Browsing by Author "Dlamini, Gcwalisile Frances"

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    Characterising skeletopathy in an animal model of Type 2 diabetes
    (2024) Dlamini, Gcwalisile Frances
    Type two diabetes (T2D) is a chronic, progressive heterogonous syndrome with a genetic and environmental origin. It is now recognized as an epidemic with a high morbidity and mortality rate. The endocrinology of type 2 diabetes (T2D) and its predisposing factors have been studied extensively, while diabetic skeletopathy has received negligible research. Previous studies report that fractures in T2D vary with specific sub regions in bones, therefore prompting our study to focus mainly on the femoral head and neck as well as the humerus head. Femoral neck fractures are the commonest, followed by the proximal femur, distal radius and proximal humerus. Susceptibility to fracture is a sequelae of poor bone remodeling. Poor bone remodeling is established at molecular and cellular levels. It depends on the activity of osteoblasts, osteocytes and osteoclasts, which are under the influence of TGF-β1, a pro-osteogenic cytokine, together with BMP3, an anti-osteogenic cytokine.T2D induced bone marrow adipocity and the accumulation of AGEs in cortical bone have also been implicated in increasing susceptibility to fracture. It is still unclear how T2D affects molecular and cellular elements that culminate in weaker bones observed in diabetic patients. In addition, it is debatable if T2D affects the skeleton at disease onset or later in the disease. Therefore, this study aimed to characterize T2D induced skeletopathy and related it to age, in the Zucker Diabetic Sprague Dawley (ZDSD) rat, using the femur and humerus. This study initially confirmed the diabetic state by monitoring animal weights, fasting blood glucose levels, and fasting oral glucose tolerance tests (OGTTs) every fortnight. Then triglyceride levels and quantified serum levels of osteoregulatory hormones such as insulin and osteocalcin were monitored. To assess oxidative stress, Malondialdehyde (MDA) serum levels were also determined by ELISA. Once diabetes was successfully induced, rats were grouped according to strain and age at termination. Termination age was at 20 weeks and 28 weeks . The Sprague Dawley (SD) rats were v the controls, while the Zucker Diabetic Sprague Dawley rats (ZDSD) were the experimental groups. These were designated as SD20WK (n=8) and ZDSD20WK (n=7) respectively. Another batch was designated as SD28WK (n=8), and ZDSD (n=15) that were terminated at 28 weeks of age. The latter were further divided into moderate diabetes (ZDSD28WK-MOD) (n=9) and severe diabetes (ZDSD28WK-SVD) groups (n=6). Bilateral humeri and femora were harvested then fixed in 10% buffered formalin. Right proximal femora and humeri were scanned using a 3D-μCT scanner (Nikon XTH 225L) to analyse trabecular morphometric parameters, cortical bone area and medullary canal area. Biomechanical strength was analyzed by three point bending tests using a universal tensile tester. Left proximal femora and humeri were processed for histology. Some sections were stained with Haematoxylin and Eosin (H&E) to assess normal histologic morphology and adipocyte quantification. Remnant sections were immunolabelled using the anti-TRAP and anti-ALP antibodies for osteocyte and osteoblast quantification respectively, to assess osteolysis and osteogenesis. Immunolocalization of AGEs, TGF-β1 and BMP3 was also conducted to investigate their role in diabetic skeletopathy. We found that diabetes affected osteoblastogenesis as measured by ALP positive cells and bone marrow adipocytes. TRAP positive osteocytes numbers were increased in the presence of T2D, suggesting an increased osteolysis. There was reduced TGFB1 expression with increased BMP3 expression. The number of AGEs immuno-positive cells as well as its extracellular expression was increased. Our finding suggest that osteoblast and osteocyte numbers are regulated by TGFβ1 and BMP3 in both bones, under the influence of AGEs. Our findings from osteometry, 3-point bending tests and Micro CT support that diabetes weakens bone. The diabetic effect results in lighter, shorter hollow bones that perform poorly under loading, as well as exhibit unfavourable trabeculae microarchitecture. Our findings confirm that T2D causes increased fragility in the proximal femur and humerus as well the mid-diaphysis. These perturbations occur early and late in the disease, and they are also exacerbated by the presence of hyperglycemia. vi We conclude that the ZDSD rat can be used as a translational model for diabetic skeletop
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    The effect of pre-pubertal exposure to di-(n-butyl) phthalate (dbp) on cell proliferation in adult male Japanese quail (Coturnix corutnix japonica) brains
    (2018) Dlamini, Gcwalisile Frances
    Neurogenesis is a process that comprises neuronal progenitor cell proliferation, migration differentiation, and integration of young neurons into existing neuronal circuits. Neurogenesis has been observed in avian species, including quail. Neuronal cell proliferation, which is part of adult neurogenesis, was investigated using cellular markers DCX and PCNA, in the hippocampus, mesopallium, nidopallium, medial striatum, medial pre-optic nucleus and ventricles of 14 week old male Japanese quail brains. This was after a 30 day pre-pubertal exposure to the environmental contaminant and endocrine disruptor di(n-butyl) phthalate (DBP), a well-known neuronal development disruptor and neuronal toxicant. Thirty birds were randomly grouped into groups of four (n=4 each) and were intragastrically fed DBP dissolved in corn-oil. The control was fed corn oil only, while three groups were fed 10 mg/bodyweight, 50 mg/bodyweight, 400 mg/bodyweight, effectively dividing them into a low medium and high dose groups. The birds were euthanised using carbon dioxide, brains were harvested and post fixed in a 1:1 mix of ethylene glycol and glycerol (antifreeze) in 0.244M PB (phosphate buffer) and stored at -20 0C. Repeated five series coronal sections of 50 µm were cut in a rostro-caudal direction using a freezing microtome. The 5th series sections were stained with cresyl violet for analysis of brain cytoarchitecture, while 3rd and 4th series sections were stained with DCX and PCNA. Intense staining was observed along the ventricles for both PCNA and DCX, and some PCNA-ir cells were present in the POM. DCX-ir cells were observed in the pallial brain areas of both treated and control groups. Statistically significant differences (p= 0.0001) in counts of DCX-ir between the control and treated groups in the five brain regions were observed, but there were no statistically significant differences (p=0.886) between the brain regions themselves across all dosage groups. DBP treatment affected DCX-ir cell counts in all brain areas under study, irrespective of brain area or dosage. There were significant differences (p=0.0067) of PCNA-ir cell counts between the treated groups and the control for the POM (p=0.0067) and PCNA-ir cell counts between the control and ventricles (p=0.0001). DBP affected cell proliferation in the ventricles and the POM.