Doctoral Degrees (Anatomy and Histology)

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Browsing Doctoral Degrees (Anatomy and Histology) by browse.metadata.advisor "Page, Benedict"

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    Cellular mechanisms involved in the recapitulation of endocrine development in the duct ligated pancreas
    (Stellenbosch : University of Stellenbosch, 2011-03) Tchokonte-Nana, Venant; Page, Benedict; Du Toit, Don F.; University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Anatomy and Histology.
    ENGLISH ABSTRACT: Diabetes mellitus is amongst the leading causes of morbidity and mortality in the world, affecting young, adult and old people. Beta cell replacement therapy for insulin delivery remains the ultimate remedy for diabetes. However, insufficient donor pancreas and the use of immunosuppressive drugs prevent the wide-spread of this therapy. Other avenues of self generated beta cells within the organ itself need to be explored. Therefore, understanding the chronobiology of cellular mechanisms in the lineage of beta cell induced neogenesis is a valuable tool in improving beta cell replacement in patients with diabetes. The aim of this study was to induce recapitulation of the morpho-genetic sequence of endocrine cells development in the pancreas of rats after the pancreatic duct ligation (PDL) procedure. Serial sections of PDL tissues of the pancreas were obtained from 78 Sprague- Dawley rats and were assessed morphologically. The immunofluorescent tissues were statistically analysed using a computerized morphometry technique. The protein expression indices of Caspase3, Insulin, Pdx1, Ngn3, NeuroD and Pax6 were quantified. The efficiency levels of coexpression of these homeodomain proteins separately with insulin were defined by the ratio of the mean value of insulin expression to the mean value of their respective protein expression. The morphological changes were characterized by the appearance of granulated acinar cells at 6 hours post-PDL and the proliferation of endocrine tissues from 84 hours through to 120 hours. The morpho-immunofluorescent evaluation showed the highest immunoreactivity of Caspase3 and Pdx1 at 6 hours, Ngn3 at 36 hours, Pax6 and insulin at 84 hours while NeuroD expression was at 120 hours. The immunohistofluorescent analysis showed that caspase3 and Pdx1 were the first to be expressed at 6 hours while the insulin and NeuroD expression appeared later at 84 hours and 120 hours, respectively. However, Pax6 expression was continuous across time periods post-PDL, while Ngn3 expression showed a peak at 36 hours. The efficiency (highest and earliest expression) of co-expression of all these homeodomain proteins with insulin was restricted between 12 hours and 24 hours. The optimal efficiency was at 12 hours by Ngn3 with insulin. A good efficiency was shown for Pdx1 with insulin, NeuroD with insulin and Pax6 with insulin at 12 hours and 24 hours, respectively. A low efficiency was observed for insulin and caspase3 co-expression at 24 hours. This study suggests that for transplantation, PDL tissues harvested at an early time post-PDL (between 12 and 24 hours) could yield a higher success rate; the study also provides evidence for a connection between morphological changes in the PDL pancreas and the protein synthesis necessary for the lineage of endocrine cell development.
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    Hyperglycaemia and its implication on the Pancreatic islet microvasculature in diabetic rat models
    (Stellenbosch : Stellenbosch University, 2020-12) Ngounou, Eleonore; Alblas, Amanda; Baatjes, Karin J.; Greyling, Linda Magdalena; Page, Benedict; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Anatomy and Histology.
    SUMMARY BACKGROUND: Despite the considerable progress made in the treatment of diabetes mellitus, vascular damage remains the leading cause of patient death. The mechanisms underlying vascular abnormalities in obesity and diabetes mellitus remain to be elucidated and may be the main cause of β-cell death. In addition, the detailed description of islet microvasculature in the pancreas is lacking in the literature; therefore, a better understanding of the characteristics of the blood vessel and the factors that maintain β-cell function is needed in clinical practice. OBJECTIVE: To describe the spatial distribution and histomorphology of islet microvasculature under the effect of hyperglycaemia in two experimental diabetic models. METHODS: Eight week old male Wistar rats (n=50) were divided into two groups that received either a standard diet (RAC) (n=20) or a high-fat diet (HFD) (n=30) for two weeks. By the end of the two weeks, altered glucose uptake was confirmed in the HFD group by an oral glucose tolerance test (OGTT). A subgroup (RAC / STZ) of the RAC group (n=10) and another (HFD / STZ) of the HFD group (n=10) then received 50 and 35mg/kg of body weight (BW) of streptozotocin (STZ) to induce type I diabetes mellitus and type II diabetes mellitus, respectively. They were kept diabetic for an additional eight weeks. The body weight and blood glucose (BGL) of the animals were recorded throughout the experimental period (88 days). Blood was collected for flow cytometry and Luminex assay before half the number of animals were sacrificed for pancreatic tissue collection for histological procedure. The remaining half was used to replicate (cast) the pancreatic vasculature by perfusion with polyurethane-based casting resin (PU4ii). Haematoxylin and Eosin (H&E) stained sections were used to assess the general morphology of pancreatic tissue. Methenamine silver and immunostaining using CD34 antibody, delineated the basement membrane and endothelial cells, respectively, of islet microvasculature. A digital camera and a nano-computed tomography (nano-CT) scanner made it possible to generate digital and 3D images. Quantitative evaluation of topographic morphometric parameters of the pancreatic vascular network in the duodenal and splenic regions of the pancreas in each experimental condition was performed using the imageJ and Volume Graphics VGStudioMax 3.0®. Reconstruction of the pancreatic vascular network was attempted using the vascular tree scale laws. RESULTS: A significant increase in the mean body weight was accompanied by a slight increase in mean BGL within 2 weeks in HFD. Streptozotocin caused the development of two diabetic models with all clinical symptoms (polyuria, polyphagia, high BGL (> 28mmol/L) and a significant decrease in body mass in both diabetic groups (26.68% and 15.54% in RAC / STZ and HFD / STZ respectively). The results of the flow cytometry and the Luminex assay validated the presence of islet vascular lesions in animals, which also justified the significant necrosis of endothelial cells, a decrease (p<0.05) in the mean percentage of the stained area of CD34 pixels in islets, and thickening of the basement membrane. The scaling law was used to obtain the relationships between 1) the length and volume of the pancreatic vascular tree up to capillary level (R2=0.693±0.053), 2) the diameter of the lumen and the blood flow in each pancreatic vascular branch (R2=0.988±0.055), and 3) the diameter and length of the branches of the vessels (R2=0.838±0.0123). CONCLUSION: This investigation has established detailed morphological features of the vasculature of the pancreas in the duodenal and splenic regions in normal and diabetic rat models. There were large differences in the structure of the pancreatic vasculature between the two regions appearing to be dictated by metabolic demand. However, there are still challenges in 3D visualisation of the capillary networks of the pancreatic vascular tree, which was the main limitation of this study.
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    An in vitro study of mesenchyme–islet cell interactions in islet neogenesis: A model for tissue replacement therapy in diabetes mellitus
    (Stellenbosch : Stellenbosch University, 2017-12) Manda, Juziel Kampando; Tchokonte-Nana, Venant; Page, Benedict; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Division Anatomy and Histology.
    ENGLISH ABSTRACT : Shortages of donor islets, immune rejection, and the need for life-long immuno-suppressors remain the clinical challenges of islet transplantation in the treatment of diabetes mellitus. An alternative to these challenges is the in vivo generation of beta cells within the patient’s pancreas. The animal model of pancreatic injury has been reported to be a potential source of islet cells for tissue replacement therapy in type 1 diabetes mellitus. However, the in vitro regenerative capacity of endogenous beta cells in this model needs more investigation. This study investigated, in vitro, the effect of pancreatic duct ligation (PDL)-induced islet/duct-mesenchymal stromal cells (MSCs) interactions on islet and duct cells development and assessed the long-term transplantation outcome of islet-mesenchymal cells isografts. Islets, duct fragments, and MSCs were isolated from post PDL tissues harvested from eighty adult male Wistar rats (250 - 300g) 24- and 120 h following duct ligation. Islets or duct fragments were cultured with or without MSCs ([Islet/MSC+ or Islet/MSC-] or [PEDC/MSC+ or PEDC/MSC-]). Development of islets and duct fragments in culture were evaluated morphologically and by immunocytochemistry using antibodies against Pdx1, Ngn3, CK7 and insulin. Islets were also transplanted with or without MSCs (Islet/MSC+ or islet/MSC-) in diabetic animals (n = 40). Isografts survival and function were evaluated by monitoring blood glucose levels, and immunohistochemistry of graft tissues were studied. Results showed activation of Pdx1+ islet cells in both cultures with or without MSCs, however, expansion of Pdx1+ cells were promoted in the presence of MSCs and this was followed by activation of Ngn3 expression and expansion of Ngn3+ cells, which was maintained in islet cells up to 4 weeks. This resulted into low levels of insulin expression in islet-like aggregates formed between the third and the fourth week. Co-culturing of duct fragments with MSC similarly resulted into maintenance of endocrine precursors that expressed Ngn3, which later formed islet-like aggregates. In cultures with MSCs, duct epithelial cells developed growth areas with cells that co-expressed CK7 and Ngn3 in periductal cells. When periductal cells formed islet-like aggregates, Ngn3 co-expressed with insulin in islet-like cell clusters closer to ducts. Transplantation of early harvested (24 h PPDL) islets showed better curative capacity than late (84 h PPDL) islets. The average glucose levels were lower throughout the 5 weeks monitoring period in 24 h PPDL transplanted rats. The average time to reverse hyperglycemia in 80% of the 24 h PPDL transplant group was 32 ± 2 days (~4.5 weeks), while only 20% in the 84 h PPDL transplant group attained normoglycemia at 61 ± 2 days (~9 weeks) (p = 0.0011) post transplantation. Graft survival rate was higher in islets co-transplanted with MSC (Islet/MSC+) compared to grafts transplanted with islets alone (Islet/MSC-). Islet morphology and distribution of beta cells was normal in Islet/MSC+ similar to the endogenous islets in the pancreas. In conclusion, MSCs promote the expansion of Pdx1+ cells and maintain the expression of Ngn3 in islet cells and duct–derived neogenetic cells. MSCs prolong graft survival and improve the capacity of early harvested post PDL islets to reverse hyperglycemia; this novel observation may be applicable to clinical transplantation.