School of Physiology (ETDs)

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Start date: 2023End date: 2023

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    The effect of two modalities of sleep disruption on immunity in healthy young female participants
    (University of the Witwatersrand, Johannesburg, 2023-07) Ajlan, Zuha; Scheuermaier, Karine; Iacovides, Stella
    Studies have shown that sleep deprivation leads to an inappropriate immune response by elevating pro-inflammatory markers, including interleukin (IL-)1, IL-6, and tumour necrosis factor (TNF-)α. This inappropriate immune activation increases the risk of developing autoimmune disorders. Despite women representing 80% of patients with autoimmune disorders and having a greater prevalence of poor sleep quality and sleep disorders, most experimental human studies investigating sleep and immunity focused on men. Therefore, this study assessed the effect of sleep fragmentation vs sleep restriction on sleep parameters. I then compared the immune response after the two types of sleep disruptions relative to a normal sleep episode and I investigated the association between sleep architecture and immune markers in healthy young women in the follicular phase of their menstrual cycle. Fourteen healthy females underwent a randomised-crossover controlled study consisting of one adaptation night and three randomised, non-consecutive sleep conditions, namely: baseline night (BN, uninterrupted 8 hours of sleep); restriction night (RN, sleep was limited to the first 4 hours of their habitual sleep episode); fragmentation night (FN, eight randomised forced awakenings through an 8-hour sleep opportunity night). Polysomnographic (PSG) sleep recordings were obtained for each condition, and plasma was collected 2.5 hours after their habitual waketime following each condition. A multiplex Luminex assay was used to measure the concentration of nine cytokines. I compared PSG-extracted sleep variables between the three experimental nights. I ran mixed models analyses testing cytokine levels in each sleep condition (RN vs. FN vs. BN) in unadjusted analyses and then adjusting for order of the condition (first vs. second vs. third experimental night), day of follicular phase of the menstrual cycle and age. I also used an unadjusted mixed model analysis to test the association between cytokine levels and each sleep variable. Total sleep time, non-rapid eye movement (NREM) and rapid eye movement (REM) were reduced in FN and RN but were lowest during RN (p<0.001). I found an effect of sleep condition on IL-8 (F = 3.40, P = 0.05) with IL-8 being lower in RN vs FN or BN. There was no effect of condition on the other cytokines in unadjusted or adjusted analyses. Lower wake after sleep onset (WASO) and higher NREM were associated with higher IL-8 concentration regardless of the sleep condition. Lower stage 2 (N2) (F = 6.28, β = -0.001, P = 0.02) and higher stage 3 (N3) (F = 7.01, β = 0.004, P = 0.01) was associated with a higher TNF-α regardless of the sleep condition. In conclusion, the study shows that acute sleep disruption alters sleep architecture and leads to an inappropriate immune activity in young healthy women. Future studies should try and investigate chronic sleep fragmentation vs chronic sleep restriction on the immune system.
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    The impact of altitude on the prevalence and characteristics of Restless Legs Syndrome
    (University of the Witwatersrand, Johannesburg, 2023) Munian, Pariska
    Restless Legs Syndrome (RLS) is a neurological sensory disorder, characterised by the irresistible urge to move due to unpleasant, deep-seated paresthesias in the legs. The urge to move usually occurs in the evening, when an individual is at rest and the sensations experienced are alleviated with movement. The prevalence of RLS in a general population ranges from 2.5 to 10%, from studies across the globe. Differences in RLS prevalence have been noted between different ethnic groups, with individuals of European ancestry exhibiting greater prevalence of RLS compared to individuals of Asian and African ancestry. The aetiology of RLS is unclear; however, there is evidence of central nervous system iron dysregulation. Low partial pressure of oxygen at high altitude, which is a large distance above sea level, may exacerbate iron dysregulation which may account for the greater prevalence of RLS at high compared to low altitude, which is an area at sea level. However, the impact of altitude on the prevalence of RLS requires further investigation and is the aim of this study. To investigate the effect of altitude on the prevalence and characteristics of RLS, a questionnaire was administered to the general South African population at two altitudes: low altitude (Durban, South Africa) and higher altitude, (1753m above sea level, Johannesburg, South Africa). The survey was completed by 1291 participants (416 at low altitude and 875 at higher altitude). Using an online questionnaire, data were collected on demographic characteristics (including age, sex and ethnicity), the Cambridge-Hopkins RLS questionnaire (to assess the presence/absence of RLS), self-reported iron deficiency, subjective measures of sleep, measures of daytime sleepiness (using the Epworth Sleepiness Scale) and levels of fatigue (using the Fatigue Assessment Scale). RLS was significantly more prevalent at the higher altitude (n = 69, 7.9%) compared to low altitude (n = 20, 4.8%), which may be due to an increase in iron dysregualtion at high altitude, resulting from the low partial pressure of oxygen. Factors associated with RLS also were exacerbated at higher altitude; these include increased RLS severity (p = 0.003), increased daytime sleepiness (p = 0.04) and decreased self-reported iron levels (p = 0.03) in individuals with RLS at higher altitude compared with low altitude. RLS was less prevalent in individuals with African ancestry than in those with European ancestry at the higher altitude (p = 0.0025). However, RLS was more prevalent in individuals with African ancestry than in those with Indian ancestry at low altitude (p = 0.0004). My data therefore support that altitude appears relevant to the pathophysiology of RLS, with high altitude presenting as a risk factor for RLS and exacerbating some characteristics of RLS
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    Investigating the toxic effects of nyaope on the brain, liver, and kidney of Sprague- Dawley rats
    (University of the Witwatersrand, Johannesburg, 2023) Mathiki, Khethani Thendo
    Background. Worldwide drug use is estimated to be 345 million, encompassing both synthetic and plant-based medications, as well as both legal and illicit opioids. The use of these drugs is on the rise and is becoming a growing concern for public health. Moreover, the emergence of heroin dominant street drugs like nyaope have gained popularity, especially in low- and middle- income countries such as South Africa. The use of nyaope exposes individuals to the risk of substance use disorders (SUDs) and the array of mental health issues, as evidenced by social and clinical studies. Nyaope, a relatively inexpensive illicit drug, is commonly found in townships and outskirts of inner cities which are predominantly saturated with African/Black ethnic communities. Its composition varies depending on the geographical area but primarily consists of ingredients like heroin, opioid derivatives, rat poison, antiretroviral drugs (ARVs), and other substances. Similar to other opioids, heroin exerts its effects on the central nervous system (CNS) by acting on opioid receptors in various brain regions, including the prefrontal cortex (PFC), nucleus accumbens, and amygdala. These regions play a critical role in regulating mood and cognitive functions. Additionally, opioids undergo processing in the liver before being excreted by the kidneys. Our work sought to evaluate the effects of acute toxicity from the unique street drug cocktail known as nyaope on the behaviour and molecular markers of the PFC, liver, and kidney in Sprague-Dawley rats, despite the abundance of material already available on opioid usage. Methods. Twenty-five Sprague-Dawley rats were sourced from the Wits Research Animal Facility following ethical clearance and habituated for a duration of ten weeks. A pilot study involving three of these rats was conducted to determine the appropriate exposure dose of nyaope. Following the pilot study, the remaining twenty-two rats were divided into two groups; nyaope-treated (n=11) and saline-treated (n=11). Nyaope-treated rats received a single dose of nyaope at 0.4 mg/kg/bw and were subsequently exposed to the Open Field Test (OFT), which assesses various behavioural indices, including locomotor activity, mood, and exploratory behaviour, using the AnyMaze video tracking system. The saline-treated rats received a single dose of physiological saline at 0.4 mg/kg/bw and underwent the same 30-minute exposure to the OFT. After this exposure, the animals were placed in their respective home cages and qualitatively observed for an additional 30 minutes. Following this observation period, the rats were anaesthetized with isoflurane and euthanized exactly one hour after exposure to nyaope or saline. Tissues from the brain, liver, and kidney were collected, and RT-PCR was conducted to assess toxicity markers, including genes that code for proteins involved in the processes of apoptosis (BAX and Bcl-2), autophagy (SQSTM1/p62), microglial repair (ANXA3), and inflammation (IL-6). In addition, plasma samples were collected and analysed using IDEXX catalyst technology to examine the plasma presence of liver toxicity markers; aspartate transferase and alanine transferase, along with the kidney toxicity marker; creatinine. Results. The qualitative findings indicated that rats treated with nyaope exhibited reduced grooming behaviour. Additionally, the nyaope-treated rats experienced a phase of heightened activity followed by extreme lethargy. In contrast, the saline-treated rats displayed consistent mobility and curious behaviour. Compared to the saline-treated rats, the nyaope-treated rats exhibited clinical signs such as tremors, a rigid tail, hypoxia, and increased diuretic behaviour. When observing the track plots of the nyaope-treated rats, they tended to favour the outer zone in a thigmotaxis pattern, with few bouts into the centre, while the saline-treated rats showed more uniform movement within the OFT apparatus. Quantitative behavioural data using the AnyMaze tracking system revealed that nyaope-treated rats had decreased locomotor activity. They covered less total distance during the test and travelled shorter distances within the centre zone compared to saline-treated rats. Nyaope-treated rats also had fewer mobility episodes and moved at slower speeds on average than the saline-treated rats. In terms of mood assessment, the nyaope-treated rats spent less time mobile overall, both in the outer and centre zones and engaged in significantly fewer grooming bouts compared to the saline-treated rats. In the assessment of exploratory behaviour, it was noted that nyaope-treated rats exhibited fewer instances of rearing, line crossing, and head entries into the centre than the saline-treated rats.Regarding the molecular assessment of the brain and kidney, there were no significant differences in the expression of molecular markers between the two groups, except for a decreased expression of Bcl-2 (p < 0.001) in the kidneys of nyaope-treated rats compared withthe saline-treated rats. Additionally, plasma expression levels of AST, ALT, and creatinine were similar between the two groups. Conclusion. These findings indicate that exposure to 0.4 mg/kg/bw of nyaope for one hour does result in behavioural changes, even though it does not immediately lead to acute molecular toxicity in the brain, liver and kidney. Conversely, nyaope exposure causes a reduction in mRNA expression of Bcl-2, suggesting that the drug induces cell and tissue damage in the kidney through apoptosis
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    Understanding the in vitro effect of TNF-α and hyperglycaemia on endothelial activation
    (University of the Witwatersrand, Johannesburg, 2023) Amoni, Joel Ikechukwu; Millen, A; Gunter, S
    Background. Inflammation is one of the main underlying mechanisms in the development of cardiovascular disease (CVD). Indeed, in diseases characterised by high-grade systemicinflammation and in comorbid conditions, such as diabetes mellitus, it has been suggested that inflammation, at least in part, contribute to the increased risk of CVD. One of the earliest signs of inflammation-induced CVD is endothelial dysfunction. However, whether inflammation promotes endothelial dysfunction via the same signalling pathways in different pathological conditions characterised by systemic inflammation is not well understood. Inflammation- induced aberrant expression of microRNA (miRNA), small non-coding RNAs that function to regulate gene expression post-transcriptionally, have been linked to impaired endothelial function. However, the mechanisms whereby miRNAs may mediate endothelial activation requires further investigation. Therefore, the aim of this study was to investigate the molecular mechanisms involved in the regulation of endothelial function in different models of inflammation. Methods. EA.hy926 immortalized endothelial cells were cultured in Dulbecco’s modified eagle’s medium (DMEM) + HAM’s F12 nutrient mix supplemented with 10% foetal bovine serum (FBS). EA.hy926 cells were exposed to tumour necrosis factor-alpha (TNF-α) at a concentration of 10ng/ml for 24 hours to induce an inflammatory response while the controls were cells exposed to plain media for 24 hours. EA.hy926 cells were also exposed to either 5mM or 30mM glucose for 72 hours, as a model of glycemia-induced inflammation, while the control cells were exposed to plain media for 72 hours. Total RNA was extracted from the cell pellets and subsequently reverse transcribed to miRNA cDNA and mRNA cDNA. Quantitative real time PCR was used to determine the relative expression of interleukin-6 (IL-6), vascular cell adhesion molecule 1 (VCAM-1), miRNA-155-5p, endothelial nitric oxide synthase (eNOS) and superoxide dismutase 2 (SOD-2). Additionally, an ELISA assay was used to determine the ratio of phosphorylated p65/total p65 in cells exposed to TNF-α (10ng/ml for 24 hours), glucose (30mM for 72 hours) and plain media controls. Results. Compared to control cells, the relative mRNA expression of the inflammatory marker IL-6 was significantly increased in the cells exposed to TNF-α (p=0.002) and 5mM (p=0.002) and 30mM (p = 0.0001) glucose, respectively. In addition, the relative mRNA expression of VCAM-1 was increased in the cells exposed to TNF-α (p <0.0001) and 30mM glucose (p =0.03) when compared to their respective controls. Interestingly, miRNA-155-5p expression was also increased in the cells exposed to TNF-α (p= 0.04), 5 mM glucose (p = 0.007) and 30 mM glucose (p = 0.02). Exposure to TNF-α, and 5 mM and 30 mM glucose resulted in increased eNOS expression compared to the control cells (p = 0.04, p=0.002 and p = 0.0002, respectively). The ratio of phosphorylated-to-total NF-κB p65 were not different in either the TNF-α exposed or glucose exposed cells compared to control cells (all p>0.05). Conclusion. These findings suggest that TNF-α and hyperglycaemia exposure resulted in endothelial dysfunction. However, hyperglycaemia caused much greater oxidative stress (increased eNOS) most likely due to glucose scavenging of nitric oxide (NO). This suggests that the underlying mechanisms of endothelial dysfunction occurring due to hyperglycaemia and inflammation may be driven by different mechanisms. This study highlights the need for further investigation into the mechanisms whereby miRNA-155-5p regulate endothelial dysfunction
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    The Epidemiology of Menstrual Pain in a South African University Population
    (University of the Witwatersrand, Johannesburg, 2023) Futi, Benedicte Malonda; Iacovides, Stella; Scheuermaier, Karine
    Dysmenorrhoea, pain associated with menstruation, is a significant public health concern among young women of reproductive age. Identifying associated risk factors for the development of dysmenorrhoea is essential to minimize the impact of monthly menstrual pain on the daily functioning of these women, both in a personal and professional capacity. However, epidemiological data on the prevalence and associated risk factors for dysmenorrhoea in South Africa are scarce. This study aimed to determine the prevalence of dysmenorrhoea and its associated risk factors in a South African university student and staff population. An online survey was distributed to all 26 public universities across South Africa. The final sample comprised data from 7280 participants, and I found a high prevalence [76.7% (95% CI, 75.7-77.6)] of moderate-to-severe dysmenorrhoea among the respondents. Factors significantly associated with increase odds of experiencing moderate to severe dysmenorrhoea included: having heavy (adjusted OR = 2.749, 95% CI 2.208-3.421; p < 0.001) menstrual flow, having a positive family history of dysmenorrhoea (adjusted OR = 1.615, 95% CI 1.346-1.938; p < 0.001), always experiencing poorer subjective sleep quality [“often” (OR= 1.595, 95% CI 1.16-2.191; p=0.004), “sometimes” (OR= 1.523, 95% CI 1.22-1.902; p=0.0002) and “rarely” (OR=2.046, 95% CI 1.596-2.623; p<0.0001)], and scoring higher on the central sensitisation inventory total score (adjusted OR= 1.033, 95% CI 1.026-1.04; p < 0.001). On the other hand, factors significantly associated with decrease odds of experiencing moderate to severe dysmenorrhoea included: older age at the time of the study (adjusted OR= 0.982, 95% CI 0.967-0.998; p= 0.0285), older age at menarche (adjusted OR = 0.938, 95% CI 0.89-0.989; p= 0.0186), having been pregnant (adjusted OR = 0.757, 95% CI 0.605-0.946; p= 0.0145), lower BMI (adjusted OR = 0.986, 95% CI 0.972-1; p = 0.044), being of European ancestry (adjusted OR = 0.698, 95% CI 0.567-0.859; p = 0.007), and having light menstrual flow (adjusted OR= 0.473, 95% CI 0.373-0.6; p < 0.001). I also found a significant impact of dysmenorrhoea on daily life, with 51.6% of respondents reporting absenteeism from school or work during menses and 88.4% of the respondents requiring pharmacological treatments, such as contraceptive pills and nonsteroidal anti-inflammatory drugs (NSAIDs), to manage their menstrual pain. The study highlights the need for increased awareness, education, and effective interventions aimed at reducing the prevalence and impact of dysmenorrhoea on women's lives. The implications of both the increased central sensitisation (CS) and the sleep-pain reciprocal relationship suggest that they could potentially lead to the development of chronic pain conditions. Future research should further explore the interventions and management strategies that could improve sleep quality and prevent the onset of central sensitisation, thus reducing the risk of developing chronic pain conditions. The findings have important implications for the management of dysmenorrhoea that can improve women's quality of life and promote better health outcomes. These findings also point towards the need to educate women about the importance of seeking medical attention for dysmenorrhoea and the potential long-term implications of untreated dysmenorrhoea