Evidence indicates attention can alter afferent inhibition, a Transcranial Magnetic Stimulation (TMS) evoked measure of cortical inhibition following somatosensory input. This measure is emerging as a valuable tool for clinical assessment of sensorimotor function. However, the reliability of the measure remains relatively low. Further, attention is capable of modifying the magnitude of afferent inhibition. Therefore, for afferent inhibition to become an assessment with translation within and beyond the research lab, the reliability of the measure must be improved. Controlling the focus of attention may be one method to improve the reliability of afferent inhibition. In the present study, two experiments were conducted. One to assess the biological effects of attention on SAI and LAI, and the other to address whether the reliability of SAI and LAI are altered in the presence of varying attentional demands. The magnitude of short- and long-latency afferent inhibition (SAI and LAI, respectively) was assessed under four conditions with varying attentional demands focused on the somatosensory input that mediates SAI and LAI circuits. Further, the reliability of SAI and LAI was assessed with and without directed attention to the relevant somatosensory input to explore whether attention to the tactile stimulation can improve intrasession and intersession reliability of these measures. Thirty individuals participated in four conditions; three conditions were identical in their physical parameters and varied only in the focus of directed attention (visual attend, tactile attend, non- directed attend) and one condition consisted of no external physical parameters (no stimulation). Reliability was measured by repeating conditions at three time points to assess intrasession and intersession reliability. Results indicate the magnitude of SAI and LAI were not modulated by varied attention. Reliability assessments demonstrated that the attention manipulations increased intrasession and intersession reliability of SAI and LAI compared to the no stimulation condition. This research exposes the influence of attention, and its impact on the reliability of afferent inhibition. By quantifying these influences, this research has identified new information to inform the design of TMS research in sensorimotor integration. / Thesis / Master of Science in Kinesiology / Attention can alter transcranial magnetic stimulation (TMS) evoked afferent inhibition. Measures of afferent inhibition are emerging as valuable tools for clinical assessments of sensorimotor function. However, the reliability of afferent inhibition remains relatively low, limiting its value in the clinic. Afferent inhibition is increased when the one’s attention is focused on the peripheral nerve stimulation used to elicit afferent inhibition. However, it is unknown whether afferent inhibition, with attention directed to somatosensory input, will improve the reliability of these measures. This is important as it suggests that changes to the methodology used to acquire afferent inhibition can improve the reliability of this measure, thereby increasing the opportunity for translation to the clinic. The goal of this study was to assess the influence of attention on afferent inhibition circuits, short afferent inhibition (SAI) and long afferent inhibition (LAI) and determine whether attention modulation would increase the reliability of afferent inhibition.

This study examines the prospects of using the electrospinning process to fabricate tissue engineering scaffolds targeting a variety of regenerative applications, with a primary focus on the production of nerve guides for the treatment of long-defect nerve injuries in the peripheral nervous system. A basic overview of the conventional electrospinning process is provided, and the utility of this fabrication scheme in the production of collagen-based tissue engineering scaffolds is demonstrated. Next, a novel modification of the basic electrospinning process is presented. This process, called two pole air gap electrospinning, was developed to produce nerve guides that exhibit an anisotropic structure that mimics the extracellular matrix of native peripheral nerve tissue. This electrospinning process makes it possible to produce macroscopic nerve guides that are cylindrical in shape and composed of dense arrays of nano- to micron-scale diameter fibers. Unlike, conventional hollow core nerve guides, these electrospun constructs lack a central lumen, hence the designation 3D (for three-dimensional) nerve guide. The fibers are nearly exclusively arrayed in parallel with the long axis of the construct. This architectural feature provides thousands of individual channels, and aligned fibers that provide guidance cues that are designed to drive regenerating axons to grow in a highly directed fashion down the longitudinal axis of the guide. To supplement the structural cues provided by the fibrillar arrays of the electrospun 3D nerve guides, an alginate-based platform designed to deliver therapeutic reagents was developed and characterized. This platform makes it possible to fabricate gradients of therapeutic reagents within the fibrillar arrays of an electrospun nerve guide. Functional and structural analyses of these constructs supplemented with or without a gradient of NGF, in a long-defect nerve injury in the rodent sciatic nerve indicate that the 3D design is superior to the gold standard treatment, the autologous nerve graft. Animals treated with the 3D grafts recovered motor and sensory function faster and exhibited far higher nerve-to-nerve and nerve-to-muscle signal amplitudes in electrophysiological studies than animals treated with autologous grafts or conventional hollow core cylindrical grafts.

The main objective of this project was to develop a method for soft neural probe insertion into a nerve. These soft probes are made of a silicon elastomer that is a few orders of magnitude less stiff than the nerve, which makes the insertion process particularly challenging. To overcome this challenge a tungsten microwire was used as an insertion shuttle to help penetrate the nerve by increasing the overall stiffness of the probe. At a first stage, the insertion process was tested on a nerve phantom (e.g. agarose gel with PDMS membrane) to control the insertion parameters and validate the insertion platform. Once the envisioned insertion method was established, the probe was implanted in a real rodent nerve. / <p>Examensarbetet är utfört vid Institutionen för teknik och naturvetenskap (ITN) vid Tekniska fakulteten, Linköpings universitet</p>

Traumatic injuries to the spinal cord (SCI) and peripheral nerve (PNI) affect several thousand people worldwide every year. At present, there is no effective treatment for SCI and despite continuous improvements in microsurgical reconstructive techniques for PNI, many patients are still left with permanent, devastating neurological dysfunction. This thesis investigates the effects of mesenchymal stem cells (MSC) derived from adipose (ASC) and dental (DSC) tissue and chitosan/microRNA-124 polyplex particles on regeneration after spinal cord and peripheral nerve injury in adult rats. Dental stem cells were obtained from apical papilla, dental pulp, and periodontal ligament. ASC and DSC expressed MSC surface markers (CD73, CD90, CD105 and CD146) and various neurotrophic molecules including BDNF, GDNF, NGF, VEGF-A and angiopoietin-1. Growth factor stimulation of the stem cells resulted in increased secretion of these proteins. Both ASC and DSC supported in vitro neurite outgrowth and in contrast to Schwann cells, ASC did not induce activation of astrocytes. Stimulated ASC also showed an enhanced ability to induce capillary-like tube formation in an in vitro angiogenesis assay. In a peripheral nerve injury model, ASC and DSC were seeded into a fibrin conduit, which was used to bridge a 10 mm rat sciatic nerve gap. After 2 weeks, both ASC and DSC promoted axonal regeneration in the conduit and reduced caspase-3 expression in the dorsal root ganglion (DRG). ASC also enhanced GAP-43 and ATF-3 expression in the spinal cord, reduced c-jun expression in the DRG and increased the vascularity of the implant. After transplantation into injured C3-C4 cervical spinal cord, ASC continued to express neurotrophic factors and laminin and stimulated extensive ingrowth of 5HT-positive raphaespinal axons into the trauma zone. In addition, ASC induced sprouting of raphaespinal terminals in C2 contralateral ventral horn and C6 ventral horn on both sides. Transplanted cells also changed the structure and the density of the astroglial scar. Although the transplanted cells had no effect on the density of capillaries around the lesion site, the reactivity of OX42-positive microglial cells was markedly reduced. However, ASC did not enhance recovery of forelimb function. In order to reduce activation of microglia/macrophages and the secondary tissue damage after SCI, the role of microRNA-124 was investigated. In vitro transfection of chitosan/microRNA-124 polyplex particles into rat microglia resulted in the reduction of reactive oxygen species and TNF-α levels and lowered expression of MHC-II. Upon microinjection into uninjured rat spinal cords, particles formed with Cy3-labeled control sequence RNA, were specifically internalized by OX42 positive macrophages and microglia. Alternatively, particles injected in the peritoneum were transported by macrophages to the site of spinal cord injury. Microinjections of chitosan/microRNA-124 particles significantly reduced the number of ED-1 positive macrophages after SCI. In summary, these results show that human MSC produce functional neurotrophic and angiogenic factors, creating a more desirable microenvironment for neural regeneration after spinal cord and peripheral nerve injury. The data also suggests that chitosan/microRNA-124 particles could be potential treatment technique to reduce neuroinflammation.

Despite surgical innovation, the sensory and motor outcome after peripheral nerve injury is incomplete. In this thesis, the biological pathways potentially responsible for the poor functional recoveries were investigated in both the distal nerve stump/target organ, spinal motoneurons and dorsal root ganglia (DRG). The effect of delayed nerve repair was determined in a rat sciatic nerve transection model. There was a dramatic decline in the number of regenerating motoneurons and myelinated axons found in the distal nerve stumps of animals undergoing nerve repair after a delay of 3 and 6 months. RT-PCR of the distal nerve stumps showed a decline in expression of Schwann cells (SC) markers, with a progressive increase in fibrotic and proteoglycan scar markers, with increased delayed repair time. Furthermore, the yield of SC which could be isolated from the distal nerve segments progressively fell with increased delay in repair time. Consistent with the impaired distal nerve stumps the target medial gastrocnemius (MG) muscles at 3- and 6-months delayed repair were atrophied with significant declines in wet weights (61% and 27% compared with contralateral sides). The role of myogenic transcription factors, muscle specific microRNAs and musclespecific E3 ubiquitin ligases in the muscle atrophy was investigated in both gastrocnemius and soleus muscles following either crush or nerve transection injury. In the crush injury model, the soleus muscle showed significantly increased recovery in wet weight at days 14 and 28 (compared with day 7) which was not the case for the gastrocnemius muscle which continued to atrophy. There was a significantly more pronounced up-regulation of MyoD expression in the denervated soleus muscle compared with the gastrocnemius muscle. Conversely, myogenin was more markedly elevated in the gastrocnemius versus soleus muscles. The muscles also showed significantly contrasting transcriptional regulation of the microRNAs miR-1 and miR-206. MuRF1 and Atrogin-1 showed the highest levels of expression in the denervated gastrocnemius muscle. Morphological and molecular changes in spinal motoneurons were compared after L4-L5 ventral root avulsion (VRA) and distal peripheral nerve axotomy (PNA). Neuronal degeneration was indicated by decreased immunostaining for microtubule-associated protein-2 in dendrites and synaptophysin in presynaptic boutons after both VRA and PNA. Immunostaining for ED1-reactive microglia and GFAPpositive astrocytes was significantly elevated in all experimental groups. qRT-PCR analysis and Western blotting of the ventral horn from L4-L5 spinal cord segments revealed a significant upregulation of apoptotic cell death mediators including caspases-3 and -8 and a range of related death receptors following VRA. In contrast, following PNA, only caspase-8 was moderately upregulated. The mechanisms of primary sensory neuron degeneration were also investigated in the DRG following peripheral nerve axotomy, where several apoptotic pathways including those involving the endoplasmic reticulum were shown to be upregulated. In summary, these results show that the critical time point after which the outcome of regeneration becomes too poor appears to be 3-months. Both proximal and distal injury affect spinal motoneurons morphologically, but VRA induces motoneuron degeneration mediated through both intrinsic and extrinsic apoptotic pathways. Primary sensory neuron degeneration involves several different apoptotic pathways, including the endoplasmic reticulum.

Apesar do desenvolvimento da tecnologia envolvendo o campo da regeneração em nervos periféricos, ainda não existe uma técnica para recuperação do tecido nervoso que apresente resultados totalmente satisfatórios, fato que desperta o interesse de vários pesquisadores em todo mundo e representa um desafio para os cirurgiões que realizam procedimentos reconstrutivos e estéticos. Embora o enxerto autólogo de nervo seja a melhor alternativa para a recuperação de nervos periféricos lesados, as técnicas que envolvem o reparo tubular têm alcançado excelentes resultados através da utilização de materiais sintéticos e biológicos, sob a forma de tubos, no reparo de extensos segmentos nervosos. Hoje, através dos avanços da engenharia tecidual, novos materiais estão em desenvolvimento, com o objetivo de aliar características microscópicas às técnicas cirúrgicas existentes. O colágeno, que é sabidamente um elemento promotor da proliferação celular e do reparo tecidual, tem sido amplamente utilizado em estudos de regeneração nervosa. Da mesma maneira, o ácido poli-láctico-poli-glicólico (PLGA), um copolímero sintético, tem apresentado algumas características favoráveis ao processo de regeneração, como biocompatibilidade e propriedades mecânicas adequadas. Com o propósito de facilitar a regeneração nervosa quando ocorre perda tecidual, uma técnica já difundida pode ser destacada: o enxerto de veia invertida, em que a veia jugular externa é utilizada ao avesso, funcionando como um tubo, criando um microambiente para a regeneração nervosa, através da exposição de elementos fundamentais da camada mais externa do vaso (túnica adventícia). Neste trabalho, agregamos como diferencial a utilização de dois tipos de membranas, especialmente desenvolvidas para fins odontológicos, que visam neoformação óssea, em um estudo que visa à regeneração de um nervo periférico misto, o nervo isquiático. As membranas de colágeno e PLGA foram colocadas na região do enxerto, sob a forma de tubo, com o objetivo de recuperar um segmento de 10 mm do nervo referido. E, por fim, aliamos a utilização de um segmento de 5 mm da veia jugular externa como tecido de preenchimento dos tubos feitos com as membranas, na tentativa de verificar a eficácia das moléculas biológicas presentes neste vaso, porém agora sendo utilizadas diretamente dentro do enxerto. Após um período de 6 semanas, o grupo que apresentou o melhor resultado foi aquele em que foi utilizada a membrana de colágeno, na forma de tubo, preenchida com a veia jugular externa. Nesse grupo, o diâmetro médio das fibras mielínicas apresentou um valor médio de 5,8 µm e espessura média da bainha de mielina de 1,65 µm. Para o período de 12 semanas, entre os grupos analisados, o maior valor médio encontrado para o diâmetro foi de 5,64 µm e 1,31 µm para a espessura, sendo que este resultado foi apresentado pelo grupo em que se utilizou a membrana de PLGA sem preenchimento com a veia jugular. Embora os valores não sejam muito inferiores, nos dois períodos, ficaram abaixo dos valores encontrados nos grupos controle normais. Em suma, acreditamos que as membranas de colágeno e PLGA, associadas à técnica de tubulização, apresentam grande potencial para serem utilizadas na regeneração de nervos periféricos. / Despite the development of technology involved in peripheral nerve regeneration, there is no technique that presents a recovery of the nervous tissue with completely satisfactory results. This fact arouses interest of several researchers around the word. Although the autologous nerve grafting is the current gold standard for the repair of large nerve gaps, over the past decades the development of artificial nerve conduits has therefore been of great interest due to the excellent results achieved. Through the advances in tissue engineering, new materials, synthetic and biological, have been used for construct nerve guides. The collagen is one of the oldest natural polymers used as a biomaterial, and it is known as a promoter of cell proliferation and of tissue repair. In the same way, the synthetic copolymer, poly lactic-co-glycolic acid (PLGA), have been used for nerve regeneration, and it have shown some favorable characteristics to nerve repair such as stability, biocompatibility, and mechanical properties. With the purpose of facilitating the regeneration in large nerve gaps, the inside-out vein graft is a widespread technique, where the vein works as a conduit, and it provides a good microenvironment for axon regeneration through the exposition of some primordial elements of its adventitial layer. In this work, we join as a differential the use of two materials, specially developed for dental purposes, which are normally used to facilitate the osteogenesis, in a nerve regeneration study. We performed the surgical procedures in the sciatic nerve, with two types of membranes (PLGA and collagen), which were used as tubes, in order to promote the regeneration of this nerve. The collagen and PLGA membranes were used with the objective of a recovery in a 10-mm sciatic nerve gap model. These tubular implants were filled with a 5 mm segment of the jugular vein in order to verify the effectiveness of some biological molecules of the adventitia layer in the nerve regeneration. After 6 and 12 weeks the graft and the distal stump were observed under light microscopy. In each studied period, the diameters of the myelin fibers, and the thickness of the myelin sheaths of the regenerated axons were measured. After 6 weeks, the group which presented the best result was the group with the collagen membrane tube filled with the external jugular vein. In this group the diameter of the myelin fibers presented an average value of 5.8 µm and an average value of the thickness of the myelin sheaths of 1.65 µm. After 12 weeks, between all the groups, the best results were found in the group where the PLGA membrane unfilled with the external jugular vein was used as a nerve guide, which presented the average diameter of 5.64 µm, and the average thickness of 1.31 µm. Although the average values werent much lower than the values found in the controls, they were beneath than the normal groups. These results proved that the collagen and the PLGA membranes have a great potential to be used as artificial nerve conduits for promoting peripheral nerve regeneration.

As lesões dos nervos periféricos, que inervam os músculos esqueléticos, evoluem para perdas da propriocepção e alterações na morfologia e função das fibras musculares, causando um impacto negativo na qualidade de vidas dos indivíduos. Tais lesões implicam em alteração na expressão de genes específicos do músculo, como por exemplo, na MyoD e Miogenina, atuantes na ativação de células satélites e reguladores da massa muscular A técnica cirúrgica de tubulização é um recurso empregado na prática clínica para tratamento de músculos que sofreram desnervação. O objetivo do presente estudo foi analisar se a técnica de tubulização com o preenchimento de gordura altera a expressão de Myod e Miogenina, a morfometria do músculo sóleo de ratos e localização da Myod e Miogenina. Para isso, 57 ratos Wistar foram separados em grupos: controle inicial (GCI); final 45 (GCF45), final 150 (GCF150), desnervado 45 dias (GD45), desnervado 150 dias (GCD150) e grupos experimentais com veia vazia 45 dias (GESP45) e 150 dias (GESP150) e com veia preenchida de gordura 45 dias (GEG45) e 150 dias (GEG150). Para os procedimentos cirúrgicos de desnervação e reinervação e coleta do músculo os animais foram profundamente anestesiados. Após os devidos tempos experimentais, os animais foram sacrificados, o músculo sóleo foi dissecado, envolvido em meio de criopreservação e estocado a -80°C. A quantificação de mRNA do MyoD e Miogenina foi realizada por amplificação por reação em cadeia de polimerase (PCR) em tempo real (RealTimePCR) e a localização da produção de Myod e Miogenina foi realizada por microscopia confocal a laser e imunofluorescência. A morfometria foi realizada em lâminas coradas com HE, observadas em microscópio ótico e calculadas pelo software Image Pro-Plus 6.2. Os resultados do presente estudo mostraram que houve aumento da expressão do Myod e Miogenina nos grupos experimentais 45 dias quando comparados ao grupo controle inicial e um decréscimo da expressão de Myod e Miogenina para os grupos experimentais com 150 dias. A área da secção transversa nos grupos experimentais com 45 dias (GESP45 e GEG45) não apresentaram diferença estatística, quando comparado com grupo desnervado 45 dias (GCD45), enquanto que o grupo experimental com preenchimento de gordura 150 dias (GEG150) obteve os melhores resultados na medida da área da secção transversal do músculo sóleo. As lâminas observadas no microscópio confocal mostram a MyoD e Miogen localizadas no mionúcleo. Concluiu-se que o uso da gordura na técnica de tubulização do nervo ciático de ratos, interfere na regeneração do músculo sóleo. / Peripheral nerve injuries can result in the loss of propioception, morphological and functional alterations of muscle fibers which causes a negative impact on the quality of life. These injuries elicit an alteration on the expression of muscle specific genes, like MyoD and Myogenin, involved in the satellite cell activation and muscle mass regulation. The vein graft tubulization is a well known technique for treatment of denervated muscle. The aim of this work was to investigate if vein graf tubulization filled with fat tissue changes the expression and localization of MyoD and Myogenin and to study if it can modify the morphometry of soleus muscle. Fifty seven Wistar rats were divided in initial control group (ICG), final control group 45 days and 150 days (FCG45; FCG150), denervated 45 days and 150 days (D45; D150) and experimental groups with vein graft 45 days and 150 days (VG45; VG150). and vein graft filled with fat tissue 45 days and 150 days (VF45; VF150). For denervation and reinervation procedures and muscle biopsy the animals were submitted to anaesthesia and after the experimental time they were euthanized. Soleus muscle was dissected, involved in criopreservation medium and stored at -80oC. It was performed RealTime polymerase chain reaction (RealTimePCR) for MyoD and Myogenin mRNA quantification. The localization of its production was analysed by laser confocal microscopy and immunofluorescence staining. The morphometric analysis were done by Hematoxilin-Eosin staining and examined at optical microscopy using the Image ProPlus 6.2 software. There was an upregulation on the expression of MyoD and Myogenin for the experimental groups at 45 days when compared to the initial control group. On the other hand, we found a downregulation on the MyoD and Myogenin expression in the same groups with 150 days. The area of transversal section in the 45 days experimental groups (VF45, VG45) did not show statistical difference compared with denervated group with 45 days (D45). Moreover, the group filled with fat tissue at 150 days (VF150) presented the best results in the transversal section area of soleus muscle. In addition, the slides analysed under confocal microscopy showed the localization of MyoD and Myogenin in the mionuclei. In conclusion, the application of vein graft filled fat tissue improves the soleus muscle regeneration.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-03-03T14:19:21Z No. of bitstreams: 2 Dissertação - Jonas Francisco Scopel - 2015.pdf: 1854226 bytes, checksum: 9d48662eb3f8662b36ce1a088c29ac1c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-03-03T14:22:35Z (GMT) No. of bitstreams: 2 Dissertação - Jonas Francisco Scopel - 2015.pdf: 1854226 bytes, checksum: 9d48662eb3f8662b36ce1a088c29ac1c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-03-03T14:22:35Z (GMT). No. of bitstreams: 2 Dissertação - Jonas Francisco Scopel - 2015.pdf: 1854226 bytes, checksum: 9d48662eb3f8662b36ce1a088c29ac1c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-02-20 / Diagnostic imaging techniques play an important role in assessing the exact location, cause, and extent of a nerve lesion, thus allowing clinicians to diagnose and manage more effectively a variety of pathological conditions such as entrapment syndromes, traumatic injuries, and space-occupying lesions. Ultrasound and nuclear magnetic resonance imaging are becoming useful methods for this purpose, but they still lack spatial resolution. In this regard, recent phase contrast x-ray imaging experiments of peripheral nerve allowed the visualization of each nerve fiber surrounded by its myelin sheath as clearly as optical microscopy. In the present study, we attempted to produce high-resolution x-ray phase contrast images of a human sciatic nerve by using synchrotron radiation propagation-based imaging (PBI). The PBIs showed high contrast and high spatial resolution, allowing clear identification of each fascicle structure and surrounding connective tissue. The outstanding result is the detection of such structures by phase contrast x-ray tomography of a thick human sciatic nerve section. This may further enable the identification of diverse pathological patterns, such as Wallerian degeneration, inflammatory infiltration and amyloid deposits. To the best of our knowledge, this is the first successful phase contrast x-ray imaging experiment of a human peripheral nerve sample. Our long-term goal is to develop peripheral nerve imaging methods that could supersede biopsy procedures. / As técnicas de diagnóstico por imagem desempenham um importante papel na determinação da localização exata, causa, e extensão de lesões de nervos periféricos, permitindo que o diagnóstico e manejo sejam mais eficazes em uma série de condições patológicas, como síndromes de encarceramento, e lesões traumáticas e expansivas. A Ressonância Nuclear Magnética e a Ultrassonografia têm se mostrado úteis neste propósito, porém possuem resolução espacial relativamente baixa. Neste sentido, experimentos recentes utilizando Radiografia por Contraste de Fase permitiram observar fibras nervosas individuais recobertas por suas bainhas de mielina de forma tão detalhada quanto a microscopia óptica. No presente estudo, procuramos obter imagens de alta resolução de nervo ciático humano, utilizando método de Imagem Realçada por Refração. As imagens demonstraram alto contraste e resolução espacial, permitindo a identificação de cada fascículo nervoso e suas respectivas bainhas conjuntivas. Resultados promissores foram obtidos através de Tomografia por Contraste de Fase de um espesso fragmento de nervo ciático humano, onde tais estruturas foram igualmente identificadas. Tais resultados poderiam possibilitar a detecção de diversas alterações patológicas, como degeneração Walleriana, infiltrado inflamatório e depósitos de amiloide. Ao nosso conhecimento, este é o primeiro experimento bem sucedido de Imagem por Contraste de Fase de nervos periféricos humanos. Nosso objetivo a longo prazo é desenvolver métodos de diagnóstico por imagem que possam substituir procedimentos de biópsia.

Background Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting toxicity with significant sequelae impacting prognosis and quality of life. The natural history and pathophysiological mechanisms of CIPN are unclear. Equally, the lack of systematic approach to diagnosis and assessments contribute to difficulty identifying at risk patients with implications on symptom burden. Effective management of CIPN is also difficult due to limited treatment options. To try and address this challenging clinical problem, this thesis aimed to adopt a translational approach to: 1) characterisation and early identification of the development of CIPN in cancer patients receiving neurotoxic chemotherapy and 2) explore topical treatment options in patients with chronic peripheral neuropathic pain. Methodology In the CIPN study, a mixed cohort of colorectal, gynaecological and lung cancer patients receiving neurotoxic chemotherapy (platinum agents and taxanes) were assessed prospectively, at baseline (prior to initiating chemotherapy), during cycles (every 3 weeks) and post-treatment (every 3 months) for up to 12 months (cumulative 261 assessments). Comprehensive longitudinal clinical characterisation consisted of the integration of quantitative sensory testing (QST), objective measure of function (grooved pegboard test), patient-reported outcomes and in vivo confocal microscopy to provide insight into the clinical course and potential psychophysical biomarkers of CIPN during and after chemotherapy. In the pilot intervention study, patients with chronic, complex cancer treatment related peripheral neuropathic pain received a single application of high concentration 8% capsaicin patch. Assessments conducted at baseline, 4 weeks and 12 weeks included patient-reported outcomes and QST with an exploratory application of in vivo confocal microscopy in a case. Results In the CIPN study, 33 patients when compared to 33 age and gender matched healthy controls displayed thermal hyperalgesia, sensorimotor impairment and increased resting heart rate prior to initiating neurotoxic chemotherapy. Characterisation of somato-sensory profile demonstrated dysfunction of the various types of primary afferent nerves (Aβ, Aδ and C). Assessing the change over time from baseline to during cycles and post-treatment follow up, revealed signs and symptoms as early as cycle 2 with an increase in the later cycles and 3 months post-treatment follow up. A greater burden was observed at 12 months in comparison to baseline. Significant changes were observed in QST parameters indicating both small and large fibre deficits. Interesting associations were observed for example with tactile deficits in the upper and lower limb and patient-reported outcomes. The repeated measures model provided an opportunity to distil the relationship between subjective and objective measures of CIPN. The subclinical findings at baseline however did not translate to obvious predictors of CIPN development. The exploratory use of in vivo confocal microscopy (45 healthy controls, 9 patients) demonstrated correlation with current assessment tools (QST). Analysis from the pilot intervention study of 20 patients revealed clinically significant improvement in pain in a subset at 4 and 12 weeks post-treatment. Conclusion Overall the combination of subjective and objective measures utilised in the prospective characterisation of this mixed cohort of cancer patients provided a useful paradigm for qualifying and quantifying the trajectory of CIPN related peripheral nerve damage and symptom burden with additional contribution from the novel in vivo confocal microscopy work. In capturing the varied spectrum of phenotypes, this approach may provide insight into the complexities of the underlying neurobiological mechanisms. The baseline subclinical sensory, motor and autonomic nerve dysfunction implicate a cancer-mediated process possibly contributing to CIPN. Although the preliminary investigation of baseline predictors of CIPN was inconclusive, thermal pain threshold warrant further investigation. These findings highlight the need to further address prediction and risk stratification in larger studies. The exploratory intervention study suggests that patients with chronic neuropathic pain may receive some benefit in pain severity, function and mood with effect continuing at 12 weeks post-treatment. This research warrants further investigation in larger cohorts.

Peripheral nerve injuries are often associated with significant loss of nervous tissue leading to poor restoration of function following repair of injured nerves. Although the injury gap could be bridged by autologous nerve graft, the limited access to donor material and additional morbidity such as loss of sensation and scarring have prompted a search for biosynthetic nerve transplants. The present thesis investigates the effects of a synthetic matrix BD™ PuraMatrix™ peptide (BD)hydrogel, alginate/fibronectin gel and fibrin glue combined with cultured rat Schwann cells or human bone marrow derived mesenchymal stem cells (MSC) on neuronal regeneration and muscle recovery after peripheral nerve injury in adult rats. In a sciatic nerve injury model, after 3 weeks postoperatively, the regenerating axons grew significantly longer distances within the conduits filled with BD hydrogel if compared with the alginate/fibronectin gel. The addition of rat Schwann cells to the BD hydrogel drastically increased regeneration distance with axons crossing the injury gap and entering into the distal nerve stump. However, at 16 weeks the number of regenerating spinal motoneurons was decreased to 49% and 31% in the BD hydrogel and alginate/fibronectin groups respectively. The recovery of the gastrocnemius muscle was also inferior in both experimental groups if compared with the nerve graft. The addition of the cultured Schwann cells did not further improve the regeneration of motoneurons and muscle recovery. The growth-promoting effects of the tubular conduits prepared from fibrin glue were also studied following repair of short and long peripheral nerve gaps. Retrograde neuronal labeling demonstrated that fibrin glue conduit promoted regeneration of 60% of injured sensory neurons and 52% of motoneurons when compared with the autologous nerve graft. The total number of myelinated axons in the distal nerve stump in the fibrin conduit group reached 86% of the nerve graft control and the weight of gastrocnemius and soleus muscles recovered to 82% and 89%, respectively. When a fibrin conduit was used to bridge a 20 mm sciatic nerve gap, the weight of gastrocnemius muscle reached only 43% of the nerve graft control. The morphology of the muscle showed a more atrophic appearance and the mean area and diameter of fast type fibres were significantly worse than those of the corresponding 10 mm gap group. In contrast, both gap sizes treated with nerve graft showed similar fiber size. The combination of fibrin conduit with human MSC and daily injections of cyclosporine A enhanced the distance of regeneration by four fold and the area occupied by regenerating axons by three fold at 3 weeks after nerve injury and repair. In addition, the treatment also significantly reduced the ED1 macrophage reaction. At 12 weeks after nerve injury the treatment with cyclosporine A alone or cyclosporine A combined with hMSC induced recovery of the muscle weight and the size of fast type fibres to the control levels of the nerve graft group. In summary, these results show that a BD hydrogel supplemented with rat Schwann cells can support the initial phase of neuronal regeneration across the conduit. The data also demonstrate an advantage of tubular fibrin conduits combined with human MSC to promote axonal regeneration and muscle recovery after peripheral nerve injury.