School of Anatomical Sciences (ETDs)
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Item Histo-morphological Perturbations in the Testes of Diabetic Sprague Dawley Rat Following Atripla® And Alcohol Co-administration(University of the Witwatersrand, Johannesburg, 2023-08) Owembabazi, Elna; Nkomozepi, Pilani; Mbajiorgu, Ejikeme FelixA decline in male fertility is increasingly becoming a major concern in this millennium. However, the associated effects of diabetes, alcohol abuse, and cART use and their interaction which are clinically important in male reproductive health have not received proper attention. Each of these conditions (diabetes, alcohol abuse, and cART use) has been shown to negatively impact the male reproductive system. Moreover, diabetes, alcohol abuse, and cART are intricately connected and thus can and occur simultaneously in an individual, a scenario that might provoke severe reproductive dysfunctions. Therefore, this study investigated the impact of diabetes, alcohol abuse, cART use, and their combinations on testicular histomorphometry, reproductive hormone profile, oxidative and inflammatory markers, germ cell proliferation and apoptosis, and androgen receptor expression. A total of forty-eight HIV naive rats (48) were divided into two main groups, non-diabetic and diabetic groups, which were further subdivided into four subgroups consisting of six rats each. The non-diabetic groups (1-4); Group 1; Control, Group 2: Alcohol treated (A), Group 3: combination antiretroviral therapy treated (cART), and Group 4: alcohol plus cART treated (A+cART). The diabetic groups (5-8); Group 5: diabetic only (DM), Group 6: diabetic treated with alcohol (DM+A), Group 7: diabetic treated with cART (DM+cART), and Group 8: diabetic treated with both alcohol and cART (DM+A+cART). The rats were fed normal rat chow and terminated after 90 days of treatment. Blood was drawn through a cardiac puncture into plain vacutainers, thereafter, animals were perfused with 0.1M phosphate buffer, and the testes harvested, weighed, and then fixed in 10% neutral buffered formalin for histology and immunohistochemistry analysis. During the experimental period, hyperglycemia, low glucose tolerance, and polydipsia were observed in the diabetic groups (5-8) only. Though, a general decrease in testis weight, volume, and size was found in all treated groups compared to the control group, a significant (p˂0.05) decrease was detected in the DM+A group only. With exception of the DM+A+cART group, the epithelial area fraction, epithelial height, and tubule area and diameter reduced significantly in all treated groups. The luminal area fraction and luminal diameter which significantly reduced in cART, DM, and DM+cART groups, were increased in A+cART. Further, connective tissue and interstitial area fractions increased significantly in all treated groups. The spermatogonia increased significantly in A, cART, DM, and DM+A+cART groups relative to control group, but reduced significantly in DM+A. However, spermatocytes, round spermatids, and elongated spermatids decreased significantly in all treated groups, with exception of spermatocytes of the alcohol group. All treated groups showed a decrease in the number of Sertoli cells relative to the control but a significant decrease was only found in the DM+A group, but all treated groups had significantly decreased Leydig cell diameter and volume. Johnsen’s testicular score was significantly reduced in A, A+cART, DM, and DM+A treated groups. Additionally, varying severity of seminiferous tubules (ST) lesions such as shrinkage of ST, lifting of epithelium, widened intercellular space, karyolysis, epithelial sloughing, ST atrophy, multinucleated giant cells, and germinal epithelium derangement were observed in the treated animal groups. Further, the thickness ST basement membrane increased significantly in cART, DM, and DM+cART groups but testis capsule thickness increased significantly in A+cART, DM+A, and DM+A+cART groups. The testicular interstitial connective tissue fibers viz. collagen, reticulin, and elastin reduced significantly in all treated groups, except for the reticulin which was non-significantly decreased in the alcohol (A) group. Furthermore, luteinizing hormone was significantly elevated in A and A+cART groups but significantly reduced in the DM+A+cART group. However, follicle-stimulating hormone increased significantly in all treated groups, with exception of DM+cART group. Testosterone levels were significantly reduced in DM, DM+A, and DM+A+cART groups, but no significant difference (p>0.05) was found in the inhibin B level in all treated groups compared to the control. In addition, the intensity and number of Sertoli and Leydig cells expressing androgen receptor were significantly reduced in all treated groups, except for the number of Sertoli cells expressing androgen receptor in the alcohol group. Expression of inflammatory markers (IL-1β, TNF-α, and IL-6) was upregulated in all treated groups, with exception of IL-1β of the A+cART group and TNF-α of cART and A+cART groups. The markers for oxidative stress (iNOS, MDA, and 8-OHDG) and apoptosis (caspase 3) were upregulated in all treated groups, with exception of MDA in the alcohol group. Though, the number of germ cells expressing proliferation marker Ki-67 was significantly reduced in all treated groups, the staining intensity was significantly increased compared to the control group. The results show that diabetes, alcohol abuse, cART, and their combinations have deleterious effects on the testicular histoarchitecture and function, which are suggested to result from the upregulation of oxidants and cytokines and androgen receptor depletion. The results further suggest that the interaction arising from a co-presence of cART and alcohol in diabetic condition could mildly diminish their independent effects due to their common cytochrome P450 metabolic pathway. This study yielded invaluable data on the contribution of cART alcohol-diabetes interaction on the male reproductive functioning/dysfunction and hopefully will help clinicians in managing reproductive challenges in patients of this category.Item Characterising skeletopathy in an animal model of type 2 diabetes(University of the Witwatersrand, Johannesburg, 2022-11) Dlamini, Gcwalisile Frances; Ndou, RobertType 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 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. We conclude that the ZDSD rat can be used as a translational model for diabetic skeletopathy at cellular and molecular level, and it can be extrapolated to humans after consideration of other factors like, basal metabolism, age, sex and skeletal loading patterns. We recommend optimal control of blood glucose levels at all stages of the disease to reduce the incidence of fractures in diabetic patients.Item Morphological changes in the lungs and air sacs of the Japanese quail (Coturnix japonica) exposed to heat stress(University of the Witwatersrand, Johannesburg, 2023-08) Abdulkadir, Abdurrahman; Reddy, DeranThe Japanese quail presents a potential protein food security alternative in rural sub-Saharan Africa because of its small size and easy husbandry. The nutritional and therapeutic value of its meat and egg makes it an interesting and better choice than chicken in some parts of the world. In addition, researchers used Japanese quail as an animal model of human genetic and developmental disorders because of its short generational interval. However, global warming threatens its welfare by propagating heat stress. The physiological response of Japanese quail under heat stress causes negative performance and sometimes instant mortality. Hence, in this study, lung of the quail under heat stress was microscopically examined as it is the most important organ for survival under heat stress. All other organs depend on the lungs for oxygen, and it is also the most crucial in evaporative cooling. A total of 38 Japanese quail were used in this study. A pilot study was conducted that used eight quails, to ensure the possibility of survival under the proposed experimental temperatures. Afterwards, 30 quails were randomly allocated — based on initial body mass— to five groups of six quails each. The control groups (CT and CT2) were maintained at a thermoneutral temperature of 25°C throughout the experiment while acute heat stress group (AH) were maintained at 38°C for 24 hours only. The chronic heat stress groups (CH1 and CH2) were maintained at 35°C for seven days and 28 days respectively. Body mass, cloacal temperature, and respiratory rate of quails were measured daily to monitor health and detect any serious ill health from heat exposure. Food and water were provided ad libitum. At the end of the experiment, all quails were terminated using an overdose of anaesthetic and lungs were harvested and processed for microscopy. Lung weight, volume and size were measured before sampling. Tissue samples were processed, and sections were cut with a microtome and stained with Mayers H&E, new pentachrome stain and Gomori’s one-step trichrome stain. Other tissue samples were triple immunolabelled with anti-α-SMA and Collagen 1 antibody and DAPI nuclear stain. Tissue samples were also processed for scanning and transmission electron microscopy. No significant difference in body mass, cloacal temperature, respiratory rate and lung parameters was found in heat-stressed quails compared with control. However, microscopic examination revealed blood congestion and excessive leakage of blood into airway of lungs in heat-stressed groups compared with the control. In addition, there was structural damage to parenchyma and blood vessels, which incites an inflammatory response causing deposition of collagen fibres in some areas of the lungs in heat-stressed groups. Interestingly, these effects occur in a time-dependent pattern. The most impact is seen in AH and CH1 groups while CH2 shows signs of recovery. In conclusion, Japanese quail lung was negatively impacted by heat stress, which can lead to instant mortality or long-term reduction of performance. Despite the evidence from this study suggesting that Japanese quail can adapt to the effects of heat stress if it survives the initial impact, a conscious effort must be made to alleviate or remove heat stress for quality outcomes.