Thesis (PhD (Physiology (Human and animal))-- University of Stellenbosch, 2007. / Definition: Stem cells are unspecialised cells with the capacity for long-term self-renewal and the ability to differentiate into multiple cell-lineages. The potential for the application of stem cells in clinical settings has had a profound effect on the future of regenerative medicine. However, to be of greater therapeutic use, selection of the most appropriate cell type, as well as optimisation of stem cell incorporation into the damaged tissue is required. In adult skeletal muscle, satellite cells are the primary stem cell population which mediate postnatal muscle growth. Following injury or in diseased conditions, these cells are activated and recruited for new muscle formation. In contrast, the potential of resident adult stem cell incorporation into the myocardium has been challenged and the response of cardiac tissue, especially to ischaemic injury, is scar formation. Following muscle damage, various growth factors and cytokines are released in the afflicted area which influences the recruitment and incorporation of stem cells into the injured tissue. Transforming Growth Factor-β (TGF-β) is a member of the TGF-β-superfamily of cytokines and has at least three isoforms, TGF-β1, -β2, and -β3, which play essential roles in the regulation of cell growth and regeneration following activation and stimulation of receptor-signalling pathways. By improving the understanding of how TGF-β affects these processes, it is possible to gain insight into how the intercellular environment can be manipulated to improve stem cell-mediated repair following muscle injury. Therefore, the main aims of this thesis were to determine the effect of the three TGF-β isoforms on proliferation, differentiation, migration and fusion of muscle progenitor cells (skeletal and cardiac) and relate this to possible improved mechanisms for muscle repair. The effect of short- and long-term treatment with all three TGF-β isoforms were investigated on muscle progenitor cell proliferation and differentiation using the C2C12 skeletal muscle satellite and P19 multipotent embryonal carcinoma cell-lineages as in vitro model systems. Cells were treated with 5 ng/mℓ TGF-β isoforms unless where stated otherwise. In C2C12 cells, proliferating cell nuclear antigen (PCNA) expression and localisation were analysed, and together with total nuclear counts, used to assess the effect of TGF-β on myoblast proliferation (Chapter 5). The myogenic regulatory factors MyoD and myogenin, and structural protein myosin heavy chain (MHC) were used as protein markers to assess early and terminal differentiation, respectively. To establish possible mechanisms by which TGF-β isoforms regulate differentiation, further analysis included determination of MyoD localisation and the rate of MyoD degradation in C2C12 cells. To assess the effect of TGF-β isoforms on P19 cell differentiation, protein expression levels of connexin-43 and MHC were analysed, together with the determination of embryoid body numbers in differentiating P19 cells (Chapter 6). Furthermore, assays were developed to analyse the effect of TGF-β isoforms on both C2C12 and P19 cell migration (Chapter 7), as well as fusion of C2C12 cells (Chapter 8). Whereas all three isoforms of TGF-β significantly increased proliferation of C2C12 cells, differentiation results, however, indicated that especially following long-term incubation, TGF-β isoforms delayed both early and terminal differentiation of C2C12 cells into myotubes. Similarly, myocyte migration and fusion were also negatively regulated following TGF-β treatment. In the P19 cell-lineage, results demonstrated that isoform-specific treatment with TGF-β1 could potentially enhance differentiation. Further research is however required in this area, especially since migration was greatly reduced in these cells. Taken together, results demonstrated variable effects following TGF-β treatment depending on the cell type and the duration of TGF-β application. Circulating and/or treatment concentrations of this growth factor could therefore be manipulated depending on the area of injury to improve regenerative processes. Alternatively, when selecting appropriate stem or progenitor cells for therapeutic application, the effect of the immediate environment and subsequent interaction between the two should be taken into consideration for optimal beneficial results.

Hung, Sze Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 186-192). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

研究目的：脊髓損傷是中樞神經系統的嚴重創傷，致殘率高。脊髓損傷後的再生修復一直是當前醫學的難題。迄今為止，脊髓損傷依然缺乏一種有效地治療方法。既往研究證明，胰島素樣生長因子-1對鼠和兔脊髓損傷有保護作用，為了進一步把這些發現應用到臨床方面，我們採用與人類生理更相近的豬只作為實驗動物，構建與臨床相似的脊髓損傷動物模型，并以此為基礎，系統性研究胰島素樣生長因子-1的脊髓保護作用，評估該治療的功效。 / 研究方法：以運動誘發電位為指導，通過直接壓迫和牽拉造成脊髓損傷。18頭猪只隨機分為3組：胰島素樣生長因子-1治療組、生長激素治療組及生理鹽水對照組。脊髓損傷后1小時、24小時及48小時經鞘內注射給藥。于術後第1天、第3天及第21天收集腦脊液檢測胰島素樣生長因子-1和生長激素濃度。連續21天使用修正的 Tarlov 評分標準對動物的運動功能進行評估。第21天處死動物並取材，檢測脊髓中NeuN, GFAP, caspase-3 的活性，并通過TUNEL染色觀察細胞凋亡情況，比較各組之間有無差別。 / 研究結果：通過這種方法建立的脊髓損傷動物模型穩定可靠，各組之間無明顯差異。鞘內給藥24小時及48小時后，腦脊液中胰島素樣生長因子-1和生長激素濃度明顯升高，術後21天檢測，其濃度恢復至基礎值。胰島素樣生長因子-1治療組的運動功能的恢復優於其它各組。與生理鹽水對照組比較，胰島素樣生長因子-1治療組可以明顯提高脊髓損傷后神經元的存活數量，抑制星形膠質細胞增生，減少細胞凋亡。而生長激素治療組僅抑制星形膠質細胞增生，其它方面與生理鹽水對照組無明顯差別。 / 結論：胰島素樣生長因子-1通過提高神經元存活數量，抑制星形膠質細胞增生，以及減少細胞凋亡促進脊髓損傷的恢復。 / Objective: Spinal cord injury is a devastating condition that leads to long-term disabilities. Currently, there is no effective treatment that minimizes spinal cord damage or enhances neurological recovery. Recent studies in rats or rabbits suggested that neurologic recovery after spinal cord injury could be improved with the administration of neurotropic hormones, such as insulin-like growth factor-1 (IGF-1). In order to apply such bench-side discovery to clinical practice, we conducted a study in a higher animal model, akin to human physiology, to evaluate the effectiveness of intrathecal injections of IGF-1to improve neurological recovery in a porcine model of acute traumatic spinal cord injury. / Methods: Traumatic spinal cord injury model was produced by controlled compression and distraction of the exposed T12 segment of the spinal cord. Eighteen pigs were randomly assigned to receive intrathecal injections of either IGF-1, growth hormone or saline at 1, 24 and 48 hours after spinal cord injury. Locomotor function was assessed daily using the validated modified Tarlov’s scale for 21 days. Spinal cord segments were then harvested and the survival of neurons, reactive astrogliosis and apoptosis were determined using neuronal-specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. / Results: Intrathecal injections of IGF-1 and growth hormone significantly increase the concentrations of the neurotropic hormones in the cerebrospinal fluid after injury (p < 0.01). These concentrations returned to baseline by 21 days after drug delivery. Motor deficits on the first day after injury were comparable between animals in the treatment and control groups. By the end of the third week, neurologic recovery was better in animals receiving IGF-1 treatment (p < 0.05). Immunohistological and western blot studies of the injured segments of spinal cord showed that treatment with both IGF-1 and growth hormone prevented reactive astrogliosis (p < 0.05) while only IGF-1 improved the survival of mature neurons (p < 0.05). IGF-1 also inhibited apoptosis after spinal cord injury (p < 0.05). / Conclusions: In our clinically relevant model of traumatic spinal cord injury in pigs, intrathecal injection of IGF-1 demonstrated beneficial effects on neurological and histological recovery. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Qinzhou. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 105-122). / Abstract also in Chinese. / Declaration of origination --- p.I / Abstract --- p.II / Acknowledgements --- p.VI / Table of Contents --- p.VIII / List of Tables --- p.XII / List of Figures --- p.XIII / Abbreviations --- p.XVIII / Chapter Part 1 --- Spinal Cord Injury: A Review --- p.1 / Chapter Chapter 1-1 --- Acute Spinal Cord Injury: Epidemiology, Socioeconomic Impact --- p.2 / Chapter 1.1.1 --- Epidemiology of Spinal Cord Injury --- p.2 / Chapter 1.1.2 --- Socioeconomic Impact of Acute Spinal Cord Injury --- p.5 / Chapter Chapter 1-2 --- Mechanisms of Spinal Cord Injury --- p.6 / Chapter Chapter 1-3 --- Putative Treatments for Spinal Cord Injury --- p.8 / Chapter 1.3.1 --- Methylprednisolone --- p.8 / Chapter 1.3.2 --- Stem Cell Therapy --- p.11 / Chapter 1.3.3 --- Riluzole --- p.11 / Chapter 1.3.4 --- Other Pharmacological Therapies for Spinal Cord Injury --- p.12 / Chapter Chapter 1-4 --- Insulin-like Growth Factor-1 for the Treatment of Spinal Cord Injury --- p.13 / Chapter Chapter 1-5 --- Summary --- p.17 / Chapter Part 2 --- Insulin-like Growth Factor-1 and Growth Hormone for Spinal Cord Injury --- p.18 / Chapter Chapter 2-1 --- Hypothesis and Objectives --- p.19 / Chapter Chapter 2-2 --- Establishment of Animal Models for Acute Spinal Cord Injury --- p.22 / Chapter 2.2.1 --- Introduction --- p.22 / Chapter 2.2.2 --- Experimental Animals --- p.22 / Chapter 2.2.3 --- Anesthesia --- p.23 / Chapter 2.2.4 --- Transcranial Electrical Motor Evoked Potential --- p.26 / Chapter 2.2.5 --- Surgery --- p.28 / Chapter 2.2.6 --- Statistics --- p.34 / Chapter 2.2.7 --- Results --- p.34 / Chapter 2.2.8 --- Discussion --- p.38 / Chapter Chapter 2-3 --- Optimal Stimulation Protocols for Transcranial Electrical Motor Evoked Potential. --- p.42 / Chapter 2.3.1 --- Introduction --- p.42 / Chapter 2.3.2 --- Methods --- p.42 / Chapter 2.3.2.1 --- Experimental Animals and Anesthesia --- p.42 / Chapter 2.3.2.2 --- Transcranial Electrical Motor Evoked Potential Recording --- p.44 / Chapter 2.3.2.3 --- Stimulation Protocol --- p.44 / Chapter 2.3.3 --- Analyses --- p.44 / Chapter 2.3.4 --- Results --- p.45 / Chapter 2.3.5 --- Discussion --- p.52 / Chapter Chapter 2-4 --- Evaluation of the Efficacy of Insulin-like Growth Factor-1 and Growth Hormone in a Porcine Model --- p.54 / Chapter 2.4.1 --- Introduction --- p.54 / Chapter 2.4.2 --- Materials and Methods --- p.54 / Chapter 2.4.2.1 --- Study Design --- p.54 / Chapter 2.4.2.2 --- Intrathecal Injection and Collection of Cerebrospinal Fluid --- p.58 / Chapter 2.4.2.3 --- Measurements --- p.58 / Chapter 2.4.2.3.1 --- Clinical Evaluation --- p.58 / Chapter 2.4.2.3.2 --- Biochemical Assessments --- p.58 / Chapter 2.4.2.3.3 --- Spinal Cord Section, Histological and Immunochemical Staining --- p.63 / Chapter 2.4.2.3.4 --- Western Blot --- p.69 / Chapter 2.4.3 --- Statistical Analysis and Sample Size Calculation --- p.72 / Chapter 2.4.3.1 --- General Analysis --- p.72 / Chapter 2.4.3.2 --- Sample Size --- p.72 / Chapter 2.4.4 --- Results --- p.73 / Chapter 2.4.4.1 --- Changes of TceMEP --- p.73 / Chapter 2.4.4.2 --- Motor Deficit after Spinal Cord Injury at Baseline --- p.75 / Chapter 2.4.4.3 --- Insulin-like Growth Factor-1 and Growth Hormone in Cerebrospinal Fluid --- p.77 / Chapter 2.4.4.4 --- Clinical Assessment --- p.80 / Chapter 2.4.4.5 --- Demyelination, Neuron Survival and Astrocyte Reaction --- p.85 / Chapter 2.4.4.6 --- Apoptosis --- p.89 / Chapter 2.4.5 --- Discussion --- p.93 / Chapter 2.4.5.1 --- Principal Findings --- p.93 / Chapter 2.4.5.2 --- Insulin-like Growth Factor-1 and Neuroprotection after Spinal Cord Injury --- p.93 / Chapter 2.4.5.3 --- Growth Hormone and Neuroprotection after Spinal Cord Injury --- p.95 / Chapter 2.4.5.4 --- Strengths and Limitations of Our Study --- p.96 / Chapter 2.4.5.5 --- Summary --- p.97 / Chapter Part 3 --- Summary and Future Directions --- p.99 / Chapter Chapter 3-1 --- Summary --- p.100 / Chapter Chapter 3-2 --- Future Directions --- p.103 / Chapter Part 4 --- References and appendixes --- p.104 / References --- p.105 / Appendixes --- p.123

CNPq / O tratamento e cuidados com úlceras de pé diabético é uma questão de saúde pública. As técnicas atuais utilizadas para o acompanhamento do processo de cicatrização de úlceras abertas são baseadas principalmente em medidas aproximadas da área da ferida e envolvem contato direto, o que representa riscos de contaminação para o paciente. Neste trabalho é proposto o uso de técnicas de visão computacional para medir não apenas a área, mas o volume de úlceras abertas, fornecendo informação objetiva sobre o processo de cicatrização para os médicos, evitando contato direto com a ferida durante o procedimento de medição. A técnica proposta envolve a aquisição de uma sequência de imagens da ferida com iluminação estruturada usando um laser de baixa potência em linha, seguida de reconstrução da profundidade a partir da geometria do dispositivo de aquisição. É identificada a região da ferida de forma semiautomática e construída uma superfície fictícia do que seria a pele natural para servir de base para o cálculo do volume, que corresponde à quantidade de tecido faltante. Experimentos controlados ex-vivo utilizando um modelo de borracha de um Pé Diabético e um joelho de porco com uma ferida artificial representando a pele humana demonstram a viabilidade da técnica proposta. / Treatment and follow-up of Diabetic Foot ulcers is a major public health issue. Current techniques used for following up the healing process of open ulcers are mostly based on approximate area measurements of the wound and involve direct contact, which poses risks of contamination for the patient. This work proposes the use of computer vision techniques to measure not just the area, but the volume of open ulcers, providing additional objective information about the healing process to physicians while avoiding direct contact with the wound during the measurement procedure. The proposed technique involves acquiring a sequence of images of the wound with structured illumination using a low-power laser beam line, followed by depth reconstruction from the geometry of the acquisition device. The wound region is identified in a semi-automatic way and a fictitious surface of what would be the normal skin surface is then used to compute the volume, which corresponds to the missing amount of tissue. Controlled ex-vivo experiments using a rubber model of a Diabetic Foot and a knuckle of pork with an artificial wound representing the human skin demonstrate the viability of the proposed technique. / 5000

CNPq / O tratamento e cuidados com úlceras de pé diabético é uma questão de saúde pública. As técnicas atuais utilizadas para o acompanhamento do processo de cicatrização de úlceras abertas são baseadas principalmente em medidas aproximadas da área da ferida e envolvem contato direto, o que representa riscos de contaminação para o paciente. Neste trabalho é proposto o uso de técnicas de visão computacional para medir não apenas a área, mas o volume de úlceras abertas, fornecendo informação objetiva sobre o processo de cicatrização para os médicos, evitando contato direto com a ferida durante o procedimento de medição. A técnica proposta envolve a aquisição de uma sequência de imagens da ferida com iluminação estruturada usando um laser de baixa potência em linha, seguida de reconstrução da profundidade a partir da geometria do dispositivo de aquisição. É identificada a região da ferida de forma semiautomática e construída uma superfície fictícia do que seria a pele natural para servir de base para o cálculo do volume, que corresponde à quantidade de tecido faltante. Experimentos controlados ex-vivo utilizando um modelo de borracha de um Pé Diabético e um joelho de porco com uma ferida artificial representando a pele humana demonstram a viabilidade da técnica proposta. / Treatment and follow-up of Diabetic Foot ulcers is a major public health issue. Current techniques used for following up the healing process of open ulcers are mostly based on approximate area measurements of the wound and involve direct contact, which poses risks of contamination for the patient. This work proposes the use of computer vision techniques to measure not just the area, but the volume of open ulcers, providing additional objective information about the healing process to physicians while avoiding direct contact with the wound during the measurement procedure. The proposed technique involves acquiring a sequence of images of the wound with structured illumination using a low-power laser beam line, followed by depth reconstruction from the geometry of the acquisition device. The wound region is identified in a semi-automatic way and a fictitious surface of what would be the normal skin surface is then used to compute the volume, which corresponds to the missing amount of tissue. Controlled ex-vivo experiments using a rubber model of a Diabetic Foot and a knuckle of pork with an artificial wound representing the human skin demonstrate the viability of the proposed technique. / 5000

Indiana University-Purdue University Indianapolis (IUPUI) / Human traumatic spinal cord injuries (SCI) are primarily incomplete contusion or compression injuries at the cervical spinal level, causing immediate local tissue damage and a range of potential functional deficits. Secondary damage exacerbates initial mechanical trauma and contributes to function loss through delayed cell death mechanisms such as apoptosis and autophagy. As such, understanding the dynamics of cervical SCI and related intracellular signaling and death mechanisms is essential. Through behavior, Western blot, and histological analyses, alterations in phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-kinase (PI3K) signaling and the neuroprotective, functional, and mechanistic effects of administering the protein tyrosine phosphatase (PTP) inhibitor, potassium bisperoxo (picolinato) vanadium ([bpV[pic]) were analyzed following cervical spinal cord injury in rats. Furthermore, these studies investigated the combination of subacute Schwann cell transplantation with acute bpV(pic) treatment to identify any potential additive or synergistic benefits. Although spinal SC transplantation is well-studied, its use in combination with other therapies is necessary to complement its known protective and growth promoting characteristics. v The results showed 400 μg/kg/day bpV(pic) promoted significant tissue sparing, lesion reduction, and recovery of forelimb function post-SCI. To further clarify the mechanism of action of bpV(pic) on spinal neurons, we treated injured spinal neurons in vitro with 100 nM bpV(pic) and confirmed its neurprotection and action through inhibition of PTEN and promotion of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling. Following bpV(pic) treatment and green fluorescent protein (GFP)-SC transplantation, similar results in neuroprotective benefits were observed. GFP-SCs alone exhibited less robust effects in this regard, but promoted significant ingrowth of axons, as well as vasculature, over 10 weeks post-transplantation. All treatments showed similar effects in forelimb function recovery, although the bpV and combination treatments were the only to show statistical significance over non-treated injury. In the following chapters, the research presented contributes further understanding of cellular responses following cervical hemi-contusion SCI, and the beneficial effects of bpV(pic) and SC transplantation therapies alone and in combination. In conclusion, this work provides a thorough overview of pathology and cell- and signal-specific mechanisms of survival and repair in a clinically relevant rodent SCI model.

Indiana University-Purdue University Indianapolis (IUPUI) / Tendinopathy is a common degenerative pathology that is characterized by activity related pain, focal tendon tenderness, intratendinous imaging changes, and typically results in changes in the histological, mechanical, and molecular properties of the tendon. Tendinopathy is difficult to study in humans, which has contributed to limited knowledge of the pathology, and thus a lack of appropriate treatment options. However, most believe that the pathology is degenerative as a result of a combination of both extrinsic and intrinsic factors. In order to gain understanding of this pathology, animal models are required. Because each tendon is naturally exposed to different conditions, a universal model is not feasible; therefore, an appropriate animal model must be established for each tendon susceptible to degenerative changes. While acceptable models have been developed for several tendons, a reliable model for the Achilles tendon remains elusive. The purpose of this dissertation was to develop an animal model of Achilles tendinopathy by investigating the individual and combined effects of an intrinsic and extrinsic factor on the rodent Achilles tendon. Rats selectively bred for high capacity running and Sprague Dawley rats underwent uphill treadmill running (an extrinsic factor) to mechanically overload the Achilles tendon or served as cage controls. Collagenase (intrinsic factor) was injected into one Achilles tendon in each animal to intrinsically break down the tendon. There were no interactions between uphill running and collagenase injection, indicating that the influence of the two factors was independent. Uphill treadmill running alone failed to produce any pathological changes in the histological or mechanical characteristics of the Achilles tendon, but did modify molecular activity. Intratendinous collagenase injection had negative effects on the histological, mechanical, and molecular properties of the tendon. The results of this dissertation demonstrated that the combined introduction of uphill treadmill running and collagenase injection did not lead to degenerative changes consistent with human Achilles tendinopathy. Intratendiouns collagenase injection negatively influenced the tendon; however, these changes were generally transient and not influenced by mechanical overload. Future studies should consider combinations of other intrinsic and extrinsic factors in an effort to develop an animal model that replicates human Achilles tendinopathy.