by Lao Jie. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 168-182). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Reorganization of the intermediate filament (IF) network during axonal regeneration is accompanied by changes in the expression of various IF proteins. An increase in expression of the neuronal IF subunit gefiltin in goldfish retinal ganglion cells (RGCs) has been linked to the unique ability of the goldfish optic nerve to regenerate following injury. Evidence suggests that the optic tectum may regulate the expression of gefiltin during regeneration. The goal of this thesis was to determine the function of the tectum in the regulation of gefiltin mRNA expression during optic fiber regeneration in the goldfish. It was found that gefiltin mRNA levels in the RGCs of animals that received an optic nerve crush (ONC group) increased by 10 days, peaked from 20 to 38 days at around 5.5-fold over normal, and declined to near normal by 115 days. In animals that had the entire tectum removed and an optic nerve crush (ETR group), gefiltin mRNA levels increased by 10 days, peaked at 20 days at around 5.5- to 6.5-fold over normal, and although they dropped slightly thereafter, they remained elevated at around 5-fold over normal for at least 115 days. When axons regenerated to the ipsilateral tectal lobe as a result of a left tectal lobe removal and left eye removal surgery the expression pattern of gefiltin mRNA paralleled that of the ONC group. It was also found that the abundance of gefiltin subunits in the retina was elevated at 30 days of regeneration in ONC and ETR animals, and that levels in the nerve were reconstituted to 80% of normal by 30 days. These results demonstrate that increases in gefiltin mRNA and protein levels during optic nerve regeneration are independent of the tectum, whereas the downregulation of gefiltin mRNA levels is entirely dependent upon the tectum. (Abstract shortened by UMI.)

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by Leung Shiu Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 137-146. / Chapter CHAPTER ONE --- Introduction --- p.1 / Chapter 1.1 --- Surgical intervention done for nerve injury --- p.1 / Chapter 1.2 --- Rehabilitation procedures after nerve injuries --- p.2 / Chapter 1.3 --- Frustrating result of recovery after nerve injuries --- p.3 / Chapter 1.4 --- Reasons for the poor results --- p.3 / Chapter 1.5 --- Objective of the study --- p.5 / Chapter 1.6 --- Hypothesis and organization of the study --- p.6 / Chapter CHAPTER TWO --- The effects of pulsed electromagnetic field on peripheral nerve regeneration --- p.8 / Chapter 2.1 --- Electrical field and nerve growth --- p.8 / Chapter 2.2 --- Experimental findings of effect of the electromagnetic field on peripheral nerve regeneration --- p.9 / Chapter 2.3 --- The diversity of interest --- p.17 / Chapter CHAPTER THREE --- Physiological effects of the pulsed electromagnetic field --- p.18 / Chapter 3.1 --- The conventional use of electromagnetic field in musculoskeletal rehabilitation --- p.18 / Chapter 3.2 --- The pulsed electromagnetic field --- p.18 / Chapter 3.3 --- Nature of the pulsed electromagnetic field with a carrier frequency of 27.12 MHz --- p.19 / Chapter 3.4 --- Therapeutic effects of the pulsed electromagnetic field --- p.20 / Chapter 3.5 --- Some experimental results of the pulsed electromagnetic field --- p.20 / Chapter 3.6 --- Discussion --- p.25 / Chapter CHAPTER FOUR --- Methology --- p.27 / Chapter 4.1 --- Experimental animals and aneasthesia --- p.27 / Chapter 4.2 --- Models of lesions --- p.28 / Chapter 4.3 --- Sample size and grouping of the experimental rats --- p.35 / Chapter 4.4 --- Pulsed electromagnetic field stimulation --- p.37 / Chapter 4.5 --- Methods of evaluating the nerve regeneration --- p.38 / Chapter 4.6 --- Statistical analysis --- p.53 / Chapter CHAPTER FIVE --- Results --- p.54 / Chapter 5.1 --- Directly repaired groups --- p.54 / Chapter 5.2 --- Crushed groups --- p.62 / Chapter 5.3 --- Artery bridge groups --- p.73 / Chapter 5.4 --- Sham operated groups --- p.84 / Chapter 5.5 --- Electron microscopic examination --- p.90 / Chapter 5.6 --- Summary of all the data --- p.94 / Chapter CHAPTER SIX --- Discuss ion --- p.96 / Chapter CHAPTER SEVEN --- Conclusion --- p.103 / Chapter 7.1 --- Restatement of the experimental objective and hypothesis --- p.103 / Chapter 7.2 --- Conclusion --- p.103 / Chapter 7.3 --- Suggestions for furthur research --- p.104 / Chapter 7.4 --- Clinical Implication --- p.105 / Chapter APPENDIX I --- Determination of the duration of survival of the experimental animal --- p.106 / Chapter APPENDIX II --- Perfusion of rats --- p.115 / Chapter APPENDIX III --- Horseradish peroxidase and tetramethvlbezindine reaction --- p.118 / Chapter APPENDIX IV --- Histology fixation --- p.120 / Chapter APPENDIX V --- Determination of the Position of the Histology Specimens --- p.121 / Chapter APPENDIX VI --- Raw Data Collected in the Experiment --- p.132 / REFERENCE --- p.137

This study aimed to better understand the clearance of myelin debris during Wallerian degeneration in the goldfish visual paths. Myelin debris was first examined immunohistochemically in the presence or absence of regenerating axons. From these preliminary experiments it was apparent that the clearance of myelin debris was not affected by regenerating axons and that the debris was removed in a differential pattern along the visual pathway. Specifically, in the distal stump of the nerve as well as in the optic tract, myelin debris had been effectively cleared by one-month postoperative, while in the cranial segment of the nerve debris persisted for at least 6 weeks after injury. The differential pattern of myelin debris in the optic nerve and tract was then analyzed qualitatively and quantitatively using thick and thin plastic sections at various time points during regeneration. The results suggested that highly activated peripheral macrophages were responsible for the effective clearance of myelin in the distal nerve stump. In the optic tract a number of cellular properties, including their unique population of astrocytes may have enhanced the rate of debris clearance. By contrast, in the cranial segment of the nerve persistent debris was found both intracellularly in phagosomes and extracellularly, suggesting that the resident phagocytes were deficient in effecting both phagocytosis and emigration. Deficient phagocytosis may be a result of the production of anti-inflammatory cytokines in this region, while the failure to emigrate is most likely due to the rigid network of astrocytes in the nerve.

No description available.

No description available.

The nexus of engineering and molecular biology has given birth to high-throughput technologies that allow biologists and medical scientists to produce previously unattainable amounts of data to better understand the molecular basis of many biological phenomena. Here, we describe the development of an enabling biotechnology, commonly known as microfluidics in the fabrication of high-throughput systems to study nerve degeneration and regeneration in the well-defined model nematode, Caenorhabditis elegans (C. elegans). Our lab previously demonstrated how femtosecond (fs) laser pulses could precisely cut nerve axons in C. elegans, and we observed axonal regeneration in vivo in single worms that were immobilized on anesthetic treated agar pads. We then developed a microfluidic device capable of immobilizing one worm at a time with a deformable membrane to perform these experiments without agar pads or anesthetics. Here, we describe the development of improved microfluidic devices that can trap and immobilize up to 24 individual worms in parallel chambers for high-throughput axotomy and subsequent imaging of nerve regeneration in a single platform. We tested different micro-channel designs and geometries to optimize specific parameters: (1) the initial trapping of a single worm in each immobilization chamber, simultaneously, (2) immobilization of single worms for imaging and fs-laser axotomy, and (3) long term storage of worms on-chip for imaging of regeneration at different time points after the initial axon cut. / text

The transplantation of primary Schwann cells (SC) has been shown to improve nerve regeneration. However, to monitor the survival of transplanted cells within the host, a stable labelling method is required. The in vitro characteristics of green fluorescent protein labelled SC (GFP SC) and their effects in an in vivo peripheral nerve injury model were investigated.   The GFP-SC were readily visualised ex vivo and stimulated significantly better axonal regeneration compared to controls. Clinical use of autologous SC for the treatment of nerve injuries is of limited use due to difficulty in obtaining clinically useful numbers. However, bone marrow mesenchymal stem cells (MSC) can trans-differentiate into SC like cells (dMSC). The in vitro and in vivo differentiation of MSC was explored, and the study extended to include the easily-accessible adipose stem cells (ASC).  In vitro, glial growth factor stimulated MSC express S100, a SC marker, and its expression is maintained following in vivo transplantation.  Similarly, untreated MSC transplanted in vivo also expressed S100, which indicates glial differentiation in response to local cytokines and growth factors. Using an in vitro model, comprising dMSC or dASC co-cultured with adult dorsal root ganglia (DRG) neurons, the capacity of the dMSC and SC like differentiated ASC (dASC) to promote axon myelination was verified: both cell types expressed transcripts for protein zero, peripheral myelin protein-22 and myelin basic protein. The potential of stem cells in nerve repair may be limited by innate cellular senescence or donor age affecting cell functionality thus it was essential to determine the effects of donor age on morphology and functionality of stem cells.  The proliferation rates, expression of senescence markers (p38 and p53) and the stimulation of neurite outgrowth from DRG neurons by stem cells isolated from neonatal, young or old rats were very similar. However, the distribution and ultrastructure of mitochondria in dMSC and dASC from young and old rats were quite different, and seem to indicate physiological senescence of the aged cells.  Given the wide-ranging influence of Notch signalling in cell differentiation, including the neural crest to a glial cell type switch, and self-renewal in mammals, its role in the differentiation of stem cells to SC was investigated. The mRNA for notch-1 and -2 receptors were expressed in the dASC, blockage of notch signaling did not affect the neurotrophic and myelination potential of dASC.  In conclusion, these findings show that GFP labelling has no deleterious effect on SC survival and function. MSC and ASC differentiated into glial-type cells acquire SC morphology, and express characteristic SC markers, and the differentiation process was independent of the Notch signaling pathway. Also, following transplantation into a nerve gap injury dMSC improve regeneration. This study established that following co-culture with DRG neurons, dMSC and dASC were able to express peripheral myelin proteins.  Also, the functional bioactivity of these cells is independent of the donor animal age. Finally, although the glial lineage differentiated aged cells characterized in this study expressed markers typical of senescence they retained the potential to support axon regeneration.

Nerve autografts are widely used clinically to repair nerve grafts. However, nerve grafts have many limitations, such as, availability of donor nerve grafts, and loss of function at donor site. To overcome these problems, we have used a tissue engineering approach to design three-dimensional (3D) agarose scaffolds containing gradients of laminin-1 (LN-1) and nerve growth factor (NGF) to mimic in vivo conditions to promote nerve regeneration in rats. To determine the effect of LN-1 gradients on neurite extension in vitro, dorsal root ganglia (DRG) from chick embryos were cultured in 3D hydrogels. A gradient of LN-1 molecules in agarose gels was made by diffusion technique. LN-1 was then immobilized to the agarose hydrogels using a photo-crosslinker, Sulfo-SANPAH (Sulfosuccinimidyl-6-[4-azido-2-nitrophenylamino] hexanoate). Anisotropic scaffolds with three different slopes of LN-1 gradients were used. Isotropic scaffolds with uniform concentrations of LN-1, at various levels, were used as a positive control. DRG cultured in anisotropic scaffolds with optimal slope of LN-1 gradient extended neurites twice as fast as DRG in optimal concentration in isotropic scaffolds. Also, in the anisotropic scaffolds the faster growing neurites were aligned along the direction of LN-1 gradient. To promote nerve regeneration in vivo, tubular polysulfone guidance channels containing agarose hydrogels with gradients of LN-1 and NGF (anisotropic scaffolds) were used to bridge 20-mm nerve gaps in rats. Nerve autografts were used as positive controls and isotropic scaffolds, with uniform concentration of LN-1 and NGF, were used as negative controls. After 4-months, the rats were sacrificed and nerve histology was done to test for nerve regeneration. Only anisotropic scaffolds and nerve autografts contained evidence of axonal regeneration. Both groups had similar numbers of myelinated axons and similar axonal-diameter distribution. However, nerve graft group performed better in functional outcome as measured by relative gastrocnemius muscle weight (RGMW) and electrophysiology. Optimization of performance of anisotropic scaffolds by varying the LN-1 and NGF concentration gradients might lead to development of scaffolds that can perform as well as nerve auotgrafts for nerve regeneration over long nerve gaps.