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A light activated approach for large gap peripheral nerve repairFairbairn, Neil G. January 2016 (has links)
Introduction: Conventional suture repair of peripheral nerves following injury is associated with several limitations such as technical difficulty, intra- and extra-neural scar formation, axonal escape and the leakage of neurotrophic factors. These limitations are particularly relevant following nerve grafting when regenerating axons must traverse two coaptation sites. Outcomes following suture repair are notoriously poor, providing large impetus for the development of alternative methods. Photochemical tissue bonding (PTB) uses visible light to create sutureless, non-thermal bonds between two closely apposed tissue surfaces stained with a photoactive dye. When used with a human amnion nerve wrap for end-to end nerve repair, this technique results in superior functional and histological outcomes in comparison to conventional epineurial suture. When initially applied to large gap injury and nerve grafting, outcomes were unsuccessful due to proteolytic degradation of amnion and photochemical bonds during extended periods of recovery. Chemical crosslinking of nerve wraps prior to PTB may improve wrap durability and efficacy of technique. This thesis provides a comprehensive three-phase assessment of the efficacy of this novel approach when applied to the repair of large gap injuries with nerve grafts. Phase 1 assesses the ex vivo biomechanical properties of nerve wraps and light activated bonds in addition to the in vivo performance of photochemically sealed crosslinked nerve wraps against several other clinically relevant fixation methods in a rodent sciatic nerve isograft model. Following major multi-limb injury and amputation, demand for autogenous nerve graft may exceed that which can be supplied by the patient. Acellular nerve allograft (ANA) is an alternative option in these circumstances although outcomes are typically inferior to autograft. Phase 2 assesses the performance of the optimum repair strategy from phase 1 against conventional epineurial suture when applied to ANA. Most studies investigating the efficacy of novel repair techniques tend to perform repairs immediately following injury, a situation that rarely occurs clinically. Delays of weeks or months are not uncommon and have been shown to have a detrimental effect on regeneration and outcome. Phase 3 assesses the efficacy of PTB when applied to delayed nerve grafting. Additional work investigating a novel imaging technique for visualizing nerve revascularisation following injury and repair has been included. Optical frequency domain imaging (OFDI) uses low power infrared light to provide real time in vivo imaging of tissue microvasculature and flow characteristics. Originally applied to the study of tumour biology, this technique may prove useful for outcome assessment in preclinical research and eventually for the assessment of nerve viability in the clinical setting. Experiments investigating the early development of a brain body interface system (BBI) for upper limb reanimation following spinal cord injury (SCI) have also been included. The ultimate aim of this project is to restore autonomous motor control in a non-human primate (NHP) using cortically driven stimulation of peripheral nerves via implantable nerve cuffs. The experiments reported in this thesis detail the development of a selective, reversible paralysis model of elbow flexion in a NHP and demonstrate selective fascicular stimulation using acute and chronically implanted nerve cuffs in rodent and murine models. Methods: Phase 1: Three candidate nerve wraps (human amnion (HAM), crosslinked human amnion (xHAM), crosslinked swine intestinal sub-mucosa (xSIS)) and 3 fixation methods (suture, fibrin glue, PTB) were investigated. Crosslinking was performed using (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS). Biomechanical tests were performed using a tensiometer. Ex vivo wrap durability was assessed using a type- 2 collagenase degradation assay. Under isoflurane anaesthesia, 110 inbred male Lewis rats had 15mm left sciatic nerve defects created and repaired with reversed isografts. 9 groups (n=10) had isografts secured by one of the aforementioned wrap/fixation combinations. PTB repairs had nerve wraps and nerve ends stained with photoactive dye (Rose Bengal) and, once nerve ends were apposed and wrapped circumferentially, the interface was illuminated with a 532nm laser. Fibrin repairs had nerve ends apposed, wrapped circumferentially and secured with Tisseel fibrin glue. Suture repairs had nerve ends apposed, wrapped circumferentially and then secured with two 10-0 nylon sutures at each coaptation site (one either side of each repair). Positive and negative control groups (n=10) were repaired with graft+suture (10-0 nylon) and no repair respectively. Phase 2: 20 sciatic nerves were harvested from Sprague Dawley rats and sent to AxoGen Inc. for decellularisation. An additional 20 male inbred Lewis rats were randomized into 2 groups (n=10). All rats had 15mm left sciatic nerve defects created and repaired with processed ANA. 1 group had nerves secured using conventional epineurial suture. The remaining group had ANA secured using photochemically sealed amnion wraps. Phase 3: 40 inbred male Lewis rats were randomized into 4 groups (n=10). All 40 rats had 15mm left sciatic nerve gaps created and reconstructed with reversed isografts harvested from donor Sprague Dawley rats. In groups 1 and 2, nerve gaps were repaired immediately with either conventional epineurial suture or photochemically sealed amnion wraps, respectively. In groups 3 and 4, repair took place 30- days following injury using either conventional epineurial suture or photochemically sealed amnion wraps, respectively. All outcomes were assessed using walking track analysis and calculation of sciatic function index (SFI). Walking track analysis and SFI was performed pre-operatively, after the 30-day delay (phase 3) and at 30-day intervals following surgery. Following sacrifice after 5-months, left (experimental) and right (control) gastrocnemius muscles were excised and weighed for calculation of muscle mass retention. Nerves were excised for histomorphometric analysis including axon count, fiber diameter, axon diameter, myelin thickness and G-ratio. For all in vivo experiments, statistical analysis was performed using ANOVA, repeated measures ANOVA and the post hoc Bonferroni test. Optical Frequency Domain Imaging (OFDI) pilot study: eight rodents were randomized into 4 groups (n=2): (1) crush injury, (2) transection and end-to-end repair, (3) transection and repair of 10mm nerve gap using contralateral autograft, (4) transection and repair of 10mm nerve gap using ANA. Under ketamine/xylazine anaesthesia, all rodents had sciatic nerves exposed through hind limb dorsolateral incisions. Imaging was performed immediately pre-injury, immediately post-injury and on post-operative days 1, 3, 5 and 7. Rodents were secured firmly to polystyrene platforms in order to reduce movement artifact during imaging Brain-Body Interface (BBI) experiments: In the upper limb of a Rhesus macaque nonhuman primate, the median nerve branch to brachialis and radial nerve branch to brachioradialis were transected, leaving elbow flexion entirely reliant on the musculocutaneous nerve. The musculocutaneous nerve was transposed into a subcutaneous position. Ultrasound guided nerve block resulted in a highly selective, reversible paralysis of elbow flexion. Under ketamine/xylazine anaesthesia, Sprague Dawley rats (n=5) and C57 Black 6 mice (n=5) had sciatic nerves exposed through dorsolateral, muscle splitting incisions. 8-channel stimulating cuff electrodes were wrapped around sciatic nerves and connected to a Tucker14 Davies stimulation/recording system. Electromyography (EMG) needle electrodes were inserted into the tibialis anterior (TA) and gastrocnemius (G) muscles to record muscle activity.
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Effect of intranasal delivery of carbon dioxide on trigeminal ganglion neurons : inhibition of neuron-glia gap junction communication and neuropeptide secretion /Kankipati, Stanka, M.K., January 1900 (has links)
Thesis (M.S.)--Missouri State University, 2008. / "August 2008." Includes bibliographical references (leaves 45-51). Also available online.
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Nerve gap repair by the use of artificial conduits and cultured cellsKalbermatten, Daniel January 2010 (has links)
Peripheral nerve injuries are often associated with loss of nerve tissue and require autologous nerve grafts to provide a physical substrate for axonal growth. This thesis investigates the use of fibrin as both a tubular conduit to guide nerve regeneration and also as a matrix material to suspend various regenerative cell types within/on poly-3-hydroxybutyrate (PHB) nerve conduits. Adipose derived stem cells (ASC) are found in abundant quantities. In this thesis the ability of rat ASC to differentiate into Schwann cells was determined and a preliminary study of the neurotrophic potential of human ASC was also investigated. Rat sciatic nerve axotomy was performed proximally in the thigh to create a 10-mm gap between the nerve stumps and the gap was bridged using the various conduits. At early time points the nerve grafts were harvested and investigated for axonal and Schwann cell markers. After 16 weeks the regenerative response from sensory and motor neurons was also evaluated following retrograde labelling with Fast Blue fluorescent tracer. Stem cells were treated with a mixture of glial growth factors and after 2 weeks in vitro the expression of Schwann cell markers was analysed by immunocytochemistry and Western blotting. ASC were cocultured with the NG108-15 neuronal cell line to determine their ability to promote neurite outgrowth. Human ASC were isolated from the deep and superficial layers of abdominal fat tissue obtained during abdominoplasty procedures. RT-PCR was used to investigate the expression of neurotrophic factors. Immunohistochemistry showed a superior nerve regeneration distance in the fibrin conduit compared with PHB. The fibrin conduit promoted regeneration of 60% of sensory neurones and 52% of motor neurones when compared with an autograft group at 16 weeks. The total number of myelinated axons in the distal nerve stump in the fibrin-conduit group reached 86% of the graft and the weight of gastrocnemius and soleus muscles recovered to 82% and 89% of the controls, respectively. In vitro studies showed that rat ASC could be differentiated to a Schwann cell phenotype. These treated cells enhanced both the number of NG108-15 cells expressing neurites and neurite length. In the same coculture model system, human superficial fat layer ASC induced significantly enhanced neurite outgrowth when compared with the deep layer fat cells. RT-PCR analysis showed ASC isolated from both layers expressed neurotrophic factors. These results indicate that a tubular fibrin conduit can be used to promote neuronal regeneration following peripheral nerve injury. There was also a beneficial effect of using a fibrin matrix to seed cells within/on PHB conduits which should ultimately lead to improved functional recovery following nerve injury. There might also be an advantage to use a simple strip of PHB rather than a conventional tube-like structure implying that single fascicle nerve grafting could be advantageous for nerve repair. The results of in vitro experiments indicate adipose tissue contains a pool of regenerative stem cells which can be differentiated to a Schwann cell phenotype and given that human ASC express a range of neurotrophic factors they are likely to be of clinical benefit for treatment of peripheral nerve injuries.
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TNF-a regulation of cell signaling in trigeminal ganglion in an in vivo model of TMJ inflammation /Damodaram, Srikanth, January 1900 (has links)
Thesis (M.S.)--Missouri State University, 2008. / "August 2008." Includes bibliographical references (leaves 101-112). Also available online.
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