Doctoral Degrees (Anatomy and Histology)

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The anatomy of seismic signalling: morphological adaptations of the hind limb in drumming and non-drumming African mole-rats (Bathyergidae)
(Stellenbosch : Stellenbosch University, 2021., 2021-03-31) Sahd, Lauren; Kotze, Sanet H.; Bennett, Nigel C.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Anatomy and Histology.
ENGLISH ABSTRACT: Hind foot drumming is one of the most common forms of seismic signalling. The family Bathyergidae (African mole-rats) is well known for exhibiting hind foot drumming during courtship and territorial behaviour. Hind foot drumming in bathyergid species arises as a result of the rapid flexion and extension of the hip and knee joints by either a single or alternating hind limbs. While the ecological context of drumming in these species has been well described, the possible morphological adaptations to enable the production of these seismic signals, have yet to be unravelled. Therefore, the primary aim of the present study was to investigate if morphologically discernible adaptations to the hind limb osteology and muscles could be determined in two drumming (Georychus capensis and Bathyergus suillus) and one non-drumming species (Cryptomys hottentotus natalensis) of African mole-rats using a variety of techniques. The gross anatomy of the hind limb was investigated by undertaking detailed dissections to determine the origin and insertion points as well as the innervation of the muscles of the hind limb. Thereafter, 32 muscles were removed from a single limb (with joint angles closest to 90°) for muscle architecture measurements which included belly length, muscle fascicle length, muscle mass and physiological cross-sectional area. The remaining soft tissue was removed from the specimens by maceration to enable the description of the osteology of the hind limb. Additionally, morpho-functional indices were used to morphometrically compare the bones between species. Twenty-one hind limb muscles were selected to undergo muscle fibre typing using myosin heavy chain slow antibody immunohistochemical staining. The amount of positive fibres was quantified to determine the total percentage of slow muscle fibres in each muscle section. Micro computed tomography (CT) scans of contrast enhanced stained specimens were used to determine accurate volumetric measurements of 26 muscles per sample in all three species as well as to assemble three dimensional reconstructions of the musculature of the limbs. Musculus gracilis anticus may play a key role in hind foot drumming as it was the only muscle that was morphologically different between the drummers and non-drummer. In the two drumming species, m. gracilis was a single muscle, whereas it was double in C. h. natalensis. Additionally, it was the only muscle to be significantly different in G. capensis and C. h. natalensis for all muscle architecture parameters analysed. Furthermore, m. gracilis anticus was the only muscle that had a significant difference in the volume as determined by the micro CT scans between the two drumming species and C. h. natalensis. However, the number of slow fibres of m. gracilis anticus was not significantly different between G. capensis and C. h. natalensis. Furthermore, the robust tibias in the drumming species, as indicated by the tibial robustness index, possibly counter the additional biomechanical load caused by the muscles (specifically m. gracilis anticus) involved with hind foot drumming. Thus, it can be concluded that there are distinct morphological adaptations to the osteology and musculature of the two hind foot drumming species.
Assessment of health status in a 20th century skeletal collection from the Western Cape
(2019-04) Alblas, Amanda; Friedling, Louise Jacqui; Greyling, Linda Magdalena; Stellenbosch University. Faculty of Medicine and Health Science. Dept. of Biomedical Sciences: Anatomy and Histology.
ENGLISH ABSTRACT: Studies on the health status of skeletal remains give insight into the standard of living and survival pattern of historic populations. Analysis of trauma and pathological conditions in human skeletal remains are important in biological anthropology, explaining patterns of malnutrition, stress, disease and trauma in a population. However, the difficulty to overcome is the fact that the majority of skeletal pathological conditions are limited in their interpretive significance, since they are nonspecific and a range of stressors can cause the lesions. By analysing multiple conditions within a known population, pathological responses for specific insults can be outlined and in return help in interpretation of the frequencies and distributions within and between populations. The Kirsten Skeletal Collection, housed at Stellenbosch University, Division of Clinical Anatomy broadly represents individuals from mainly low socio-economic communities of different population groups in the Western Cape, dating throughout the 20th century. The aim of this study was to macroscopically and radiologically examine adult individuals (n=624, nmales=438, nfemales=186) in this collection for skeletal markings that included malnutrition (diet and metabolic deficiencies), osteoarthritic lesions, neoplasms, infective diseases and antemortem trauma lesions to be used as baseline information in further anthropological studies on the people of the Western Cape region. Statistically, the prevalence of specific diseases or trauma were correlated between the sexes, three different age-at-death and population groups as well as three different time periods throughout the 20th century using two-way frequency-tests and correspondence analyses. During the 20th century, many factors resulted in poverty, and "non white” people, namely the South African Black (SAB) and South African Coloured (SAC) population groups, was especially disadvantaged by the laws introduced by the ruling political party. The influx of people from rural areas during World War II to work in the manufacturing industry resulted in the already overcrowded, unsanitary informal settlements around the Cape Peninsula to be flooded, influencing the disease patterns in the communities. The results demonstrated that the lowest prevalence of metabolic deficiencies, iron deficiency anaemia (porotic hyperostosis), growth arrest signs (Harris’ lines), infections such as tuberculosis, osteomyelitis and non-specific periosteal reactions were observed in the South African White (SAW) population group. This confirms that higher socio economic societies, that escaped the unsanitary conditions associated with poor housing and overcrowding environments, were more succesfully buffering themselves from malnutrition and exposure to pathogens. Better dental health as well as dental fillings were also more associated with the SAW population group that had unrestrained access to dentists and health care facilities. In contrast, the ‘non-white’ population groups, that were supressed during the Apartheids regime, demonstrated a high prevalence of malnutrition, metabolic deficiencies, tuberculosis and trauma lesions. The difference between the higher and lower social categories was especially recognised during the late time period when the Apartheid laws of population group segregation among others, started to show results in the 1960 and 1970. Later, during the 1980s and 1990s, political unrest caused by the supressed majority, and the world due to the Apartheid laws, resulted in sanctions and lower economic opportunities for South Africa. A notable higher frequency of infective markings on bone was observed during the late time period in the study, an indication of the successful use of antibiotics during the last decades of the 20th century, which provided more time for lesions to manifest on bones due to the increased life-span of people. Studies on skeletal collections rely on the assumption that the remains represent a past community, population group or populations from a specific region and can be used as a valid comparative reference for reconstructing different aspects of skeletal biology of past people that lived in that population. The Kirsten Skeletal Collection represents adult age groups between 18 and 100, three population groups, both sexes, various time periods over the 20th century as well as known cause of death and last known residence. However, this skeletal collection relies on body donations or retention of adult unclaimed or family donated bodies under the statues of the Inspector of Anatomy, and therefore, resulted in a biased sample. This bias is perceived in the fact that the Kirsten Skeletal Collection have an overrepresentation of males, aged individuals and people with lower socio-economic status. Although three major South African population groups are represented, suggesting depiction in population variation, it is highly unequal, especially representing the overpresented heterogenous mixed population group (SAC) that lived in and around the northern townships of Cape Town. Despite limitations, in general this study of the Kirsten Skeletal Collection may represent many of the traits in the population at that time and may be useful in future studies on honours, masters and PhD level to refine region- and population specific reference data and play a supportive role in research and training of specialists. These data to be collected and interpreted in future studies, include estimation of demographic parameters (age, sex, ancestry origin) as well as human variation, trauma biomechanics and pathological conditions. If the existing predispositions of the collection are acknowledged and accounted for by the use of suitable methodology, the Kirsten Skeletal Collection holds much potential to become a valuable resource for future research projects in osteology and related fields.
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.
The effect of an in utero high fat diet on the expression of transcription factors and glucose sensing in the developing rat pancreas
(Stellenbosch : Stellenbosch University, 2005-12) Cerf, Marlon Eugene; Du Toit, D. F.; Louw, J.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Anatomy and Histology.
ENGLISH ABSTRACT: A high fat diet (HFD) reduces beta-cell mass, impairs glucose signalling and is involved in the development of Type 2 diabetes. Malnutrition during gestation is hypothesized to irreversibly damage beta-cell development. The transcription factors Pdx-1 and Pax 4 are involved in islet cell development. Pdx-1 is reported to regulate expression of GLUT-2, glucokinase (GK) and the insulin gene. Aims The aim of this study is to investigate, in the neonatal and weanling rat, the effect of exposure to a HFD in utero and/or lactation on weight, glucose and insulin concentrations, islet cell development, pancreatic transcription factors and glucose sensing genes. Methods Neonatal and weanling rats were exposed to a maternal HFD for defined periods of gestation and/or lactation. After termination, pups were weighed and glucose and insulin concentrations determined. mRNA expression of Pdx-1, Pax 4, GLUT-2 and GK was quantified by LightCycler PCR. Pancreatic sections were immunostained for insulin and glucagon (islet cell development), and for Pdx-1, GLUT-2 and GK (beta-cell function) followed by image analysis. Results: Exposure to an in utero HFD throughout gestation resulted in hyperglycaemic pups with reduced beta-cell volume and number, Pdx-1 and GK immunoreactivity. In contrast the alpha-cell volume, number and size were augmented in neonates exposed to a HFD throughout gestation. Most weanlings were hyperglycaemic and hypoinsulinaemic. In some weanlings, reduced beta-cell number and beta- and alpha-cell size was observed. Pdx-1 mRNA was overexpressed in weanlings exposed to a maternal HFD for the final week of gestation or throughout both gestation and lactation, but reduced in those only exposed throughout lactation. Pax 4 mRNA was reduced in weanlings exposed to a maternal HFD for the first or final week of gestation, throughout gestation or throughout lactation. In most of the weanlings, GLUT-2 mRNA expression was reduced whereas immunoreactivity for GLUT-2 was increased. Both GK mRNA expression and immunoreactivity were reduced in most of the weanlings. Conclusions • Exposure to an in utero HFD throughout gestation induced hyperglycaemia in neonates. The reduced Pdx-1 expression appears to play a role in the compromised beta-cell development, and concomitant with the reduced GK levels, contributes to the hyperglycaemia in these neonates and may make them susceptible to beta-cell failure. • In most weanlings exposed to a HFD in utero and/or during lactation the hyperglycaemia and hypoinsulinaemia suggest compromised beta-cell function. The GK mRNA expression and immunoreactivity were reduced thereby impairing glycolysis which would result in reduced insulin secretion contributing to the hyperglycaemia. Furthermore, beta-cell development is adversely affected by the HFD in some weanlings. This would contribute to reduced beta-cell function and may eventually result in beta-cell failure. GLUT-2 immunoreactivity was increased in some, suggesting a compensatory adaptative mechanism to restore glucose homeostasis. • A maternal HFD has adverse effects both in neonates and weanlings on beta-cell development, transcription factor and glucose sensing gene expression and induced hyperglycaemia and hypoinsulinaemia in some of the offspring. Ways to ameliorate the HFD-induced attenuation of key beta-cell genes to ensure normal beta-cell function are important for future research in Type 2 diabetes.
A histological and morphometric assessment of endocrine and ductular proliferation in the adult rat pancreas using an occlusive pancreatic duct ligation model
(Stellenbosch : Stellenbosch University, 2000-03) Page, Benedict J. (Benedict John); Du Toit, Don F.; Wolfe-coote, Sonia A.; Stellenbosch University. Faculty of Medicine & Health Science. Dept. of Biomedical Sciences.
ENGLISH ABSTRACT: Diabetes Mellitus (DM) is synonymous with "B-cell failure". Ligation of the pancreatic duct distally to its confluence into the bile duct has been shown to induce endocrine tissue regeneration from a number of probable sources. The cells responsible for regeneration are supposed to possess either dormant pluripotent stem cell ability and/or the plasticity to undergo metaplasia to form new and surplus endocrine tissue able to replace pathologically and/or experimentally compromised pancreas. The sequence of events (cell lineage) during this process of neogenesis, has been the source of controversy for quite some time as various studies suggest that the cell lineage differs from in vivo and in vitro studies, according to experimental model and species of laboratory animal. The object of this study was to utilise an established experimental laboratory animal model to study islet morphological changes, neogenesis and or both in vivo. Further aims of the study were to determine the extent, sequence and magnitude of pancreatic duct ligation (PDL) induced endocrine neogenesis/morphogenesis in a laboratory rat model using occlusive pancreatic duct ligation. PDL's were performed on six groups of 25 normal adult Sprague-Dawley (SD) rats (300g+) according to the method of Hultquist and Jonsson (1965). Experimental animals were sacrificed at 12 hr intervals from day one post-PDL to day 10 and every 24 hrs thereafter to day 14 as described by Wang, Klëppel, Bouwens (1995). Animals received BrdU (a thymidine marker and cell proliferation indicator) 50mglkg intraperitoneally as described by Wang et al. (1995), one hour prior to removal of the pancreas after which it was fixed in Bouin's solution and histologically processed. Seven consecutive 3-6 urn thick serial sections were sequentially stained with H & E, insulin (I), glucagon (G), somatostatin (ST), pancreatic polypeptide (PP), neuropeptide tyrosine (NPY) and peptide tyrosine tyrosine (PYY). Immunolabeling was done according to the method of Guesdon, Temynck , Avrameas (1979). Double immunolabeling for BrdU and each pancreatic peptide was performed on the sections on days 3,5, 7, 9 and 11 as described by Wang et al (1994). Cellular transformation between one and 3Yz days was characterised by simultaneous total deletion and/or transdifferentiation of the acinar compartment and the appearance of immunoreactive cells for I (11.53 ±1.5%), G (1.85 ±0.8%), pp (1.50 ±0.09%), and ST (1.96 ±0.24%). Changes in the endocrine composition in existing islets, occurred along a pathway that saw PP- and ST-cells invading the islet core, islet mantle glucagon deletion and random appearance of all endocrine cell types within the inter-islet interstitium on day 3Yz. Days 4 to 6Yz saw further endocrine expansion while days 7 to 14 were distinguished by islet reconstitution and consolidation. NPY immunoreactivity appeared on day 4Y2 and persisted intermittently throughout while PVV first appeared on day 4 and disappeared after day 7Yz. The results suggest that PDL firstly induced the development of endocrine tissue distributed haphazardly throughout the space previously occupied by acinar parenchyma. Secondly, the appearance of insulin is preceded by the appearance of PP, glucagon and somatostatin by 24-hours. A still to be determined proportion of the ligation induced endocrine formation appeared to be associated with existing islets, resulting in a number of very large islets, some of which might have secretory access through the glomerularlike capillary network known to occupy the islet core. The remainder appeared to form separate "new" islets, which have a dubious access to the blood stream. In conclusion, if it is true that the pancreas can regenerate some of its endocrine tissue then it has potential clinical implication for the stabilising of diabetes mellitus. Ligated exocrine pancreatic tissue, devoid of its acinar component, has been shown to contain notable quantities of insulin positive cells. This presents intriguing possibilities as an alternative for donor tissue, usually obtained from rat foetuses, during foetal rat pancreas transplantation studies. Pancreas tissue harvested from duct ligated rats could replace the foetal tissue currently used in the treatment of experimental diabetes mellitus in laboratory animals in this laboratory.
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.
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.
A morphological assessment of the health status of a cadaver population at the Faculty of Health Sciences, Stellenbosch University, with special reference to tuberculosis (TB) Lesion distribution
(Stellenbosch : Stellenbosch University, 2014-12) Geldenhuys, Elsje-Marie; Kotze, Sanet; Burger, Elsie Helena; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Anatomy and Histology.
ENGLISH ABSTRACT: Tuberculosis (TB) is a chronic pulmonary infection characterized by granulomatous inflammation, caseating necrosis and a propensity to develop fibrosis and cavitations. Pulmonary TB (PTB) lesions may develop in a variety of ways and can be grouped into primary, secondary, progressive primary and miliary TB based on their pathogenesis and morphological appearance. The Western Cape Province, South Africa, has a high TB burden with increasing TB notification rates. At Stellenbosch University (SU), approximately 90% of cadavers used for medical dissections come from impoverished communities where TB is a major health problem in terms of morbidity. The aim of the present study was to assess the health status of a cadaver population (n=127) at the Faculty of Medicine and Health Sciences (FMHS), SU, with special reference to TB lesion distribution and prevalence. For this study, full-body digital X-rays of 127 cadavers (87 males; 40 females; average age, 47.1 years) were obtained with the Lodox® Statscan® Imaging System after embalming and prior to dissection. A complete pathology report of six organ systems including the skeletal system was used in combination with histological examination, molecular analysis and radiological findings to investigate the prevalence and association between TB and systemic pathology. Samples for histological purposes were removed from organs with pathology lesions. For molecular studies, five different nucleic acid extraction methods were used to extract DNA from the formalin-fixed paraffin-embedded cadaver samples. Pulmonary samples were subjected to a line probe assay (LPA) and polymerase chain reactions (PCR) to determine mycobacterial genotypic distribution. Two independent radiologists examined the chest X-rays and their findings were compared with the pulmonary findings. PTB was a common finding in the cadaver population (76.4%) with males more commonly affected. A female predilection was observed for extrapulmonary TB. Statistically, TB was associated with pulmonary pathology, including pneumonia and bronchiectasis. Systemic pathology commonly encountered in the present study included neoplasms, coronary artery disease, colonic diverticula, hepatic triaditis, cirrhosis, glomerulosclerosis, pyelonephritis and a variety of healed maxillofacial and appendicular skeletal fractures. Extracted nucleic acid concentrations, as determined with the NanoDrop® spectrophotometer, ranged between 10ng/μl and 1000ng/μl. The standard salting-out method was found to be the most cost-effective and therefore the preferred method for nucleic acid extraction. The HAIN® MTBDRplus® kit was effective in determining the presence of mycobacterial species belonging to the Mycobacterium tuberculosis complex (MTBC). The sensitivity to first-line drugs could not be determined as a result of DNA degradation. Spoligotyping was unsuccessful, as incomplete and unidentifiable hybridization of the 43 spacers occurred. The RD105 and MUB02/RD105 PCR results were non-reproducible and non-specific. Pulmonary cavitation and pleural thickening were the only findings that were positively correlated with the radiological findings (p<0.05). To our knowledge, this is the first study to extensively investigate TB and systemic pathology including histopathology, molecular techniques and postmortem radiology in cadavers from low socio-economic backgrounds from a high TB burden area. This study therefore provides a more complete and thorough understanding of the prevalence, distribution and morphology of TB lesions as well as the association between TB and systemic pathology.
Preclinical assessment of the immunosuppressive properties of an anti-CD4 monoclonal antibody (MAB) in an allogeneic foetal rat pancreatic transplantation model
(Stellenbosch : Stellenbosch University, 2004-12) Muller, Christo John Frederick; Du Toit, D. F.; Bouic, Patrick J. D.; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Anatomy and Histology.
ENGLISH ABSTRACT: Introduction Despite advances in insulin therapy, the side effects associated with diabetes mellitus still remain. Pancreas transplantation has benefited diabetics with end-stage renal failure by reversing the diabetic state and preventing or reversing the progression of diabetes associated diseases. Currently the side effects associated with lifelong immunosuppression preclude pancreas transplantation as a viable treatment option for both type I and II diabetics. In the laboratory, transplanted rat foetal pancreata have been shown to be able to reverse the clinical signs of streptozotocin-induced diabetes in an isogeneic model. Reversal of diabetes by allogeneic foetal rat pancreas transplantation, although possible has proved to be more difficult due to fierce rejection of the grafts and the diabetogenic effects of conventional immunosuppressants. Aims One of the goals, focus and intentions of this laboratory study in rodents, is to contribute new information to the scientific literature. The potential to “reverse” the diabetic state by allogeneic foetal pancreatic transplantation, was the main stimulus for this study. Methods Foetal pancreata of 16-18 days gestation were transplanted into a surgically prepared renal subcapsular space. Immunosuppressive protocols used to prevent rejection of the allogeneic foetal rat pancreata included donor specific transfusion (DST), cyclosporine [a calcineurin inhibitor (CsA)], mycophenolate mofetil [a purine syntase inhibitor (MMF)], and a mouse anti-rat CD4 monoclonal antibody (W3/25). Immunosuppressants were used as monotherapies and in combination. Results Isogeneic foetal rat pancreas transplantation resulted in the growth and development of mature insulin producing islets of Langerhans at the site of engraftment. Allogeneic foetal pancreatic transplantation without immunosuppression resulted in complete rejection of the grafts at 14 days post-transplantation. Histological assessment of allografts at 14 and 30 days post-transplantation showed that CsA was able to prevent acute rejection in our rat models although graft scores and survival were improved if CsA was combined with MMF. Intraperitoneal anti-CD4 monoclonal injections were well tolerated, and if given daily effectively prolonged graft survival up to 30 days. Combining DST with anti-CD4 and CsA induction therapy provided long-term graft survival without daily immunosuppression. This combination, together with allogeneic foetal rat pancreas transplantation, was effective in reversing the clinical signs of experimentally induced diabetes. To my knowledge these are the first published results in which reversal of streptozotocin induced diabetes was achieved by fully MHC mismatched foetal rat pancreatic transplantation. Conclusion Foetal rat pancreatic transplantation is a potential source of endocrine replacement, which, with effective immunosuppression allows for the development of functional islets able to reverse the clinical signs of experimentally induced diabetes in an allogeneic rat model. An unique immunosuppressive protocol, with potential clinical relevance in the human, combines anti-CD4 mAb, CsA and DST induction therapy, which alleviates the burden of daily immunosuppression and associated side effects.

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