CP, and lymphoid tissue at acute stage of injury is different for males and females, is altered at 7 days and 21 days post-injury and appears to resolve by a chronic time point. In CIPN animal models, we demonstrated the immune profile of the meninges and CP is minimally affected, however, there were numerous significant changes in lymphoid organs. Together, results from this thesis provide comprehensive characterisation of immune cell profile in the meninges and CP in EAE and peripheral neuropathy models. Further research is warranted to ascertain precise mechanisms underlying their involvement.

Anticancer therapies are commonly prescribed for breast cancer and other solid tumors. While cytotoxic agents are therapeutically efficacious in treatment of many cancers, they often lead to significant toxicities. One of the main non-hematological dose-limiting toxicities for cytotoxic chemotherapies is sensory peripheral neuropathy. Currently, there are no effective therapeutic strategies to prevent or treat chemotherapy-induced peripheral neuropathy (CIPN) due to the lack of understanding in the mechanisms underlying patient susceptibility. This dissertation presents a pharmacogenetic approach to aid in the identification of critical genetic networks that may explain the development of CIPN. With the use of genome-wide association studies conducted from clinical samples of breast cancer patients treated with microtubule-targeting agents, we highlighted the role of sphingosine lipid signaling in microtubule-targeting agent (MTA)-induced peripheral neuropathy (PN). Functional studies using human sensory neurons derived from induced pluripotent stem cells show that inhibiting such pathway attenuates paclitaxel-induced neuronal injury. Additional genes also involved in Rho GTPase signaling were further revealed from a next-generation exome sequencing study on patients treated with MTA chemotherapy. Future efforts are needed to explore how disruption in this signaling pathway develops on the molecular level and subsequently leads to manifestation of CIPN. While these studies have focused on revealing insights of the pathophysiology underlying CIPN, we also investigated the use of early patient-reported symptoms and genome-wide data for the prediction of dose-limiting neuropathy events. We show that early patient-reported outcomes can be used as a tool to predict risk for dose-limiting toxicity events. Future studies aimed at determining whether other types of genomic and clinical information may help improve such predictions. Together, the work presented herein provide novel insights to mechanisms underlying the development of CIPN in efforts to discover novel strategies for CIPN prevention and treatment.

Limited information exists on the effects of self-reported exercise on subjective and objective measures of chemotherapy-induced peripheral neuropathy (CIPN). In this study, we evaluated for differences in demographic and clinical characteristics, as well as subjective and objective measures of CIPN and balance among 290 cancer survivors with CIPN who were classified into one of three exercise (Ex) groups (i.e., NoEx, LessEx, RecEX) based on the recommended level of >150 minutes per week. Survivors completed self-report questionnaires and underwent a sensory examination and balance testing. Compared to RecEx group (34.8%), survivors in the NoEx group (20.7%) had less education, were less likely to be married/partnered, had a lower household income, a higher level of comorbidity, and a poorer functional status. No differences were found among the three exercise groups in duration of CIPN, pain intensity scores, or changes in light touch, cold and pain sensations. However, compared to the RecEx group, survivors in the NoEx group had higher vibratory thresholds and worse scores on objective measures of balance. Based on our “real world” findings, clinicians can recommend walking as a therapeutic option, as well as referrals to physical therapy for additional balance and strength training in survivors with CIPN.

Childhood Acute Lymphoblastic Leukemia (ALL) is the most common childhood malignancy in the United States, accounting for approximately 30% of pediatric cancer diagnoses each year. Over the past several decades, the 5-year survival rate for ALL has risen to nearly 90% due to incorporation of modern treatment protocols that employ vincristine (VNC) as the frontline antineoplastic agent. Unfortunately, such improved outcomes have drawbacks, as VNC, along with several other chemotherapeutic agents produce neurotoxic effects and can result in chemotherapy-induced peripheral neuropathy (CIPN) in patient populations of all ages. While CIPN has been well studied clinically and experimentally in adults, little is known about the consequences of chemotherapy exposure in early life. Given that childhood ALL diagnoses typically occur around 2-3 years of age, a time at which the developing nervous system may be differentially vulnerable to peripheral insults, this dissertation characterizes for the first time, a rodent model of pediatric vincristine-induced peripheral neuropathy.

This thesis explores the morphological changes that occur following nerve injury using ultrasound (US). The rationale was that US may provide useful insights into the pathophysiological mechanisms underlying neuropathy in metabolic conditions like diabetes and chronic kidney disease(CKD), and that US parameters may serve as biomarkers for the early detection of neuropathy and possibly as a means of monitoring treatment efficacy. Sonographic studies were undertaken in 101 patients with carpal tunnel syndrome (CTS), of which 22.5% had normal nerve conduction studies(NCS). 26% of these NCS-negative patients had abnormal scans, indicating that alterations in nerve morphology may precede electrophysiological changes. Doppler US was used to assess intraneural blood flow(INBF), and the sonograms were analysed using PixelFlux®. 49% of the CTS cohort had detectable INBF compared to none in the control group, thus supporting the assertion that sonographic detection of INBF using current US technology is pathological. Having demonstrated nerve enlargement and hypervascularity in entrapment neuropathy, further studies were conducted in patients with CKD before and after haemodialysis. Peripheral nerves of CKD patients were larger (p

Chemotherapy-induced peripheral neuropathy (CIPN) is a devastating side effect of multiple classes of antineoplastic drugs used to treat cancer. These agents cause degeneration of the peripheral axons of sensory, motor and autonomic system neurons, leading to symptoms like loss of sensation, spontaneous pain, muscle weakness and incontinence. Developing treatments for CIPN has been difficult because the mechanisms by which antineoplastic agents cause degeneration of post-mitotic neurons are unknown. In recent years, some mechanistic details of CIPN have begun to be uncovered using animal models. However, there is little evidence to support these same processes occurring in humans. Therefore, I set out to develop and characterize a human cellular surrogate model for CIPN using induced pluripotent stem cell (iPSC)-derived neurons. I examined phenotypic changes induced in sensory, motor and cortical neurons by treatment with the common neuropathy-causing chemotherapeutic agents paclitaxel, vincristine and bortezomib. Despite their distinct mechanisms of antineoplastic actions, all three drugs caused axonal toxicity in the iPSC-derived neurons. By developing a spot culture method for examining neurite degeneration over time using iPSC-derived neurons, I was able to conclusively demonstrate that these chemotherapeutics disrupt axons through two distinct effects; growth cone stalling and axon degeneration. Utilizing this novel culture system, I examined whether the mechanism of chemotherapy induced neurotoxicity proposed in animal studies could be demonstrated in this humanized model. I found evidence of post-translation modifications in tubulin and of mitochondrial dysfunction congruent with those previously reported, although the timing of the mitochondrial alterations indicates it is likely secondary to axon degeneration. I also found clear evidence of caspase activation, but this does not appear to be necessary for the degeneration to occur since its inhibition did not rescue the phenotype. Instead, my findings that exogenous nicotinamide adenine dinucleotide or inhibition of the kinase DLK can both reduce the observed neurotoxicity in human neurons treated with chemotherapeutic agents indicates that the axonal degeneration is Wallerian-like. These findings in human cells further our understanding of the pathobiology of CIPN and provide a humanized preclinical model that may be suitable for the identification and development of CIPN modifying therapies in the future.

Painful peripheral neuropathy is a common dose-limiting side effect associated with cisplatin treatment. Cisplatin is unable to cross the blood-brain barrier, and its neurotoxicity is limited to the peripheral nervous system (PNS). In the PNS, Schwann cells are an essential component supporting dorsal root ganglion (DRG) neuron viability, and impairments in Schwann cell biology contribute to cisplatin-induced painful neuropathy. We explored the role of Schwann cell-derived exosomes in the development of cisplatin-induced hyperalgesia. Consistent with our previous reports, daily injection of cisplatin (1 mg/kg, i.p.) for 7 days produced mechanical hyperalgesia in C3H/HeN mice. To investigate the impact of exosome signaling in the development of cisplatin-induced hyperalgesia, exosomes isolated from the sciatic nerves of cisplatin-treated mice were injected intrathecally into naïve mice for 5 consecutive days (7 g of total protein/10 l, i.t.). Mechanical hyperalgesia was observed after the second injection of exosomes, mimicking the effect of cisplatin alone and supporting the involvement of integrated exosome signaling in hyperalgesia produced by cisplatin. Intrathecal administration of Schwann cell-derived exosomes activated microglia, and analysis of exosomal content indicated mediators of neuronal sensitization at the central level. Collectively, our results indicate that Schwann cells affected by cisplatin contribute to mechanical hyperalgesia and exosomes are an important signaling mediator for glia-neuronal communication.

BACKGROUND: Containing a brain for signal processing and decision making, and a peripheral component for sensation and response, the nervous system provides higher organisms a powerful method of interacting with their environment. The specific neurons involved in pain sensation are known as nociceptors and are the source of normal nociceptive pain signaling to prompt appropriate responses. Though acute hypersensitization can be advantageous by encouraging an organism to allow an injured area to heal, chronic pain conditions can be pathological and can markedly reduce quality of life. While a variety of genes have been associated with congenital pain conditions, two rare cases examined in this study have not had their mutated genes identified. Potassium voltage-gated channel subfamily H member 8, or KCNH8, is involved in regulating action potential production and propagation, and has not been linked with pain processing of any kind to date. Here, a male patient evaluated at Boston Children’s Hospital contains a novel single-base KCNH8 mutation and possesses an extremely low sensitivity to cold temperatures and mechanical pain, but a higher sensitivity to warmer temperatures. A separate protein, intersectin-2, or ITSN2, normally functions in clathrin-mediated endocytosis and exocytosis. A second patient at Boston Children’s Hospital expresses a previously-unseen point mutation in ITSN2 and experiences erythromelalgia, characterized by episodes of intense pain and red, swollen limbs during ambient warm temperatures. Through the use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing, this study will produce these specific genetic mutations in mouse lines to explore their effects on mammalian behavior. OBJECTIVES: This project employs two transgenic mouse models to study the behavioral phenotypes associated with rare potentially damaging mutations in KCNH8 and ITSN2 exhibited in the human patients. Through these experiments, a greater understanding of neural pain signaling and sensitivity changes can occur. METHODS: The differences in temperature preference of KCNH8 and ITSN2 mutant mice compared to wild type mice lacking these mutations was studied using thermal plates under cold and warm conditions. Direct application of acetone and von Frey filaments to mouse paws was used to study cold and mechanical sensitivity. Further testing of stamina, anxiety, coordination, and strength were also evaluated. RESULTS: A marked decrease in sensitivity to von Frey stimulation (p

Batten disease or neuronal ceroid lipofuscinoses (NCLs) is one of the most common reasons for childhood dementia, characterized by brain atrophy, blindness, mental decline and premature death. Although animal model neurological research is traditionally conducted using small rodents, large animal models are becoming more popular for several reasons. Longitudinal monitoring of disease progression and individual variations are essential in the development of translational medicine and whereas small rodents have relatively short life spans and different brain structures compared with humans, sheep provide an excellent model for long-term studies of diseases and their treatment. Three naturally occurring ovine models are currently being utilised to increase knowledge about different types of NCL and to aid in development of treatment strategies for translation to humans. These sheep models include a CLN5 Borderdale, a CLN6 South Hampshire and a CLN6 Merino model. This thesis contains investigations of techniques for longitudinal in vivo monitoring that can be correlated with the progression of neuropathological events in all three ovine NCL models, both in the natural progression of the diseases and in trials of potential therapeutic regimes. Previous studies have described the neuropathological development of disease in the CLN5 Borderdale and the CLN6 South Hampshire models. In the present study similar investigations were conducted with the CLN6 Merino model, and the three ovine models compared. The general development of neuropathology in CLN6 Merino sheep follows that of the other two models. Common themes are neuronal loss from the cerebral cortex that proceeds in a regional pattern, with neuroinflammation, neurogenesis and ubiquitous accumulation of storage material within the cells. Immunohistochemistry indicated possible cross-regulation between the CLN5 and CLN6 proteins. In vivo techniques are essential in the monitoring of progressive diseases such as the NCLs. Neuroimaging through computed tomography (CT) and magnetic resonance imaging (MRI) are suitable tools to describe neuropathological changes without the need to sacrifice animals. Here, current techniques and protocols for CT scanning in ovine NCL research were reviewed and improved. The congruence of brain size and intracranial volume (ICV) in ovine NCL was established and techniques for the monitoring of brain size in therapeutic trials were validated and correlated with neuropathological changes. Furthermore, 3-dimensional reconstruction was used to monitor regionality of the cortical changes and this could be correlated with findings from post examination. CT based surgery planning was used to improve accuracy of intracereboventricular gene therapy delivery and reduce duration of the surgical procedure. Pilot studies on the use of MRI as a longitudinal in vivo monitoring modality in ovine NCL research were conducted and these indicated that MRI will yield enhanced detail compared with CT and that MRI measurements can be closely correlated with the neuropathological changes observed at post mortem examination. However, some specific technical issues were identified and will need to be addressed in the future. These include image quality, fit of templates and an important need to establish ovine-specific image acquisition protocols. Blindness in ovine NCL results from atrophy of photoreceptor cells in the retina as well as changes in the central visual pathways. Treatment should aim not only to treat the atrophy of the brain, but also to prevent or halt the development of blindness. Previous trials have indicated the need for ocular delivery of therapy to achieve this. As for neurodegeneration, in vivo monitoring of retinal pathology is essential for the longitudinal assessment of treatment efficacy. Here, protocols were established for the use of repeated electroretinography (ERG) to monitor the development of blindness in the CLN5 Borderdale and the CLN6 South Hampshire models. The results show that ERG is an invaluable tool for monitoring the progression of retinal degeneration in ovine NCL. Furthermore, it was established that the course of development of retinal changes differs between the two ovine models that were investigated. The measurements enabled some degree of differentiation between the retinal and central components of the blindness in ovine NCL. Finally, it was shown that the in vivo monitoring tools established throughout the studies described in this thesis are invaluable for the assessment of treatment efficacy in gene therapy trials for ovine NCL. The measures evaluated here were shown to correspond with other clinical measures of treatment efficacy, such as behavioural observations, maze-testing and neuropathology. Key words: Neuronal ceroid lipofuscinosis, Batten disease, lysosomal storage disease, large animal models, sheep, neuroimaging, neurodegeneration, neuroinflammation, gene therapy, computed tomography, magnetic resonance imaging, electroretinography, in vivo monitoring.

This thesis employed systems modeling and engineering design methods in order to study the `thermal grill illusion' (TGI), a perceptual illusion in which a spatial configuration of warm and cool elements produce a paradoxical pseudo-burning sensation. The motivation for this study was derived from the possibility to develop new methods for assessing peripheral sensory deficits affecting the sense of touch, associated with peripheral neuropathy. Thermal grill stimuli, consisting of spatial configurations of alternating warm and cool elements, are non-injurious and can elicit rapid and unambiguous perceptual responses, whose absence might provide a reliable indicator of sensory loss, although this has not been previously investigated, and is only indirectly addressed in this thesis. An integrated custom electrothermal display was optimized for delivering thermal grill stimuli to the body. In order to validate the display technique, a thermodynamic model accounting for heat exchange (diffusion) through the skin was developed and the model predictions were compared with thermal perception. I calibrated and assessed the approach in perceptual experiments with healthy human subjects.

Diabetes is a huge economic and clinical burden to the health care system in the U.S. However, the incremental effect of diabetes complications has not been well documented in the literature. In addition, diabetes-related hospitalization discharge status has not been thoroughly examined. This study was carried out with two objectives: the primary objective of this study was to examine the association between the presence and severity of diabetes-related complications and diabetes-related hospitalization costs and discharge status. The secondary objective of this study was to explore how the presence and type of diabetes influenced complications-related hospitalization costs and discharge status. This study was a cross-sectional study with secondary data analysis of the 2010 to 2012 National Inpatient Sample (NIS). The study sample consisted of four disease cohorts: diabetes-related hospitalization cohort, peripheral lower-extremity (PLE) diseases-related hospitalization cohort, kidney diseases-related hospitalization cohort, and coronary atherosclerosis-related hospitalization cohort. In each cohort, hospitalization cost was calculated by multiplication of the total hospitalization charge by the cost-to-charge ratio. Hospitalization discharge status was dichotomized as routine discharge and non-routine discharge. Two statistical models were fit: a generalized linear model with gamma distribution and log link was used for modeling hospital inpatient cost; multivariate logistic regression was adopted for modeling hospitalization discharge status. The covariates in the regression model included complications and other variables identified by the Andersen’s behavioral model of health service utilization. The diabetes-related hospitalization contained 804,192 discharge records. The incremental cost of severe PLE was approximately $6,849. Severe PLE and less severe PLE increased diabetes-related hospitalization cost by 66.6% and 1.35% respectively. Comorbid kidney diseases were not found to be associated with an apparent increase in diabetes-related hospitalization cost. The incremental cost of severe coronary atherosclerosis was approximately $1,200. Severe PLE was also associated with increased odds of having non-routine discharges after hospitalization. The final peripheral lower extremity (PLE) diseases-related hospitalization cohort contained 219,752 discharge records. The presence of type 1 diabetes was associated with a 12% decrease in PLE-related hospitalization cost, while the presence of type 2 diabetes was associated with a 3.85% decrease in PLE-related hospitalization cost. The association between diabetes and PLE-related hospitalization cost was modified by age. PLE-related hospitalization cost was highest among patients younger than 65 years and without diabetes ($17,075). Diabetes was also significantly associated with hospitalization discharge status. PLE patients with diabetes were 1.37 times more likely to have a non-routine discharge compared to PLE patients without diabetes. The presence of type 2 diabetes was associated with even greater odds (a 26.2% increase) of having non-routine discharges. The final cohort of kidney diseases-related hospitalization contained 504,320 discharge records. The association between diabetes and kidney diseases-related hospitalization cost was also modified by age. The incremental cost of type 1 diabetes to kidney diseases-related hospitalization was $922 for patient below 65 years and $1,320 for patient age 65 years or older. Among patients below 65 years, the kidney diseases hospitalization cost for patients with type 2 diabetes was $785 lower than that for patients without diabetes. However, among patients age 65 years or older, the cost for patients with type 2 diabetes was only $84 lower than the patients without diabetes. The presence of type 1 diabetes was associated with a 33.2% increase in the odds of having non-routine discharges; while the presence of type 2 diabetes was associated with a 20.2% increase in the odds of having non-routine discharges. The coronary atherosclerosis-related hospitalization cohort contained 546,488 discharge records. The association between diabetes and coronary-atherosclerosis related hospitalization cost was also modified by age. The incremental cost of type 1 diabetes was $1,796 among hospitalized coronary atherosclerosis patients age 65 years or older, while the cost for patients with type 1 diabetes comorbidity was $550 lower than patients without diabetes among those age below 65 years. Type 1 diabetes was associated with a 35.4% increase in the odds of having non-routine discharges, while type 2 diabetes did not have any significant effect on coronary atherosclerosis-related hospitalization discharge status. In sum, this study found that severe peripheral lower-extremity diseases and severe coronary atherosclerosis are associated with significant increases in diabetes-related hospitalization cost. In addition, severe PLE was also associated with increased likelihood of having non-routine discharges after diabetes-related hospitalization. The impact between diabetes and complications-related hospitalization cost varies by age group and diabetes type. Among patients with PLE-related hospitalization, cost was highest among patients age

Great strides have been made with regard to neurogenesis in the enteric nervous system (ENS), rapidly following in the wake of recent revelations about the neurogenic properties of the central nervous system (CNS). As the ENS is more exposed, highly complex, and vulnerable to a variety of developmental, acquired, and multisystemic disorders, there is a sense of urgency for studies to address the potential within the gut to restore neurons that are injured or lost. It is the intricacies of the ENS and yet unclear relationships between agonists and embryonic precursors that have made demonstrating the arrival of new neurons in mature gut difficult under steady-state conditions. This thesis demonstrates that inducible models of a wide range of insults to the gut have yielded crucial information about ENS neurogenesis, while in vivo experimentation under steady-state conditions has proven inconsistent. Specifically, the signaling pathways of Ret and PTEN have revealed the existence of a ‘natural block’ that normally regulates neurogenesis and keeps proliferation well controlled. Furthermore, the overwhelming effects of serotonin agonism on neuron density in response to injury have uncovered an essential role of neuronal transdifferentiation by enteric glial cells that extends beyond what was once understood to be a largely homeostatic role. The influence of extrinsic innervation of the gut will also be explored, physiology of which is important for both the utility of gut microbiota and the role of Schwann cell progenitors in the development of the ENS. Therefore, this thesis will outline ENS organization and function, as well as describe common pathologies that serve as the foundation upon which neurogenesis is investigated. Critical inducible models to which chemical and molecular agonists are applied will be examined in detail, as it is through these models that therapeutics and biomedical engineering can be optimized in order to correct the pathophysiology of enteric neuropathies that as of now are still treated with surgical intervention.

This thesis explores the pathophysiology of axonal dysfunction in diabetes, utilizing excitability techniques which provide information on axonal ion channel function in human subjects. The rationale was that excitability studies may be useful to determine the mechanisms underlying axonal dysfunction in diabetic peripheral neuropathy (DPN) and that it may serve as a biomarker of incipient neuropathy and possibly as a means of monitoring treatment efficacy. Excitability studies were initially undertaken in 54 patients with type 2 diabetes (T2DM), and demonstrated a relationship between neuropathy-specific-quality-of-life and excitability markers that reflect activity of persistent Na+ conductances. These changes occurred concurrently with progressive axonal depolarization with increasing neuropathy severity. Further studies were then undertaken to explore these mechanisms in type 1 diabetes (T1DM). Assessment of sensory and motor excitability in 30 patients suggested membrane depolarization in sensory and motor axons. Mathematical modelling demonstrated that these changes were due to reduced nodal Na+ and K+ conductances and abnormal Na+/K+pump activity. Having demonstrated prominent changes in axonal function in T1DM, studies were conducted to explore the basis for these changes. The possibility that different forms of insulin administration may have differing effects on axonal function was considered. Axonal function was assessed in two separate cohorts of T1DM patients: those treated with continuous subcutaneous insulin infusion (CSII) and a second cohort who received multiple daily insulin injections (MDII). The studies demonstrated abnormalities of axonal function in MDII-treated patients. In contrast, CSII-treated patients had normal axonal function. The final series of studies explored the effect of glycaemic variability on axonal function in T1DM. The relationship between glycaemic variability and axonal excitability was assessed in 12 T1DM patients, using a continuous glucose monitoring system. Patients were studied at three different glucose ranges and glycaemic variability was separately measured over a 48-hour period at the time of testing. The studies demonstrated that acute glucose level did not correlate with axonal dysfunction. However, glycaemic variability was strongly correlated with neurophysiological parameters, suggesting that it is an important determinant of axonal dysfunction in T1DM.

This thesis examined the pathophysiology of peripheral neuropathy across the spectrum of chronic kidney disease (CKD) using nerve excitability techniques. The purpose of these studies was to establish the prevalence and impact of neuropathy in moderate severity CKD, to investigate the causative role of potassium in the development of axonal dysfunction and to provide an evidence base concerning the differential effects of various therapies on neuropathy in this patient group. Initial studies were undertaken in CKD patients receiving different forms of treatment, namely high-flux haemodialysis and haemodiafiltration. Compared to high-flux haemodialysis, patients who received haemodiafiltration demonstrated greater normality of nerve excitability prior to dialysis, across a single dialysis session and at long-term follow-up. In keeping with previous studies, pre-dialysis K+ was strongly correlated with measures of axonal depolarisation. Subsequent studies were undertaken to investigate whether potassium had a causal role in mediating axonal depolarisation using a modified dialysis intervention. This study demonstrated that axonal depolarisation remained unchanged for as long as serum potassium remained elevated, despite the significant removal of other dialysable uraemic toxins. Furthermore, mathematical modelling analysis revealed that nerve excitability abnormalities in dialysis patients were profoundly worse than that expected for normal axons exposed to similarly high potassium concentrations. Studies undertaken in renal transplant patients demonstrated differences in outcomes that were dependent on the form of post-transplant immunosuppression. Immunosuppressive regimens based on calcineurin inhibition were associated with a greater severity of neuropathy and nerve excitability abnormalities compared to patients receiving alternative immunosuppressive therapies. In the final set of studies nerve function was assessed in diabetic and non-diabetic CKD patients and a disease control group diabetic patients without CKD. Studies in the control group demonstrated nerve excitability abnormalities in type 1 diabetic patients prior to neuropathy onset. In CKD patients, peripheral neuropathy was highly prevalent and was strongly correlated with measures of physical function and quality of life. Additionally, this study revealed that diabetic-CKD is associated with a more severe neuropathy phenotype. In total these findings demonstrate the impact of neuropathy and highlight the need for early detection and management of this condition in all stages of CKD.

Advancing age and diabetic peripheral neuropathy (PN) are both associated with an increased risk of fall-related injuries, especially when walking on uneven surfaces, but little is known about the effect of even a single unexpected underfoot perturbation on gait kinematics. The challenge of establishing the effects of such a perturbation arises from the difficulty of isolating the stimulus-response relationships due to carryover effects from earlier perturbations. To address this challenge a custom perturbing shoe was invented to present a single unexpected medial or lateral underfoot perturbation during level gait. The shoe was used to test the primary hypotheses that both age and increasing severity of PN would affect the kinematic response to these perturbations, chiefly by affecting myoelectric latencies, ankle proprioceptive thresholds, and ankle and hip muscle strength capacities. We recruited 42 older subjects with and without PN and 26 healthy young subjects. We measured manual reaction times, unipedal balance times, lower extremity strength capacities, gait kinematics, lower extremity electromyographic latencies and ground reaction forces during gait trials with underfoot perturbations randomly presented in 16 of 60 gait trials. The results showed that hip strength was a significant predictor of unipedal stance time (R2 = 0.73). Hip abduction/adduction and ankle inversion strengths explained almost 70% of the variation in gait speed over an uneven surface. The perturbing shoe proved a reliable method for affecting step kinematics following an unexpected underfoot perturbation. In healthy young adults, the single perturbation affected the kinematics of up to four of recovery steps but not pelvic displacements. Despite exhibiting EMG responses on the first recovery step, healthy older adults chose to alter their lateral pelvic displacements, but not recovery step kinematics. Subjects with moderate PN did not display early EMG or step kinematic responses, but larger lateral pelvic displacements than the healthy older adults. These results suggest that with advancing age and PN, subjects employ a stance limb hip response rather than an ankle response to recover from a single underfoot perturbation. These results suggest that future interventions for patients with moderate PN should aim to increase maximum hip strength and rate of strength development.

This thesis examines the pathophysiology underlying chemotherapy-induced neurotoxicity. Oxaliplatin, utilised in the treatment of colorectal cancer, produces prominent neurotoxicity. Clinical and specialised neurophysiological studies were undertaken to determine the full spectrum of oxaliplatin-induced neuropathy. These studies demonstrated acute alterations in Na+ channel-related parameters in sensory axons post-infusion, suggesting that an acute Na+ channelopathy may contribute to the development of neuropathic symptoms. Patients were also assessed longitudinally, revealing a characteristic pattern of sensory axonal abnormalities, which developed progressively across treatment and occurred earlier than changes in conventional parameters of nerve function. Importantly, excitability abnormalities were predictive of neuropathy severity at treatment completion. Studies in a subset of oxaliplatin-treated patients with Lhermitte's phenomenon (electric shock-like sensations on neck flexion) confirmed that the extent of excitability change was associated with the severity of neuropathy. To further define mechanisms, studies were undertaken before and immediately following oxaliplatin infusion. These studies established that oxaliplatin affected transient Na+ channel inactivation processes more significantly than persistent Na+ channel conductances in both motor and sensory axons. Studies utilising natural activity to produce axonal hyperpolarization established that changes in the recovery cycle of excitability post-oxaliplatin were greatly enhanced. These studies established that oxaliplatin-induced changes were membrane-potential dependent, supporting the hypothesis that oxaliplatin modulates axonal Na+ channel inactivation. To examine the long-term sequelae of oxaliplatin treatment, follow-up studies were undertaken, revealing chronic sensory neuropathy in the majority of patients. Importantly, sensory axonal excitability measures prospectively obtained during treatment were predictive of neurological outcomes at long-term follow-up, confirming that excitability techniques provided a sensitive biomarker of oxaliplatin-induced neuropathy. To contrast these findings, a cohort of paclitaxel-treated patients were assessed, revealing an entirely different basis for neurotoxicity related to damaged axonal transport systems. Paclitaxel produced early and progressive changes in stimulus threshold and maximal sensory amplitudes in the absence of focal changes in individual axonal conductances. In conclusion, studies in this thesis have established abnormalities in selective axonal Na+ channel-dependent properties in oxaliplatin-treated patients which preceded the development of neuropathy. Axonal excitability techniques may be utilised as early and sensitive biomarkers of oxaliplatin-induced neurotoxicity, yielding important insight into pathophysiological mechanisms of neuropathy.

The study of human balance and gait is a complex area that involves a large number of biological systems, including the musculoskeletal, somatosensory, vestibular, visual, and central nervous systems. In contrast, computational models used for simulating motion of the human body tend to be relatively simple, especially with respect to the feet. Clinical research, however, has begun to more closely examine the mechanical and sensory contributions of the feet in balance and gait, leading to a disparity between the state of clinical research and models used for simulation. A model with a more complex foot would aid in the clinical diagnosis and treatment of motor control disorders, improvement of prostheses, and development of functional electrical stimulation for recovery of lost motor function. This dissertation presents a computational model of a human with a more complex foot, which uses four rigid and connected segments to represent the heel, forefoot, and toes. Derivation of physical parameters, equations of motion, actuation based on human musculature, and control based on proprioception, i.e. body segment positions and velocities, will be discussed. Computation of ground reaction forces under the heel, forefoot, and toes will also be addressed. Simulations focusing on the role of the toes and toe muscles in static balance, forward leaning, and tip-toe stading will be presented. Contributions by the vestibular system will also be considered.

The incidence of diabetes in the United States and globally threatens to reach epidemic proportions: it is the leading cause of peripheral neuropathy in developed nations, afflicting up to half of the approximately 15 million Americans with diabetes. Diabetes is a metabolic and vascular disease, but the exact pathogenesis of diabetic neuropathy remains elusive. While mechanical, metabolic, vascular and morphological factors have all been associated with permanence of damage to the peripheral nervous system, no temporal ordering of these changes has been established. The present work attempts to correlate changes observed at the organism scale and molecular scale of diabetic peripheral nerve extracellular matrix with tissue-scale changes. Our overarching hypothesis that mechanical factors, evidenced by changes in nervous tissue material properties, protein expression, and morphology, play a role in this permanence. Two clinical studies on type-I-diabetes animal models were conducted to establish the extent of these tissue changes: a 16-week genetically diabetic BioBreeding rat model and a 12-week streptozotocin-induced diabetic Sprague-Dawley rat model with a weight control group. This original work represents the first in the literature to (1) observe enlarged epineurial collagen fibril diameters in diabetic rats with atomic force microscopy and (2) quantify the full mechanical constitutive response of whole rat sciatic nerve by measuring elastic modulus, ultimate tensile strength, yield behavior, and stress relaxation behavior at both physiological and failure levels in a diabetic peripheral neuropathy animal model. Additionally, this work extends previous research on post-translation expression levels of types I, III, and IV collagen in endoneurium and perineurium/epineurium in a diabetic peripheral neuropathy animal model. Two microscale composite models are presented for extracellular matrix mechanics of peripheral nerve. The first models elastic inclusions in a viscoplastic matrix at multiple loading modes and rates. It revealed elevated von Mises stress at phase boundaries. These stresses grew even larger in the stochastic composite. The second microscale model, which represents peripheral nerve as a fibrous-bundle pressure vessel, captures changes seen in constitutive response in the presence of diabetes and is consistent with literature values seen for elevated endoneurial fluid pressure in metabolically initiated peripheral neuropathies.

Cultures of dorsal root ganglia (DRG) can achieve neuronal maturation with axons, making them useful for neurobiological studies. They have not, however, previously been used to investigate subcellular events that occur following exposure to neuropathy-inducing organophosphorus (OP) esters. Recent studies in other systems demonstrated alterations of ATP concentrations and changes in mitochondrial transmembrane potential (DYm) following exposure to neuropathy-inducing OP compounds, suggesting that mitochondrial dysfunction occurs. The present dissertation proposed an investigation using chick embryo DRG cultures to explore early mechanisms associated with exposure to these toxicants. This approach uses an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-10 day old chick embryos, and grown for 14 days in minimal essential media (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then treated with 1 mM OP compounds, or the DMSO vehicle control. OP compounds used were phenylsaligenin phosphate (PSP) and mipafox, which readily elicit OPIDN in hens, and paraoxon, which does not cause OPIDN. Confocal microscopic evaluation of neuronal populations treated with PSP and mipafox showed opening of mitochondrial permeability transition (MPT) pores, and significantly lower mitochondrial tetramethylrhodamine fluorescence, suggesting alteration of mitochondrial structure and function. This supports our conclusion that mitochondria are a target for neuropathy-inducing OP compounds by inducing mitochondrial permeability transition. For further evaluation of mitochondrial function, mitochondrial respiratory chain reactions were measured. In situ evaluation of ATP production measured by bioluminescence assay showed decreased ATP concentrations in neurons treated with PSP and mipafox, but not paraoxon. This low energy state was present in several levels of the mitochondrial respiratory chain, including complexes I, III and IV, although complex I was the most severely affected. For morphological studies, the media containing the aforementioned toxicants was removed after 12 hours, and cultures maintained for 4 to 7 days post-exposure. Morphometric analysis of neurites in DRG was performed by inverted microscopy, using a system that was entirely computerized. Morphometric estimation of neurites treated with mipafox or PSP but not with paraoxon suggested that reversible axonal swelling at day 4 post-exposure had reversed by 7 days post-challenge. Ultrastructural alterations were described by electron microscopy. Damage to neurons was more severe following exposure to PSP and mipafox, with mitochondrial swelling and rarefaction of microtubules and neurofilaments observed within the cytoplasm. This study supports others that suggested mitochondria are a primary target for neuropathy-inducing OP compounds. We suggest that mitochondrial permeability transition (MPT) induce abrupt changes in mitochondrial membrane potentials, altering the proton gradient across the mitochondria membrane, decreasing ATP production within the cell. In addition, reduction in ATP production can be related to specific-complex alteration of the mitochondria respiratory chain following neuropathy-inducing OP compounds. The profound ATP depletion and the induction of MPT can induce the release of apoptotic factors and intramitochondrial ions, leading to axonal damage observed later in the course of OPIDN. This study provides evidence that chick DRG cell cultures are an excellent model to study early structural and functional features of OPIDN. It is likely that the alteration in energy lead to ultrastructural defects in these cells. These early events can contribute to alteration in neuronal ATP production previously reported in OPIDN. Cultures of dorsal root ganglia (DRG) can achieve neuronal maturation with axons, making them useful for neurobiological studies. They have not, however, previously been used to investigate subcellular events that occur following exposure to neuropathy-inducing organophosphorus (OP) esters. Recent studies in other systems demonstrated alterations of ATP concentrations and changes in mitochondrial transmembrane potential (DYm) following exposure to neuropathy-inducing OP compounds, suggesting that mitochondrial dysfunction occurs. The present dissertation proposed an investigation using chick embryo DRG cultures to explore early mechanisms associated with exposure to these toxicants. This approach uses an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-10 day old chick embryos, and grown for 14 days in minimal essential media (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then treated with 1 mM OP compounds, or the DMSO vehicle control. OP compounds used were phenylsaligenin phosphate (PSP) and mipafox, which readily elicit OPIDN in hens, and paraoxon, which does not cause OPIDN. Confocal microscopic evaluation of neuronal populations treated with PSP and mipafox showed opening of mitochondrial permeability transition (MPT) pores, and significantly lower mitochondrial tetramethylrhodamine fluorescence, suggesting alteration of mitochondrial structure and function. This supports our conclusion that mitochondria are a target for neuropathy-inducing OP compounds by inducing mitochondrial permeability transition. For further evaluation of mitochondrial function, mitochondrial respiratory chain reactions were measured. In situ evaluation of ATP production measured by bioluminescence assay showed decreased ATP concentrations in neurons treated with PSP and mipafox, but not paraoxon. This low energy state was present in several levels of the mitochondrial respiratory chain, including complexes I, III and IV, although complex I was the most severely affected. For morphological studies, the media containing the aforementioned toxicants was removed after 12 hours, and cultures maintained for 4 to 7 days post-exposure. Morphometric analysis of neurites in DRG was performed by inverted microscopy, using a system that was entirely computerized. Morphometric estimation of neurites treated with mipafox or PSP but not with paraoxon suggested that reversible axonal swelling at day 4 post-exposure had reversed by 7 days post-challenge. Ultrastructural alterations were described by electron microscopy. Damage to neurons was more severe following exposure to PSP and mipafox, with mitochondrial swelling and rarefaction of microtubules and neurofilaments observed within the cytoplasm. This study supports others that suggested mitochondria are a primary target for neuropathy-inducing OP compounds. We suggest that mitochondrial permeability transition (MPT) induce abrupt changes in mitochondrial membrane potentials, altering the proton gradient across the mitochondria membrane, decreasing ATP production within the cell. In addition, reduction in ATP production can be related to specific-complex alteration of the mitochondria respiratory chain following neuropathy-inducing OP compounds. The profound ATP depletion and the induction of MPT can induce the release of apoptotic factors and intramitochondrial ions, leading to axonal damage observed later in the course of OPIDN. This study provides evidence that chick DRG cell cultures are an excellent model to study early structural and functional features of OPIDN. It is likely that the alteration in energy lead to ultrastructural defects in these cells. These early events can contribute to alteration in neuronal ATP production previously reported in OPIDN.

The serine/cysteine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) has been used both to promote and to protect against neuropathic events of organophosphorus-induced delayed neuropathy (OPIDN) in hens (Lotti et al., 1991; Veronesi et al., 1985; Pope and Padilla, 1990; Pope et al., 1993). This study expands upon this work by correlating clinical and neuropathological findings in these modifications of OPIDN. To provide appropriate models of OPIDN, single phenyl saligenin phosphate (PSP) dosages of 0.5, 1.0, or 2.5 mg/kg were administered to adult hens. PMSF (90 mg/kg) was given either 4 hours after or 12 hours prior to PSP administration. Clinical signs and pathologic changes in the biventer cervicis nerve (El-Fawal et al., 1988) were monitored. PSP alone, 2.5 mg/kg, ellicitated severe OPIDN (terminal clinical score 7.5 & ± 1.0 [0-8 scale]; neuropathology score 2.7 ± 0.3 [0-4 scale, based on myelinated fiber degeneration]). PMSF given 12 hours prior to PSP gave complete protection (clinical and neuropathology scores of 0; p