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.Item A documentation of the morphology of the lungs of the Sprague Dawley rat after chronic heat exposure(University of the Witwatersrand, Johannesburg, 2023) Peya, Yolanda; Reddy, DeranClimate change is a natural phenomenon that is exacerbated by mul4ple human ac4vi4es in the environment. These ac4vi4es lead to a rise in global temperatures. This increases the occurrence of heatwaves which pose a major threat to ecosystems, biodiversity, and the conserva4on of species on Earth. This study, aimed to document the effects of chronic heat exposure on the lungs of the Sprague Dawley rat using histological and immunofluorescence techniques. Twenty-four (24) rats were assigned to three groups; each exposed to different temperatures; (i) group A (n = 8) exposed to 25 °C (ii) group B (n = 8) exposed to 33 °C and (iii) group C (n = 8) exposed to 34 °C. This was done for a period of 4 weeks and the rats were euthanised therea`er. Methods employed included Intratracheal instillation, and histological and immunofluorescence techniques. This study found that rats in group A (25 °C) displayed no structural adapta4ons in comparison to groups B and C. Enlarged blood vessels with thick smooth muscle and prominent elas4c fibres, bronchus-associated lymphoid 4ssue (BALT), and increased collagen deposi4on were observed in group B (33 °C). These indicate the ac4va4on of immune responses and adapta4on to new environmental temperatures. In group C (34 °C), the lung morphology was severely damaged. There was an increased loss of epithelial integrity, oedema, pulmonary alveolar proteinosis (PAP), and a severe decrease in the lung- to-surface area ra4o. The significant changes caused by the 1 °C temperature difference between groups B (33 °C) and C (34 °C) illustrate the detrimental effects of chronic heat stress. It is evident that increasing environmental temperatures due to climate change is harmful to life and immediate interven4ons are necessary to combat rising temperaturesItem Characterizing Luminal A breast cancer heterogeneity and in vitro response to hormone therapy(University of the Witwatersrand, Johannesburg, 2024) Gallant, SimoneBreast cancer is the most prevalent form of cancer diagnosed amongst women worldwide, responsible for a mortality rate of 6.9% and responsible for 684,996 deaths. Breast cancer is the most heterogeneous disease characterised by variations in genomic, epigenomic transcriptomic and proteomic profiles. The limited research on intratumoural heterogeneity in breast cancer and hormone therapy is the motivation for our study to further aid in understanding stemness markers influencing luminal A breast cancer and the effects hormone therapy has on biomarkers associated with breast cancer. In our study, we optimised modified essential 8 media to culture sorted cell populations in optimal conditions without differentiation ensuring stemness markers are maintained. Magnetic cell sorting was used to separate cells based on stemness markers CD133 and CD44. To verify these sorted markers flow cytometry was performed. The evaluation of the effects hormone therapy had on biomarkers was performed via immunocytochemistry and analysed using cell profiler. Our study revealed significant differences between subpopulations in MCF7 and T47D cell lines. It emphasizes the importance of CD44 and CD133’s role in tumour progression and its possible influence in hormone therapy. Our findings show that in populations with both stemness markers present in T47D cell line there is a reduction in progesterone receptor expression when treated with Tamoxifen. We also noticed the difference between population and hormone therapy impact on these changes. Thus, stemness markers are vital in tumour progression and the interaction of biomarkers and hormone therapy. However future research in the biological process and pathway activation is needed to further understand the intricacies of CD44 and CD133 mechanism of action as well as its association to biomarkers common pathwaysItem Effects of binge alcohol consumption on the development of the femur of adolescent Sprague Dawley rats(University of the Witwatersrand, Johannesburg, 2024) Mngoma, Ndabenzinhle Ronald; Bhika, AkaashniExcessive alcohol consumption adversely affects bone metabolism, thus resulting in reduced bone length, density, and strength. While excessive alcohol consumption is an established risk factor for osteoporotic fractures, there remains a dearth of information in literature regarding bone effects of binge alcohol consumption in adolescents. Therefore, our study aimed to examine the effects of binge alcohol consumption in an acute and chronic binge model, on the development and growth of the adolescent femur. Forty-eight Sprague Dawley rats (24 male and 24 female) aged 7 weeks were randomly allocated to one of the 4 treatment groups (n= 12/group) receiving binge alcohol (3g/kg of 20% alcohol) or caloric equivalent of maltose dextrin (pair-fed control), via oral gavage. The treatment groups were; A1, receiving alcohol on 3 alternating days for one week, C1, receiving the caloric equivalent of maltose dextrin in the same manner as A1 (acute), A4 and C4 received treatments in the same manner as A1 and C1 for four consecutive weeks (chronic). Trabecular morphometry in both the proximal and distal epiphysis, and cortical dimensions were assessed by using three-dimensional Micro- Focus X-ray Computed Tomography (3D-μCT) and Volume Graphics Studio® software. The morphology of the epiphyseal growth plate was examined by Haematoxylin and Eosin staining, whereas Ki-67 immunostaining was employed to quantify the proliferation of chondrocytes in the proliferative zone of the growth plate. A three-point bending test was employed to examine the effects of alcohol on bone strength. Results showed that binge alcohol consumption causes thinner trabeculae that are more widely spaced and with a smaller bone to volume ratio (BV/TV). However, the tensile strength was similar in the alcohol exposed rats and paired fed groups in male rats, whereas it appeared improved in female rats exposed to alcohol. A binge model also affected the number of chondrocytes in the proliferative zone negatively. All the adverse changes observed in the osseous tissue in the current study were shown in the male rats. Our study found alcohol to have no adverse effects on female rats, which could be due to hormonal differences.”Item Impact of Cannabidiol and Tamoxifen treatment on cell death and cell survival in breast cancer in vitro(University of the Witwatersrand, Johannesburg, 2024) Mahasha, Mahlatse Fortunate; Augustine, TanyaThe main non-psychotropic component of Cannabis sativa, Cannabidiol (CBD), alleviates breast cancer treatment-associated side effects but its effects with standard therapy remain unclear. In breast cancer, CBD has been shown to exhibit anti-cancer properties by inducing apoptosis and pro-death autophagy. This study aimed to investigate the effects of combined CBD and Tamoxifen treatment on metabolism, cell death, and cell survival mechanisms in luminal-A breast cancer cell lines MCF7 and T47D. The CBD concentration relative to IC50 was established by testing a range of CBD concentrations: 5 μM, 7 μM, and 10 μM, at 24 h, 48 h, and 72 h using the neutral red cell viability assay. The scratch assay was used to determine the effects of the concentrations on migratory capacity. Two models of treatment were used, single-dose treatment (model 1) and daily-replacement treatment (model 2), and appropriate controls were included. Treatment with 2 μM Tamoxifen and 5 μM CBD for 48 h was determined to be the optimal treatment condition. The MTT assay was performed, and the absorbance ratio indicative of cell proliferation was calculated. The ability of the cells to metabolize the drug components was examined through an assessment of CYP450 reductase (CPR) enzyme activity. The mRNA and protein expression levels of three autophagic markers; BECN1, LC3B, and p62, were investigated using qPCR and immunocytochemistry, respectively. Friedman’s Anova (p<0.05) and Kruskal Wallis (p<0.05) post hoc tests were used to statistically analyse the data. Combined CBD and Tamoxifen treatment showed the greatest decrease in the proliferation of MCF7 cells and T47D cells compared with all other treatments across both treatment models, with the daily- replacement treatment model (model 2) showing more efficacy thus suggesting that combined treatment may inhibit cell proliferation. CYP450 enzyme reductase activity was higher in T47D cells compared with MCF7 cells in both treatment models suggesting increased metabolic activity and susceptibility to combined treatment. However, in the daily replacement model, MCF7 cell CPR activity could not be ascertained, suggesting either prodrug availability or reduced CPR activity. Further analysis is required in this regard. For immunolocalization, optimization was conducted in late 2021 and all three antibodies showed clear and expected immunolocalization but when the experiments were repeated early 2022, immunofluorescence was reduced (P62 and BECN1), with LC3B not detectable. P62 and BECN1 were expressed in both MCF7 and T47D cells across both treatment models although BECN1 expression was not sufficient to be quantified and assessed statistically. LC3B protein levels could not be accurately quantified irrespective of the treatment model used. Low amounts of target mRNA in MCF7 cells resulted in undetermined Cq values of LC3B, P62 and BECN1 genes across both treatment models. In T47D cells, Cq values of target genes were determined across both treatment models and the fold change in gene expression indicated that combined CBD and Tamoxifen treatment effectively upregulates target genes albeit not significantly (LC3B, P62 and BECN1) with the single-dose treatment model (model 1) compared with the daily replacement model. Both the immunofluorescence and qPCR experiments would be required to be repeated to ensure conclusive results. The findings of this study nevertheless indicate that combined CBD and Tamoxifen treatment may inhibit tumour growth, but tumour cells may be able to evade cell death pathways resulting in tumour cell survivalItem Assessment of disability resulting from degenerative joint disease in a southern African skeletal population(University of the Witwatersrand, Johannesburg, 2023) Gavin, Jessica Robyn Brinkworth; Carrasco, Lorena Nunez; Meyer, Anja; Keyes, CraigAge-related skeletal changes, like degenerative joint disease (DJD), are often used to estimate age in forensic settings, yet these changes also reflect the living experience of individuals as they progress through life. This study set out to assess the impact of DJD in a sample of southern African deceased individuals and the subsequent effects on these individuals’ Activities of Daily Living (ADLs). A novel scoring system was created, translating DJD frequency and severity into how the individual was potentially physically disabled. A sample of 150 southern African individuals between the ages of 35 and 90 years were assessed for signs of DJD in each of the major joints, both for the peripheral (TMJ, shoulder, elbow, wrist, finger, hip, knee, ankle and toe joints) and axial (cervical, thoracic and lumbar vertebral joints) regions. The severity of DJD was then translated into various ADLs (transferring, eating, talking, object manipulation, walking and posture changes: head rotation; twisting and bending) that may have been affected. Socio-economic Status (SES) was included as this would have a significant impact on the types of DJD and subsequent ADL impairment seen, as well as how these individuals may have been impacted within their respective contexts. Results for DJD severity and frequencies indicated that the shoulder most often presented with the most severe DJD score with 42.67% of the pooled sample presenting with a score of 3. It was also interesting to note that most of the upper peripheral joints were affected, with the entire sample presenting with some form of DJD in the elbow, specifically. For the axial skeleton, severity scores were much more variable across different vertebral regions, with the highest severity percentage per region was seen in the thoracic vertebra (5%). In general, females presented with higher severity scores for DJD across all joints, with the TMJ (47%) and toe (22%) joints being statistically higher in females. When controlling for population affinity and sex the same trend was observed in the white sample, specifically the white females (TMJ = 22%). Black males; however, presented with higher severity frequency of DJD in the axial skeleton, specifically the lumbar vertebrae (32%). The white cohort showed very little correlation with age in relation to the TMJ, whereas this was true for the elbow and ankle in the southern African black individuals. This study indicated moderate impacts in transferring (53%), walking (51%), and eating (41%) activities for all individuals. Females presented with significantly higher ADLs for all activities except posture changes whereas problems with eating and talking were more often seen in southern African white individuals. Differences between different socio-economic status groups were also noticed with the lower SES group showing increased levels of impairment across most of the ADLs which may relate to activity differences when age is controlled for. The focus of this research was to provide deeper information into impairment and disability caused by DJD. To conduct research on individuals were their lives and stories were limited and underexplored. This research highlights the need to continue studies on skeletal remains of individuals impaired by DJD, with focus on trends on joint and activity limitations within past and present contextsItem The effects of ibogaine on myelination in Sprague Dawley rats(University of the Witwatersrand, Johannesburg, 2024) Govender, Demi NatishaIntroduction: The growing opioid epidemic is a worldwide issue which is prevalent in South Africa with the use of opioid cocktails such as nyaope. A possible solution to this problem is the use of psychedelic assisted psychotherapy. Ibogaine is a psychedelic that has been shown to curb addiction cravings and have neuroplastic effects in the brain. Ibogaine is extracted from the root bark of a West African plant and has shown to have neuroplastic effects in the brain. We investigated whether these antiaddictive properties are due to remyelination of the brain’s white matter. Methods: This study uses qPCR and western blotting to determine how myelin specific proteins and genes such as CNPase (CNP), Myelin Basic Protein (MBP) and Proteolipid Protein (PLP) are affected by morphine (opioids) and ibogaine. The experimental rat groups included a saline, morphine and ibogaine only controls, a combination morphine and ibogaine and a second combination morphine and ibogaine which included a 3 day withdrawal after ibogaine injection. Results: CNP protein was increased in the second morphine ibogaine group (p<0,0001) and the CNP mRNA fold expression was increased in the first morphine ibogaine group compared to the second morphine ibogaine group (p=0,0343). The 18,5 kDa isoform of MBP had increased expression in the ibogaine control (p=0,0384) and second morphine ibogaine group (p=0,0037). PLP shows increased protein expression in the second morphine ibogaine group when compared to the first group (p=0,0464). There is decreased PLP mRNA expression in the ibogaine control group when compared to morphine control (p=0,0033), first morphine ibogaine (p<0,0001) and second morphine ibogaine groups (p=0.003) Conclusion: Ibogaine may cause remyelination following demyelination by morphine. A consistent trend in the data shows that the myelin proteins were increased after the 3 days after administration of ibogaine following chronic morphine administration compared to 1 day after administration of ibogaine. This suggests that remyelination takes between 24-72 hours before it begins to produce new myelin around the axons due to ibogaine. These results also shows that CNP and MBP increase in expression earlier than PLP and are good markers for early remyelination. This is consistent with increase in CNP mRNA expression for CNP seen in the first morphine ibogaine but not the second group revealing an immediate effect on mRNA but a delay in protein expressionItem The role of human papilloma virus (HPV) in oropharyngeal squamous cell carcinoma: a clinicopathologic and molecular analysis(2024) Disenyane, DineoContext: Human papillomavirus (HPV) is an established aetiology in a subset of oropharyngeal squamous cell carcinoma (OPSCC). However, literature on the association between HPV and OPSCC in the African context is lacking. Objective: To determine the association of HPV in OPSCC and to correlate it with the clinicopathologic and molecular analysis. Design: A total of 60 OPSCCs were evaluated with HPV in-situ hybridisation (HPV ISH) and immunohistochemistry (IHC) for p16, p53 and Ki67. This was correlated with the site of occurrence of the tumours, the patients' smoking and alcohol usage and microscopic features of tumours (NKSCC;n=30 and KSCC;n=30). Results: HPV DNA was found in 24/60 (40%) cases. HPV16, was positive in 18/60 (30%) cases and HPV6 in 8/60 (13.3%) tumours. Two of the 60 (3.3%) cases were positive for both HPV16 and HPV6. HPV positive (+ve) tumours occurred more in males (n=22; 73%) than females (n=7; 23%); median age: 62.5 years. The primary tumour sites were the tonsil (n=7; 11.6%) and posterior tongue (n=6; 10%). Of the NKSCCs, 11 (36.7%) were HPV16+ve and 8 (26.7%) HPV6+ve. Seven (23.3%) KSCC were HPV16+ve and none (0%) HPV6+ve. Of the 24 HPV+ve tumours, 13 (54.1%) were p16+ve and 14 (58.3%) were p53+ve. Three (12.5%) tumours exhibited the molecular phenotype: p16+ve in > 70% of tumour cells, a low (25%) proliferation index. Conclusion: HPV positive OPSCC is seen more in older male patients. The distinct molecular phenotype, (p16 positive, low p53, high KI67) highlights tumours that are of true viral aetiology. In this study, the low p16 IHC stain sensitivity towards HPV ISH positive tumours infers that p16 is less reliable when used alone as a surrogate marker for HPV associated OPSCC. Therefore, in OPSCC with a high suspicion for HPV and which display a basaloid and non-keratinising morphology, a negative p16 IHC stain should be followed by other molecular techniques such as HPV ISH.
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