i MORPHOLOGICAL EVALUATION OF THE HEART, KIDNEY AND LIVER FOLLOWING SIMVASTATIN TREATMENT OF A MOUSE MODEL OF ADOLESCENT ALCOHOLISM Makgotso Nchodu 1325876 A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, School of Anatomical Sciences, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine. Johannesburg, 2023. i DECLARATION I, Makgotso Nchodu hereby declare that this dissertation is my own work, unaided work. It is being submitted for the degree of Master of Science in Medicine in the Faculty of Health Sciences at the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at any other University. Makgotso Nchodu Signed on the 5th day of May 20 23 at Johannesburg ii DEDICATION In memory of my grandmother Elizabeth Zwane 1945 - 2016 For instilling the love and value of education, thank you. iii ACKNOWLEDGEMENTS I would like to thank God for his grace and mercy throughout this journey. 1. My supervisor: Dr Oladiran Olateju. Thank you for your excellence supervision. The knowledge and skills you have shared with me are invaluable and very much appreciated. 2. My parents: Tseko Nchodu and Phindile Nchodu. Thank you for your patience and overwhelming support. Thank you for reminding me to pray and to have faith, you believed in me even when I did not believe in myself. Your unconditional love is a gift from God. 3. My brother: Katlego Nchodu. Thank you for always checking up on me and reminding me to be fearless. 4. My friends: Nosipho Zwane, Nicole Mlauzi and Nolwazi Hlabangane. 5. Ms Hasiena Ali. Thank you for your guidance and assistance with optimization. 6. My co-workers: Robin du Preez, Sabiha Latiff and Alice Efuntayo. 7. National Research Foundation: For funding my research. iv ABSTRACT Individuals that begin drinking during adolescence are more likely to become alcohol addicts, resulting in the development of alcohol-related diseases such as alcoholic cardiomyopathy, renal tubulointerstitial fibrosis and steatohepatitis. This study investigated the protective capabilities of simvastatin against alcohol-induced damage on the heart, kidney, and liver of adolescent mice administered chronic alcohol. Fifty four–week old C57BL/6J mice (F = 5; M = 5) were assigned to each experimental group: (i) NT; no treatment; (ii) ALC; 2.5 g/Kg/day of 20% alcohol (iii) SIM; 5 mg/Kg/day (iv) ALC+SIM5; 5 mg/Kg/day of simvastatin followed by 2.5 g/Kg/day of 20% alcohol (v) ALC+SIM15; 15 mg/Kg/day of simvastatin followed by 2.5 g/Kg/day of 20% alcohol. Lower dosage of simvastatin was more effective against alcohol-induced myocardial hypertrophy in females while a higher dosage of simvastatin was more effective in males. Both simvastatin concentrations significantly reduced alcohol-induced myocardial fibrosis in the females but only the low simvastatin dosage was effective in the males. ALC+SIM5 improved inflammation only in the females. Alcohol increased the area of the renal corpuscles and glomeruli, the collagen and TNF-α distributions. 5 mg simvastatin was more effective against renal hypertrophy in both males and females. Both doses of SIM were effective against renal inflammation. Both concentrations of simvastatin were not beneficial in stimulating hepatocyte regeneration except for 15 mg simvastatin in males. Only a higher dose of simvastatin prevented alcohol effect on hepatic collagen distribution. Both concentrations of simvastatin following a chronic alcohol were not beneficial against alcohol-induced inflammation in the liver. v TABLE OF CONTENTS DECLARATION i DEDICATION ii ACKNOWLEDGEMENTS iii ABSTRACT iv TABLE OF CONTENTS v-vii LIST OF FIGURES viii-ix LIST OF TABLES x NOMENCLATURE xi-xiii CHAPTER 1: INTRODUCTION 1 1.1 Intro: Adolescent alcoholism 2-3 1.2 The heart 4-5 1.3 The kidney 6-7 1.4 The liver 8-9 1.5 Rationale 10 1.6 Aims and specific objectives 11 CHAPTER 2: MATERIALS AND METHODS 12 2.1 Animal study 13-14 2.2 Experimental study 14 vi 2.2.1 Preparation and administration of simvastatin and alcohol 14-16 2.2.2 Animal perfusion and organ harvesting 18 2.3 Tissue processing and morphometry of the heart 19-22 2.4 Tissue processing and morphometry of the kidney 22-24 2.5 Tissue processing and morphometry of the liver 24-26 2.6 Statistical analysis 26 CHAPTER 3: RESULTS 27 3.1.1 Animal and organ masses 28 3.1.2 Blood alcohol concentration 29 3.2 The heart 30 3.2.1 General morphology of the cardiomyocytes 30 3.2.2 Measurements of the cardiomyocyte area and diameter 35 3.2.3 Measurement of collagen distribution 39-40 3.2.4 Measurement of TNF-α distribution 41 3.3 The kidney 43 3.3.1 General morphology of the renal corpuscles and tubules 43 3.3.2 Morphometry of renal corpuscle area 49 3.3.3 Morphometry of the glomerular area 49-50 vii 3.3.4 Morphometry of the area of the urinary space 50-51 3.3.5 Measurement of collagen distribution 56 3.3.6 Measurement of TNF-α distribution 56-57 3.4 The liver 62 3.4.1 General morphology of the liver 62 3.4.2 Density of the hepatocytes 66 3.4.3 Measurement of collagen distribution 67 3.4.4 Measurement of TNF-α distribution 67-68 CHAPTER 4: DISCUSSION 73 4.1 The heart 74-77 4.2 The kidney 77-80 4.3 The liver 81-84 CHAPTER 5: CONCLUSION AND RECOMMENDATIONS 85 5.1 Conclusion and recommendations 86 CHAPTER 6: REFERENCES 87-94 APPENDICES 95 Ethical clearance certificate 95 Turnitin report 96 viii Buffer and fixative recipes 97 LIST OF FIGURES Figure 3.1: Histological section of the ventricles of the heart and the layers of the heart wall. 31 Figure 3.2: Histological section of area and length measurements and morphology of the cardiomyocytes from the left ventricle. 32 Figure 3.3: Histological section of collagen distribution in the left ventricle. 33 Figure 3.4: Histological section of TNF-α distribution in the left ventricle. 34 Figure 3.5: Cardiomyocyte area boxplots. 37 Figure 3.6: Cardiomyocyte diameter boxplots. 38 Figure 3.7: Left ventricle collagen distribution boxplots. 40 Figure 3.8: Left ventricle TNF-α distribution boxplots. 42 Figure 3.9: Histological section of horizontal section of the kidney. 44 Figure 3.10: Histological section of kidney ultrastructure morphology. 45 Figure 3.11: Histological section of renal corpuscle and renal tubule measurements. 46 Figure 3.12: Histological section of collagen distribution in the kidney. 47 Figure 3.13: Histological section of TNF-α distribution in the kidney. 48 Figure 3.14: Renal corpuscle area boxplots. 53 Figure 3.15: Glomerular area boxplots. 54 Figure 3.16: Urinary space area boxplots. 55 ix Figure 3.17: Renal collagen distribution boxplots. 59 Figure 3.18: Renal tubule TNF-α distribution boxplots. 60 Figure 3.19: Renal corpuscle TNF-α distribution boxplots. 61 Figure 3.20: Histological section of liver parenchyma morphology. 63 Figure 3.21: Histological section of hepatic collagen distribution. 64 Figure 3.22: Histological section of hepatic TNF-α distribution. 65 Figure 3.23: Hepatocyte cell density boxplots. 70 Figure 3.24: Hepatic collagen distribution boxplots. 71 Figure 3.25: Hepatic TNF-α distribution boxplots. 72 x LIST OF TABLES Table 2.1: Summary of the different experimental groups 17 Table 3.1: Summary of animal and organ masses 28 Table 3.2: Summary of the morphometries of the cardiomyocyte area and the diameter 35 Table 3.3: Summary of the morphometries of the renal ultrastructure 52 Table 3.4: Summary of the collagen and TNF-α distributions in the kidney 58 Table 3.5: Summary of hepatocyte morphometries, collagen and TNF-α distributions 69 xi NOMENCLATURE α: Alpha α-SMA: Alpha-Smooth Muscle Actin β: Beta %: Percentage ADH: Antidiuretic hormone ALC: Alcohol only ALC+SIM5: Alcohol and simvastatin 5 mg ALC+SIM15: Alcohol and simvastatin 15 mg AREC: Animal Research Ethics Committee BAC: Blood alcohol concentration BB: Brush border BD: Ballooning degeneration CCl4: Carbon tetrachloride DNA: Deoxyribonucleic acid eNOS: Endothelial nitric oxide synthase Endo: Endocardium Epi: Epicardium xii F: Fibrosis FDA: Food and drug administration GL: Glomerulus H & E: Haematoxylin and eosin HCl: Hydrochloric acid KC: Kupffer cells Klf2: Krüppel-like Factor 2 LPS: lipopolysaccharide LV: Left ventricle LVW: Left ventricular wall MT: Masson’s Trichrome Myo: Myocardium NaOH: Sodium hydroxide NHLS: National Health Laboratory Service NS: Not signficant NT: Non-treatment PB: Phosphate buffer PFA: paraformaldehyde xiii RAS: Renin-angiotensin system RC: Renal corpuscle RT: Renal tubule RV: Right ventricle RVW: Right ventricular wall SD: Standard deviation SIM: Simvastatin 5 mg TNF-α: Tumour necrosis factor alpha TGF-β: Transforming growth factor beta TGF-β1: Transforming growth factor beta 1 WRAF: Witwatersrand Research Animal Facility 1 CHAPTER 1: INTRODUCTION 2 Chapter One 1.0 Introduction 1.1 Adolescent alcoholism Adolescence is the period of growth and development that occurs during puberty, and it is characterized by physical, psychosocial, and psychological changes (Ramsoomar, et al., 2013; Marshall, 2014). During this growth phase, young people tend to experiment with alcohol resulting in heavy episodic drinking, especially where there are lapses in policies to regulate access to alcohol (Ramsoomar, et al., 2013; Marshall, 2014). In addition, factors such as low socio- economic status, peer pressure, and psychological trauma are among the many reasons why adolescents may experiment with alcohol (Ramsoomar, et al., 2013; Marshall, 2014). Alcohol is the most socially acceptable drug globally (Dawodu et al., 2019). Unfortunately, South Africa is one of the countries with the most harmful drinking patterns (Ramsoomar et al., 2013) therefore, the increasing rate of alcohol misuse among adolescents in South Africa is a major health concern which is at a high cost to the government (Letsela et al., 2019). Binge drinking, also referred to as heavy episodic drinking is classified as the consumption of 5 or more drinks on one occasion for males and 4 or more drinks on one occasion for females (Morojele & Ramsoomar, 2016). Chronic alcohol consumption is described as a pattern of abstaining from alcohol use followed by heavy episodic drinking (Morojele & Ramsoomar, 2016). Binge drinking among youth (15-24 years) was reported to have increased from 29% to 31%, from 1998 to 2005 (Chauke et al., 2015). Approximately 33.5% of male and 23.7% of female South African high school learners were reported to binge drink by the South African Youth Risk Behaviour Survey 2008 (Swart et al., 2015). The South African Department of Education 3 estimated alcohol consumption among high school learners to be 28% and approximately 12% of the youth in South begin drinking at the age of 13 years (Maserumule et al., 2019). Alcohol misuse is greater for male than female adolescents (Olsson et al., 2016). Adolescents who misuse alcohol are more likely to engage in sexual activities at an early age (Letsela et al., 2019) which often promotes having multiple sexual partners (Letsela et al., 2019). The effects of these are multi-faceted which include sexually transmitted diseases, relationship- related violence, teenage pregnancy, rape, mental illness, and post-traumatic stress disorders (Letsela et al., 2019). In addition, prolonged or chronic alcohol consumption followed by abstinence may sensitize receptors that regulate excitability and memory which may result in aggravated withdrawal symptoms such as headaches, sweating, anxiety, insomnia, and nausea (Olsson et al., 2016). Individuals that begin drinking during adolescence are also more likely to become alcohol addicts (Olsson et al., 2016). Chronic alcohol consumption could make an individual become violent, a sex ‘predator’ (i.e., rapist), a criminal, be involved in a road accident-causing harm, disability, or death to both the perpetrator and the citizens (Ramsoomar et al., 2013; Matzopoulos et al., 2014; Swart et al., 2015). According to Swart et al. (2015), alcohol-related violence is prevalent among South African adolescents during an altercation between/amongst intoxicated individuals. Apart from the social economic impact of alcohol abuse, alcohol misuse is among many factors contributing to the burden of disease in several countries (Ramsoomar et al., 2013; Olsson et al., 2016; Bertscher et al., 2020). Chronic alcohol increases the incidence of infectious and cardiovascular diseases (Matzopoulos et al., 2014; Obad et al., 2018)which are also a burden on the health systems as huge funds are allocated to caring for alcohol-related diseases (Matzopoulos et al., 2014; Obad et al., 4 2018). Even though the South African alcohol industry is beneficial to the economy (e.g., job creation and profits) (Matzopoulos, et al., 2014), the uncontrolled and unregulated access to alcohol (especially in the townships where adolescents can easily buy alcohol) are detrimental and costly to the government (Matzopoulos, et al., 2014). The lapse in policies guiding access to alcohol promotes abnormal drinking patterns and behaviours, especially in adolescents (Olsson, et al., 2016). This has thus provoked an interest in public health to find solutions to tackle alcohol use disorders in adolescents (Ramsoomar et al., 2013; Marshall, 2014; Olsson et al., 2016). It is also crucial to address alcohol use during the early stage of development to prevent the development of alcohol-related diseases (e.g., cardiovascular diseases, diabetes, liver cirrhosis, and renal failure, etc.) later in life (Ramsoomar et al., 2016; Marshall, 2014). 1.2 The heart Chronic alcohol increases the incidence of cardiovascular diseases (Matzopoulos, et al., 2014; Obad, et al., 2018) which is a huge financial burden on the health systems (Matzopoulos, et al., 2014; Obad, et al., 2018). Chronic alcohol consumption changes the cardiac function and structure which may result in the weakening and loss of cardiomyocytes (Piano, 2016). In addition, the mitochondria, plasma membrane, ribosomes, and receptors of the cardiomyocytes are also damaged due to the high reactivity, low molecular mass, high diffusion rate, and the ability of alcohol to easily cross membranes (Fernández-Solà, 2020). Alcohol also disrupts signalling processes, activates apoptosis, and decreases heart contractility (Obad, et al., 2018; Fernandez- Sola, 2020). Likewise, the repair mechanism of cardiomyocytes is hindered by alcohol (by reducing regeneration and proliferation of cardiomyocytes) while promoting cardiac lesions (by increasing apoptosis and necrosis of cardiomyocytes), the outcome of which leads to myocardial fibrosis (Fernandez-Sola, 2020) and ventricular dysfunction (Wang et al., 2005). Alcohol is also 5 known to induce inflammation and promote the secretion of cytokines such as tumour necrosis factor-α (TNF-α) (Obad, et al., 2018) which is a pro-inflammatory cytokine that regulates myocardial homeostasis by signalling inflammation and cell abnormality (e.g., apoptosis) (Obad, et al., 2018). An increased level of TNF-α in chronic alcoholism is an indication of cardiac pathologies, fibrosis, and necrosis (Obad et al., 2018; Schumacher & Naga Prasad, 2018). With the prevalence of chronic alcoholism (especially in adolescents) and its associated cardiovascular diseases, it is important to investigate interventions that may mask or reduce the damaging effects of alcohol on the heart tissues. One of such interventions that is gaining popularity, due to its anti-inflammatory and immune-regulatory properties, is simvastatin (Cahyawati, 2019; Gao et al., 2015; Xiao et al., 2016). It is an FDA–approved drug, primarily, used for lowering blood cholesterol (hypercholesterolaemia) (Thabit et al., 2014; Liu et al., 2016; Morse et al., 2018; Murphy et al., 2020). It also prevents coronary heart disease (Atef et al., 2019; Murphy et al., 2020), prevents cardiomyocyte death (MacDougall et al., 2017), myocardial inflammation and fibrosis (Sun et al., 2015; Xiao et al., 2016; Lee, et al., 2019). Simvastatin stimulates autophagy by inhibiting the mTOR signalling pathway, reducing damage to the cardiomyocytes, and stimulating their regeneration (Gao, et al., 2015), and may prevent cardiac hypertrophy through an antioxidant mechanism which inhibits Rac1 geranylgeranylation (Zhou & Liao, 2009). Simvastatin also prevents myocarditis (Skrzypiec-Spring et al., 2021) and improves endothelial functions (Cahyawati, 2019; Xiao et al., 2016) as well as inhibiting TGF-β1 pathway which triggers the development of interstitial fibrosis and cardiomyocyte hypertrophy (Xiao et al., 2016). Simvastatin may be beneficial against alcohol-induced myocardial damage by preventing or potentially reversing the structural alterations caused by chronic alcohol use. Thus, this study 6 investigated the protective capabilities of simvastatin against alcohol-induced damage on the heart of adolescent mice administered chronic alcohol. Preventing and/or reversing alcohol-induced damage could alleviate the financial burden caused by alcohol-related diseases (Matzopoulos, et al., 2014). 1.3 The kidney The kidneys play an important role in the regulation of blood pressure (Leal et al., 2017). They are also responsible for metabolizing, detoxifying, and excreting alcohol, or its metabolites from the body (Wu et al., 2021) and which makes the kidneys highly vulnerable to damage by alcohol (Brzóska et al., 2003). Due to the functions of the kidneys, damage or a loss of function could negatively affect the functions of other organs, especially the heart (Rikalo & Romanenko, 2018)and it is a fact that chronic alcohol consumption increases the incidence of cardiovascular diseases (Matzopoulos, et al., 2014; Obad, et al., 2018). Chronic alcohol increases blood pressure through various mechanisms, one of which is the activation of the renin-angiotensin system (RAS) in the kidney which raises the systemic blood pressure, causes glomerular hypertension and vasoconstriction (Leal et al., 2017). The activated intrarenal RAS induced by chronic alcohol also promotes renal damage by altering the morphologies of the glomeruli, the tubules, and the renal blood vessels (Das & Vasudevan, 2008; Hu et al., 2018; Leal et al., 2017). At the same time, it prolongs hypertension which is detrimental to the heart and other organs (Fard et al., 2022; Leal et al., 2017). This inter-relationship shows that the severity of a cardiovascular disease increases the probability of renal failure and vice versa (Hu et al., 2018). Unfortunately, chronic alcohol consumption is a social problem amongst adolescents. They tend to experiment with alcohol resulting in heavy episodic drinking patterns, especially in places where there are lapses in the regulation of access to alcohol (Ramsoomar et al., 2013; Marshall, 2014; Dawodu et al., 2019). 7 These adolescents often become alcohol addicts later in life thus creating social, economic, and health problems (Ramsoomar et al., 2013; Matzopoulos, et al., 2014; Swart et al, 2015; Olsson, et al., 2016; Bertscher et al., 2020). Alcohol-induced renal damage changes the morphologies of the renal structures e.g., the glomeruli and the renal tubules causing renal functions such as glomerular filtration and tubular reabsorption to fail (Edelstein et al., 1997; Epstein, 1997; Das and Vasudevan, 2008; Rikalo and Romanenko, 2018; Wu et al., 2021). At the same time, chronic alcohol inhibits the function of antidiuretic hormone (ADH) in the kidneys thus resulting in the loss of water from the body (Epstein, 1997). Likewise, extracellular matrix deposition (i.e., renal fibrosis) between the renal tubules and the surrounding capillaries may increase in response to chronic alcohol thus delaying the oxygen supply and nutrients to the renal tubular cells (Wu et al., 2021). Like the heart, tumour necrosis factor-alpha (TNF-α) is also produced in the kidney by vascular endothelial, mesangial, and renal tubular epithelial cells (Mehaffey and Majid, 2017; Fard et al., 2022). The basal concentration of TNF-α is considerably low or undetectable under normal conditions but it sporadically increases at the onset of inflammation which makes the TNF-α an indicator of renal failure or disease as well as helping to trigger pathways for rescue or repair (Mehaffey & Majid, 2017). In addition, the ability of TNF-α to induce apoptosis may be beneficial or detrimental to the renal tissues as apoptosis may contribute to the pathogenesis of renal diseases or it may trigger cell proliferation to compensate for cell death (Ortiz, 2000). Simvastatin targets Bcl-XL, which inhibits apoptosis by preventing the release of cytochrome C from the mitochondria and targets the cell-survival signalling pathway survivin/NF-κB/p65, therefore, protecting renal tissue from lipoprotein-induced apoptosis (Nezic, et al., 2020). Simvastatin has been reported to 8 significantly reduce the fraction area of myofibroblasts and reduced myofibroblast expansion by decreasing the expression of α-SMA, which is a marker for the expansion of myofibroblasts (Cahyawati, et al., 2017). 1.4 The liver The liver metabolizes about 90% of alcohol and ensures that it is removed from the body (Hassan et al., 2015; Obad et al., 2018; Hu et al., 2020). Alcohol metabolism affects the regulation of lipids in the body (Jeon and Carr, 2020) by reducing the oxidation of fatty acids, increasing the production of fatty acids, and inhibiting the transportation of triglycerides from the liver (Cohen & Nagy, 2011; Sengupta et al., 2021). These often cause triglycerides and fatty acids to accumulate in the hepatocytes, a condition called steatosis (fat deposit). Steatosis may progress into alcoholic steatohepatitis in chronic alcoholism which is characterized by inflammation, cell death, and increased levels of liver enzymes (e.g., alanine aminotransferase and aspartate aminotransferase (Cohen and Nagy, 2011; Celli and Zhang, 2014; Duly et al., 2015; de Lucca et al., 2018; Jeon and Carr, 2020; Sengupta et al., 2021). Chronic alcohol also induces hepatic stress which then impairs liver functions as well as promotes the death of hepatocytes (Cohen and Nagy, 2011). Enlargement of hepatocytes (i.e., hydropic swelling or ballooning degeneration) is also common in steatohepatitis where the cells appear pale with a distended cytoplasm (Celli & Zhang, 2014; Hassan et al., 2015). As the alcohol damage continues, the hepatic stellate cells are activated resulting in an increase in collagen deposition and the loss of hepatic sinusoids – a condition called liver fibrosis (Cohen and Nagy, 2011). In a situation where alcohol continues to be consumed in excess, liver fibrosis may 9 eventually progress to a life-threatening condition called cirrhosis (Cohen & Nagy, 2011; de Almeida et al., 2020). Most cells including the Kupffer cells and the hepatic stellate cells produce reactive oxygen species for normal cell function or in response to pathogens (Gandhi, 2012). Following an excessive consumption of alcohol, the Kupffer cells usually become sensitized to endotoxins which cause more Kupffer cells to be activated (Cohen and Nagy, 2011). This invariably leads to the upregulation of reactive oxygen species causing the death of hepatocytes and hepatic failure (Cohen and Nagy, 2011). The activated Kupffer cells also produce TNF-α which at a non- pathological level is essential for suppressing apoptosis but promotes the proliferation and regeneration of hepatocytes (Cohen and Nagy, 2011). Based on these, it is true that chronic alcohol sensitizes the liver thus increasing its susceptibility to damage by a secondary risk factor (Cohen and Nagy, 2011). The diverse effects that statins have on the diseases of the cardiovascular system (Matzopoulos et al., 2014; Obad et al., 2018) are being proposed to be effective in modifying the progression of hepatic diseases. Several observational studies have reported on the effectiveness of statins in reducing portal hypertension, preventing hepatic dysfunction, preventing the development of hepatic carcinoma, and improving the survival of hepatocytes (la Mura et al., 2013). It has also been shown that the ability of statins to improve vascular remodelling and reactivity makes it effective in preventing the development of cirrhosis (Kaplan, 2018). Likewise, simvastatin improves hepatic steatosis by inducing autophagy and maintaining lipid homeostasis in the liver (Atef et al., 2019). Simvastatin also improved the function of endothelial tissue in hypercholesterolemic patients using the nitric oxide synthase dependent pathway (Gu et al., 2019; MacDougall et al., 2017; Zhou & Liao, 2009) as well as preventing inflammatory responses by 10 inhibiting the production of harmful by-products and excess free radicals in liver cirrhosis (Gu et al., 2019). Oxidation of alcohol by the aldehyde dehydrogenase in the liver may induce an overproduction of TNF-α, causing inflammation but simvastatin reduces the severity of hepatic inflammation through its ability to inhibit the upregulation of TNF-α expression as well as nitric oxide (Atef et al., 2019; Kolovou et al., 2006). 1.5 Rationale In reviews by Kolovou et al. (2006) and Fang et al., (2019), Simvastatin was reported to increase the production of nitric oxide to stabilize inflammation in the liver, heart, and kidney in response to acute alcohol consumption (Kolovou et al., 2006; Fang et al., 2019). Simvastatin was reported to prevent the production and secretion of TNF-α (Kolovou et al., 2006; Fang et al., 2019). This responsive mechanism provides these organs with protection against oxidative injury and ischaemia (Kolovou et al., 2006). Therefore, Simvastatin may be helpful in preventing and potentially reversing the functional and structural alterations in these organs induced by early alcohol consumption as often seen in adolescent alcoholics (Kolovou et al., 2006; Fang et al., 2019). Alcoholism and alcohol-related diseases increase the financial burden on the government (Matzopoulos et al., 2014). Preventing and/or reversing alcohol-induced damage could alleviate the burden on the health system (Matzopoulos, et al., 2014) improve the socio-economic status of the country and ultimately improve the quality of life of sufferers (Matzopoulos, et al., 2014). It is thus important to further explore the beneficial effects of Simvastatin in preventing alcohol-related damages in the heart, kidney and liver using a mouse model of adolescent alcoholism. 11 1.6 Aim and specific objectives The aim of the study is to explore the protective effects of Simvastatin against alcohol- induced damages in the heart, kidney, and liver in a mouse model of adolescent alcoholism by assessing the morphological and morphometrical changes in the different organs. The specific objectives are: A. To evaluate the protective effects of Simvastatin (reduced hypertrophy, fibrosis and inflammation) against alcohol-induced damage on the heart of adolescent chronic alcoholic mice using histology (H+E and MT) and immunohistochemistry (TNF-α) techniques. B. To evaluate the protective effects of Simvastatin (reduced glomerular swelling, fibrosis and inflammation) against alcohol-induced damage on the kidney of adolescent chronic alcoholic mice using histology (H+E and MT) and immunohistochemistry (TNF-α) techniques. C. To evaluate the protective effects of Simvastatin (improved hepatocyte density, reduced fibrosis and inflammation) against alcohol-induced damage on the liver of adolescent chronic alcoholic mice using histology (H+E and MT) and immunohistochemistry (TNF- α) techniques. 12 CHAPTER 2: MATERIALS AND METHODS 13 Chapter 2 2.0 Materials and methods 2.1 Animal study This study forms part of a broad study on the beneficial properties of the Simvastatin against alcohol-induced damage in some selected organs e.g., brain, sciatic nerve, long bones, heart, kidneys, and livers, harvested from adolescent mouse that were administered a prolonged alcohol consumption. This study was approved (Ethical Clearance No: 2019/11/63/C) by the Animal Research Ethics Committee (AREC) of the University of the Witwatersrand Johannesburg, South Africa. The mice were supplied by the National Health Laboratory Service (NHLS), Johannesburg and they were housed in the animal facility of the Witwatersrand Research Animal Facility (WRAF) of the Faculty of Health Sciences, University of the Witwatersrand. Ten (10) (50% sex ratio) C57BL/6J mice were assigned to each experimental group: (i) Non-treatment group (no administration of alcohol or simvastatin); (ii) Alcohol only group (intraperitoneal injection of 20% alcohol (2.5 g/Kg) in saline water); (iii) Simvastatin only group (5 mg/Kg oral dose in sterile water); (iv) Alcohol + Simvastatin 5 (5 mg/Kg oral dose in sterile water followed by intraperitoneal injection of 20% alcohol (2.5 g/Kg) in sterile saline water) (v) Alcohol + Simvastatin 15 (15 mg/Kg oral dose in sterile water followed by intraperitoneal injection of 20% alcohol (2.5 g/Kg) in sterile saline water). All treatments were performed for 28 consecutive days. Throughout the experimentation, the animals were handled and treated humanely. At the end of the treatment period, the mice were sacrificed using Euthanaze (sodium pentobarbital, 80 mg/Kg, i.p.) before trans-cardially perfusing with 0.9% cold saline followed by 4% paraformaldehyde in 14 0.1 M phosphate buffer (PM). The heart, kidneys and liver were immediately removed, weighed, immersed in 10% buffered formalin, and then stored at 4 °C before further processing. 2.2 Experimental study The animals were randomly allocated into five experimental groups and then allowed to acclimatize for one week, following which alcohol, simvastatin or appropriate controls were administered to the mice starting from postnatal day 28 (at 4 weeks old) to postnatal day 56 days (at 8 weeks old) according to Table 2.1. Alcohol was administered intraperitoneally while Simvastatin was administered via oral gavage. Both procedures were performed with utmost care by the trained staff of WRAF to reduce introducing stress into the animal. Mice of the same sex and belonging to the same experimental group were housed together in a group of five mice per cage (cage dimensions: 200 × 200 × 300 mm) and kept under a reversed 12–hour day/ 12–hour dark cycle (with the light switched off from 06:00 – 18:00). For this study, the period of adolescent in the mice was taken as (period between 3 – 8 weeks old) (Lespine and Tirelli, 2017). 2.2.1 Preparation and administration of simvastatin and alcohol Simvastatin (Cat no: 1612700 Merck, South Africa) was prepared according to McKay et al. (2004). 80 mg simvastatin was weighed and dissolved in a solution containing 1 ml 0.1 M sodium hydroxide (NaOH) and 1 ml absolute alcohol under a gently stirring for 1 hr at room temperature until the simvastatin had completely dissolved. The stock solution was prepared every fifth day and they were aliquoted and stored in – 4 °C until use. A serial dilution of the stock solution was then used to prepare the high concentration (i.e., 4 mg/ml in distilled water) and the low concentration (i.e., 1 mg/ml in distilled water) of simvastatin from which the final dosages (i.e., 15 mg/Kg/per day and 5 mg/Kg/per day respectively) were determined (Christensen et al., 15 2006; Mohammadi et al., 2015). These solutions were prepared daily and filtered sterilized using a 0.20–µm sterile filter under a sterile condition. Any unused solution per day was discarded. Using a serial dilution, a pharmacological grade absolute alcohol (96%) (Sigma–Aldrich, South Africa; Cat no: SAAR2233510LP) was diluted in saline (0.9% NaCl) into 20% alcohol solution which was then administered intraperitoneally to the mice at a dose of 2.5 g/kg (Maldonado-Devincci et al., 2010). The alcohol solution was also prepared daily, and the solution was filtered sterilized using a 0.20–µm sterile filter under a sterile condition. Alcohol was administered to the alcohol group (ALC) intraperitoneally and within 30 min after administering simvastatin to the alcohol + simvastatin 5 mg or 15 mg (ALC+SIM5 or ALC+SIM15) experimental groups. The injection site was alternated daily to reduce introducing stress or discomfort in the animal. To control for stress that may arise from handling or the administration of drugs in the mice, the mice in the non-treatment group (NT) were not administered alcohol or simvastatin and they were not handled throughout the experimental period (28 days) except for daily weighing or periodical cleaning of the cages which were performed by WRAF trained personnel. Alcohol was also not administered to the simvastatin 5 mg group (SIM) as it served as a control for the effect of simvastatin. Food and water were provided ad libitum to the mice, except in the NT and SIM groups, where it was withheld for 2 hrs post-intraperitoneal injection to partially control for the reduction in feeding during the period of peak alcohol intoxication in the alcohol- treated mice (Haycock and Ramsay 2009). On the last day of administration of alcohol or simvastatin, 50 μl of saphenous blood was collected through saphenous vein bleeding on the left hind legs of all the mice in all the experimental groups except in the NT group to determine the level of blood alcohol concentration (BAC) in each mouse (Bielawski and Abel, 1997). Blood was collected from the SIM group to 16 equilibrate the experimental groups, but BAC analysis was not performed on the blood from this group because they were not administered alcohol. Blood was collected, within 30 mins after the administration of alcohol, in heparinized capillary tubes and then stored at – 80 ºC before further processing. Prior to the determination of BAC, the blood in the capillary tubes were stored at 4 ºC overnight before being centrifuged with Vivaspin500© 100 μm membrane tubes (Biotech, South Africa) at 5000 rpm for 15 minutes to separate the serum. The serum was extracted and then the BAC was determined using an EnzyChrom™ Ethanol Assay Kit (BioVision, South Africa) according to the manufacturer’s recommendations. 17 Table 2.1: Summary of the different experimental groups Groups Experimental groups Abbreviations of groups No. of mice per experimental group Dose of administered substances Duration of administration (days) Female Male 1 No treatment NT 5 5 No alcohol, no Simvastatin To control for the procedures and treatments. 2 Simvastatin (5 mg/Kg) SIM 5 5 5 mg/Kg per day (in distilled water) administered by oral gavage. To obtain a baseline effect of simvastatin 28 3 Alcohol ALC 5 5 2.5 g/Kg alcohol (in saline) administered intraperitoneally 28 4 Alcohol + 5 mg/Kg SIM ALC+SIM5 5 5 Daily oral dose of 5 mg/Kg SIM (in distilled water) followed by 2.5 g/Kg alcohol (in saline) administered intraperitoneally 28 5 Alcohol + 15 mg/Kg SIM ALC+SIM15 5 5 Daily oral dose of 15 mg/Kg SIM (in distilled water) followed by 2.5 g/Kg alcohol (in saline) administered intraperitoneally 28 TOTAL 25 25 18 2.2.2 Animal perfusion and organ harvesting At the end of the experimental period, the mice were euthanized using Euthanaze (sodium pentobarbital, 80 mg/Kg, i.p.). Thereafter, the mice were transcardially perfused with 4% paraformaldehyde (in 0.1 M phosphate buffer, PB) (PFA). The thoracic and abdominal regions were cut open, then the heart, kidneys and liver were carefully harvested. They were dipped twice (in quick succession) in PB to wash-off any blood on the organs and thereafter a dry filter paper was used to mop-off any fluid on the organs. They were weighed and then post-fixed for 24 hr in 4% PFA in 0.1 M PB before further processing. For the removal of the sciatic nerve, the carcasses were placed in a supine position on a dissecting board. The skin on the left hind limb was reflected to expose the muscles of the hind limb. The thigh muscles were then split longitudinally to expose the whole length of the sciatic nerve. The nerve was gently lifted using a forceps and the section of the nerve overlying the hip joint and at its bifurcation at the popliteal fossa was carefully cut and immediately fixed in 10% buffered formalin (in 0.1M PB) and stored at 4 ºC overnight before further processing. 19 2.3 Tissue processing and morphometry of the heart The PFA–fixed heart was cut midway across the ventricles. The lower portion of the ventricles was processed for histology using an automated processing machine (Microme STP 120, Thermo Fisher Scientific, Walldorf, Germany) before embedding in paraffin wax. It was sectioned horizontally across the ventricles at 2 µm thickness using a microtome (Leica Biosystems RM2125 RTS, China). Serial sections were performed for haematoxylin and eosin (H&E) or Masson’s trichrome (MT) staining as well as TNF-α immunolabelling. For haematoxylin and Eosin or Masson’s Trichrome staining, sections were cleared in xylene to remove the paraffin wax and then hydrated in a graded series of alcohol (70%, 95% and 100%). The sections were washed in a running tap water for 5 minutes and stained with H&E (for general morphology and morphometry of the cardiomyocytes) for 5 mins or with MT (for determination of collagen deposition) for 5 mins. Thereafter, sections were dehydrated in a graded series of alcohol and then cover-slipped with entellen. For the TNF-α immunolabelling, sections were also cleared in xylene to remove the paraffin wax and hydrated in a graded series of alcohol (70%, 95% and 100%). An antigen retrieval was performed by immersing the sections in citrate buffer at 60 °C overnight. The sections were washed three times, thereafter, endogenous peroxidase activity was blocked by immersing the sections in 1% hydrogen peroxide, 49.5% methanol and 49.5% PB for 30 minutes. It was subsequently washed twice in PB before incubating in a blocking buffer solution (5% normal goat serum in PB at room temperature) for 30 minutes to block unspecified binding sites. Thereafter, the sections were incubated overnight at 4 °C in the primary antibody (1:250, mouse anti-TNF-α ab220210, Abcam, in PB). After the incubation period, the sections were washed twice in PB before incubating in the secondary antibody (1:1000 dilution of biotinylated goat anti-mouse IgG, 20 BA-9200-1.5, Vector labs, in PB). Following this, the sections were again washed twice in PB and then incubated for 1 hr in an avidin-biotin solution (25 µl advin + 25 µl biotin + 1.25 ml PBS, 1:125; Vector Labs). The sections were further washed twice in PB and then immersed in a DAB working solution containing 0.05% DAB (3,3’-Diaminobenzidine), 2 ml Tris HCl, 29 µl cold distilled water and 1 µl hydrogen peroxide for 10 mins and the reaction was terminated by adding an equal volume of PB. The slides were rinsed in running tap water before counterstaining with haematoxylin. Thereafter, the sections were dehydrated in a graded series of alcohol, cleared in xylene and cover-slipped with entellen. To rule out non-specific staining of the immunohistochemical protocol, tests were conducted on the sections where the primary or the secondary antibody was omitted. In both cases no staining was observed (results not shown). For the morphometries of the cardiomyocytes, images of the myocardium along the length of the left ventricular wall of the H&E–stained sections were taken using an Olympus EP50 camera (Serial No. 3H23424, Japan) attached to an Olympus BX41 microscope (Model BX41TF, Olympus Corporation, Tokyo, Japan) at times 100 objective lens (under oil immersion). The left ventricular wall was identifiable as the larger of the two ventricular walls. The inter-ventricular wall was not considered in this study. With the scale set on the software, the area, and the diameter of cardiomyocytes (Harash et al., 2019) were measured from the digitized images using an EPview software (EPview 1.3, Build 19857). The diameter of the long axis of a cardiomyocyte was measured across the level of the nucleus with the line passing through the nucleolus using the straight-line tool of the software (Baudouy et al., 2017; Harash et al., 2019). In addition, the border of cardiomyocyte was carefully traced using the polygonal tool of the software and then the area of each cardiomyocyte was analysed by the same software (Baudouy et al., 2017; Harash et al., 21 2019). The field of view was changed by moving the microscope stage to prevent duplicating measurements. In total, a minimum of 100 cardiomyocytes were measured per mouse. To determine the distribution of collagen deposition in the heart tissue, digitized images of the left ventricular wall of the MT–stained sections were taken using a Carl Zeiss Axiocam 208 color camera (Serial No. 5318003446, China) attached to a Carl Zeiss Axioskop 2 microscope (Serial No. 804161, Germany) at times 40 objective lens. The images were subsequently saved in a JPEG file format. Similar to the cardiomyocyte morphometry, the field of view was changed by moving the microscope stage along the length of the left ventricular wall. The distribution of collagen within the myocardium was determined using the deconvolution plugin settings on an ImageJ software (ImageJ 1.53q/Java 1.8.0_322, Oracle America Inc.) (Chen et al., 2017; Latiff and Olateju, 2022). The 24-bit RGB format was selected as a requirement for the deconvolution plugin setting in the ImageJ software where the green component on the processed image indicated collagen stain (Chen et al., 2017; Latiff and Olateju, 2022). Collagen distribution on each image was quantified using the threshold tool on the ImageJ software which was adjusted until all the collagen (i.e., green) stain had been highlighted (Chen et al., 2017; Latiff and Olateju, 2022). The percentage collagen distribution per image was calculated as the threshold area divided by the area of the image. Similar to the analyses used for the MT-stain sections, the percentage distribution of the TNF-α positive immunolabelling of the myocardium was performed using digitized images at 40 times objective lens. The field of view was changed, and the digitized images were also saved in the JPEG file format before processing with ImageJ software. Using the 24-bit RGB format, the DAB staining was selected for the deconvolution plugin setting on ImageJ software (Balzano et al., 2020) where the brown component was identified as the DAB staining (Balzano et al., 2020). 22 The distribution of TNF-α immunolabelling in each image was quantified by adjusting the threshold tool of ImageJ until all the DAB stain had been highlighted (Balzano et al., 2020). Thereafter, the percentage distribution of the TNF-α immunolabelling was determined by dividing the threshold area by the area of the image. 2.4 Tissue processing and morphometry of the kidneys The PFA–fixed right kidney was cut horizontally at its equator and then the inferior half of the tissue processed for H&E and MT staining as well as TNF-α immunolabelling using the same antibody and dilutions as described in 2.3. For the kidney, the sections were made at 5 µm thickness. Serial sections were prepared for haematoxylin and eosin (H&E) (for general morphology and morphometries of renal corpuscles and the glomeruli), Masson’s trichrome (MT) (for evaluating collagen deposition i.e., fibrosis around the renal tubules and the renal corpuscles) or TNF-α immunolabelling with incubation in primary antibody (1:250, mouse anti-TNF-α, ab220210, Abcam, in PB) (for quantifying the expression of TNF-α in the renal tissue). For the morphometries of the renal corpuscles and the glomeruli, images along the length of the renal cortex of the H&E–stained sections were taken using a Carl Zeiss Axiocam 208 colour camera (Serial No. 5318003446, China) attached to a Carl Zeiss Axioskop 2 microscope (Serial No. 804161, Germany) at times 63 objective lens. The field of view was randomized by moving the microscope stage to prevent duplicating measurements. With the scale set on the ImageJ software (ImageJ 1.53q/Java 1.8.0_322, Oracle America Inc.), the areas of the renal corpuscle and the glomerulus were measured from the digitized images by tracing the boundary of the parietal layer of the Bowman’s capsule and the boundary of the glomerulus using the freehand tool of the 23 software before the areas were then analysed. The area of the Bowman’s space was then calculated by subtracting the area of the glomerulus from the area of the renal corpuscle (Fernandes et al., 2019). To determine the collagen distribution in the kidney tissue, digitized images of the MT– stained sections were taken using a Carl Zeiss Axiocam 208 color camera attached to a Carl Zeiss Axioskop 2 microscope at times 40 objective lens. The images were subsequently saved in a JPEG file format. Similar to the renal corpuscle morphometry, the field of view was also randomised by moving the microscope stage along the length of the renal cortex. The distribution of collagen within the kidney tissue was determined using the deconvolution plugin settings on the ImageJ software (Chen et al., 2017; Latiff and Olateju, 2022). The 24-bit RGB format was selected as a requirement for the deconvolution plugin setting in the software where the green component on the processed image indicates collagen stain (Chen et al., 2017; Latiff and Olateju, 2022). Collagen distribution on each image was quantified using the threshold tool on the software which was adjusted until all the collagen (i.e., green) stain had been highlighted (Chen et al., 2017; Latiff and Olateju, 2022). The percentage collagen distribution per image was calculated as the threshold area divided by the area of the image. Like the analyses used for the MT-stain sections, the percentage distribution of the TNF-α positive immunolabelling of the renal tubules or the renal corpuscles were performed using digitized images at 63 times objective lens. The field of view was also randomised, and the digitized images saved in the JPEG file format. Using the 24-bit RGB format, the region of interest (ROI) manager was used to select the area of the renal corpuscle or the tubules and the size of the ROI (620368 µm2) was kept constant throughout the analyses (Balzano et al., 2020). The ROI was then duplicated so that only the areas of the renal corpuscle and the tubules were used for 24 deconvolution. The DAB staining was selected for the deconvolution plugin setting on the ImageJ software where the brown component was identified as the DAB staining. The distribution of TNF- α immunolabelling in each ROI was quantified by adjusting the threshold tool of ImageJ until all the DAB stain had been highlighted (Balzano et al., 2020). Thereafter, the percentage distribution of the TNF-α immunolabelling was determined by dividing the threshold area by the area of the region of interest. 2.5 Tissue processing and morphometry of the liver The PFA–fixed left lateral lobe (the largest lobe) of the liver was cut and processed for H&E and MT staining as well as TNF-α immunolabelling using the same antibody and dilutions as described in 2.3. For the liver, the sections were made at 10 µm thickness across the flatter surface of the liver. Serial sections were performed for haematoxylin and eosin (H&E) or Masson’s trichrome (MT) staining as well as TNF-α immunolabelling. For the morphometries of the hepatocytes, images along the length of the liver of the H&E– stained sections were taken using a Carl Zeiss Axiocam 208 color camera (Serial No. 5318003446, China) attached to a Carl Zeiss Axioskop 2 microscope (Serial No. 804161, Germany) at times 10 objective lens. With the scale set on the software, the number of hepatocytes per grid were counted (Matsuo et al., 2016) from the digitized images using the ImageJ software (ImageJ 1.53q/Java 1.8.0_322, Oracle America Inc.). A grid (31560 µm2) was placed on the image, and then the hepatocytes were counted (Matsuo et al., 2016). Cells that were touching the boundaries of the grids were not counted. The field of view was changed by moving the microscope stage at every 1–mm interval to prevent duplicating measurements. 25 To determine the distribution of collagen deposition in the liver tissue, digitized images of the length of the liver of the MT–stained sections were taken using a Carl Zeiss Axiocam 208 color camera (Serial No. 5318003446, China) attached to a Carl Zeiss Axioskop 2 microscope (Serial No. 804161, Germany) at times 40 objective lens. The images were subsequently saved in a JPEG file format. Similar to the liver morphometry, the field of view where images were taken was also randomised by moving the microscope stage at every 1–mm interval along the length of the liver. The distribution of collagen within the liver tissue was determined using the deconvolution plugin settings on an ImageJ software (ImageJ 1.53q/Java 1.8.0_322, Oracle America Inc.) (Chen et al., 2017; Latiff and Olateju, 2022). The 24-bit RGB format was selected as a requirement for the deconvolution plugin setting in the ImageJ software where the green component on the processed image indicates collagen stain (Chen et al., 2017; Latiff and Olateju, 2022). Collagen distribution on each image was quantified using the threshold tool on the ImageJ software which was adjusted until all the collagen (i.e., green) stain had been highlighted (Chen et al., 2017; Latiff and Olateju, 2022). The percentage collagen distribution per image was calculated as the threshold area divided by the area of the image multiplied by 100. Similar to the analyses used for the MT-stain sections, the percentage distribution of the TNF-α positive immunolabelling of the length of the liver was performed using digitized images at 40 times objective lens. The field of view was randomised similar to the H&E–stained sections and the digitized images were also saved in the JPEG file format. Using the 24-bit RGB format, the H-DAB staining was selected for the deconvolution plugin setting on ImageJ software (Balzano et al., 2020) where the brown component was identified as the DAB staining (Balzano et al., 2020). The distribution of TNF-α immunolabelling in each image was quantified by adjusting the threshold tool of ImageJ until all the DAB stain had been highlighted (Balzano et al., 26 2020). Thereafter, the percentage distribution of the TNF-α immunolabelling was determined dividing the threshold area by the area of the image and then multiplying by 100. 2.6 Statistical analysis A descriptive statistic using mean (± SD) and median were performed. The data was not normally distributed, thus the median of all measurements in the different experimental groups were compared using a Kruskal–Wallis test followed by a Dunn’s post hoc test. All statistical tests were performed using a PAST freeware data analyser (version 4.03; Germany) and graphs were plotted using the Excel software (Word Office Pro, USA). Statistical difference of 5% was regarded as significant for all the statistical analyses. 27 CHAPTER 3: RESULTS 28 Chapter 3 3.0. Results 3.1.1 Animal and organ weights Table 3.1: Summary of animal and organ weights Experimental Group Animal mass (mg) Mean±SD Heart mass (mg) Mean±SD Combined kidney mass (mg) Mean±SD Liver mass (mg) Mean±SD NT 16.7 ± 2.78 0.25 ± 0.078 0.882 ± 0.470 0.347 ± 0.476 SIM 16.6 ± 2.139 0.214 ± 0.088 0.704 ± 0.493 0.614 ± 0.546 ALC 16.3 ± 2.263 0.296 ± 0.040 1.179 ± 0.333 0.136 ± 0.040 ALC+SIM5 16 ± 2.345 0.245 ± 0.103 1.026 ± 0.616 0.343 ± 0.346 ALC+SIM15 15.6 ± 1.595 0.261 ± 0.100 1.01 ± 0.494 0.327 ± 0.041 29 3.1.2 Blood alcohol concentration The average blood alcohol concentration across the ALC, ALC+SIM5 and ALC+SIM15 ranged from 27.02 to 509.76 mg/dL. The average blood alcohol concentration at the time of termination in the ALC was 244.14 mg/dL, 253.36 mg/dL in the ALC+SIM5, and 182.51 mg/dL in the ALC+SIM15. The BAC values confirm the presence of alcohol in the blood of the animals in the ALC, ALC+SIM5 and ALC+SIM15 experimental groups. 30 3.2 The heart 3.2.1 General morphology of the cardiomyocytes There were no changes observed in the morphology of the cardiomyocytes across all treatment groups. The layers of the ventricular wall were present in all treatment groups. The cardiomyocytes were elongated, with a nucleus and a nucleolus. There were changes in the size of the cardiomyocytes that were observed across the treatment groups. The cardiomyocytes in the ALC appeared enlarged compared to the other treatment groups. The myocardium in the ALC has a significantly increased collagen deposition compared to the other treatment group. The myocardium in the NT showed little to no TNF-α expression while the myocardium in the ALC showed intense TNF-α expression. 31 Figure 3.1: Representative micrographs of the ventricles of the heart demosntrating the layers of the heart wall of a heart from the NT treatment group. LV= left ventricle, RV= right ventricle, LVW= left ventricular wall, RVW= right ventricular wall, Epi= epicardium, Myo= myocardium, Endo= endocardium. 32 Figure 3.2: Representative micrographs of cardiomyocytes in the left ventricle. A.1 and B.1 demonstrate how the area and lengths of the cardiomyocytes were measured; A.2 and B.2 show the same cells in the left ventricular wall. A.1 and B.1. Area and length measurements of the cardiomyocytes from the left ventricle, A.2. cardiomyocyte from left ventricular wall of NT heart, B.2. cardiomyocyte from left ventricular wall of ALC heart. NT= non-treatment group, ALC= alcohol only group. 33 Figure 3.3: Representative micrographs of the heart demonstrating the levels of collagen in the left ventricular walls of (a) a control heart and (b, c, d) treated hearts. a. Myocardium from the NT left ventricle showing little collagen distribution, b. Myocardium from the ALC left ventricle showing increased collagen deposition, c. Myocardium from the ALC+SIM5 left ventricle showing little collagen distribution, d. Myocardium from the SMI left ventricle showing no collagen distribution. Collagen stain [arrow]. NT= non-treatment group, ALC= alcohol only group. 34 Figure 3.4: Representative micrographs of the heart demonstrating the levels of inflammation in the left ventricular walls of (a) a control heart and (b, c, d) treated hearts. a. Myocardium from the NT showing minimal TNF-α expression as indicated by the faint brown DAB stain, b. Myocardium from the ALC showing intense TNF-α expression as indicated by the darker shade of brown DAB stain, c. Myocardium from the ALC+SIM15 showing a moderate degree of TNF-α expression as indicated by the brown DAB stain, d. Myocardium from the SIM showing minimal TNF-α expression as indicated by the faint brown DAB stain. NT= non-treatment, ALC= alcohol only group. 35 3.2.2 Measurements of the cardiomyocyte area and diameter The descriptive statistics for the cardiomyocyte diameter and area across the different experimental groups and for both sexes are shown in Table 3.2. In the females, the cardiomyocyte area and the diameter was the highest in the ALC but was lowest in the ALC+SIM5. A Kruskal-Wallis test revealed that the cardiomyocyte area and diameter was significantly different across the experimental groups (P<0.000). A Dunn’s post hoc revealed that the area of cardiomyocytes in any paired groups was significantly different (P<0.000) (Fig. 3.5 and Fig. 3.6). The significant increase in cardiomyocyte area and diameter in the ALC compared to the NT validates the damaging effect of chronic alcohol on the cardiomyocytes. Alcohol-induced cardiomyocyte hypertrophy was significantly reduced in the ALC+SIM5 and in the ALC+SIM15. In addition, 5 mg simvastatin (ALC+SIM5) seems to be more effective against alcohol-induced cardiac hypertrophy than 15 mg simvastatin (ALC+SIM15). In the males, the cardiomyocyte area or diameter was also highest in the ALC but was lowest in the ALC+SIM15 (Table 3.2). A Kruskal-Wallis test revealed that the cardiomyocyte area or diameter was significantly different across the experimental groups (P<0.000). A Dunn’s post hoc revealed that the area of cardiomyocytes in any paired group was significantly different (P<0.000) (Fig. 3.5 and Fig. 3.6). The significant increase in cardiomyocyte area or diameter in the ALC compared to the NT also validates the damaging effect of chronic alcohol on the cardiomyocytes of adolescent mouse. Alcohol-induced cardiomyocyte hypertrophy was significantly reduced in the ALC+SIM5 and in the ALC+SIM15. In contrary to the findings in the females, 15 mg simvastatin (ALC+SIM15) seems to be more effective against alcohol-induced cardiac hypertrophy than 5 mg simvastatin (ALC+SIM5) in the males. 36 Table 3.2: Summary of the morphometries of the cardiomyocyte area and the diameter Cardiomyocyte area Cardiomyocyte diameter Collagen distribution TNF-α distribution No of animals No of cardiomy ocytes assessed Mean ± SD (µm) Median (µm) No. of cardiomyocytes assessed Mean ± SD (µm) Median (µm) No of images assessed Mean ± SD (µm) Median (µm) No of image s assess ed Mean ± SD (%) Median (%) Female NT 5 500 229.94 ± 95.27 226.31 500 28.66 ± 8.02 28.00 43 4.66 ± 2.95 4.57 20 8.74 ± 2.63 8.91 SIM 5 500 189.27 ± 79.37 180.17 500 24.47 ± 7.11 23.95 45 8.75 ±3.73 8.40 39 9.94 ± 6.25 8.69 ALC 5 500 346.21 ±105.54 329.83 500 35.51 ± 8.44 35.36 39 13.37 ±4.77 13.62 40 36.40 ± 4.35 37.03 ALC+SIM 5 5 500 170.91 ±76.23 156.28 500 23.16 ± 7.07 22.11 43 9.70 ±7.04 7.83 41 9.83 ± 3.84 10.02 ALC+SIM 15 5 500 179.15 ± 72.10 168.46 500 26.37 ± 7.38 25.50 41 4.32 ±2.67 3.81 41 24.50 ± 13.93 22.17 Male NT 5 500 270.75 ± 85.52 259.13 500 30.07 ± 7.85 29.73 43 8.25 ±3.66 8.84 40 11.58 ± 6.54 10.10 SIM 5 500 210.69 ± 78.77 193.97 500 28.00 ± 8.44 26.50 37 7.32 ±4.04 6.32 32 14.78 ± 8.03 13.67 ALC 5 500 289.32 ± 104.75 265.80 500 32.95 ± 9.96 31.41 38 13.96 ±4.69 13.52 40 39.13 ± 6.55 40.46 ALC+SIM 5 5 500 223.74 ± 85.25 213.88 500 25.27 ± 7.06 24.50 39 4.78 ±3.03 3.57 40 31.28 ± 11.08 30.87 ALC+SIM 15 5 500 184.02 ± 65.11 173.10 500 25.55 ± 6.89 24.94 39 13.70 ±5.93 13.84 40 24.72 ± 9.71 25.86 37 Figure 3.5: Box plots of the cardiomyocyte area across the different experimental groups and for both sexes. In both sexes, cardiomyocyte was significantly higher in the ALC than in the other groups thus confirming alcohol-induced myocardial hypertrophy. Lower dosage of simvastatin was more effective against alcohol-induced myocardial hypertrophy in females while a higher dosage of simvastatin concentration significantly reduced alcohol-induced myocardial hypertrophy in males. This seems to indicate a sex-specific differences in the effects of simvastatin against alcohol-induced myocardial hypertrophy. NT = Non-treatment; SIM = Simvastatin; ALC = Alcohol; ALC+SIM5 = 5 mg simvastatin and alcohol; ALC+SIM15 = 15 mg simvastatin and alcohol. NS = Not significant at P>0.05. 38 Figure 3.6: Box plots of the cardiomyocyte diameter across the different experimental groups and for both sexes. In both sexes, cardiomyocyte was significantly higher in the ALC than in the other groups thus confirming alcohol-induced myocardial hypertrophy. Lower dosage of simvastatin was more effective against alcohol-induced myocardial hypertrophy in females while both doses of simvastatin concentration significantly reduced alcohol-induced myocardial hypertrophy in males (p = 0,623). This seems to indicate a sex-specific differences in the effects of simvastatin against alcohol-induced myocardial hypertrophy. NT = Non-treatment; SIM = Simvastatin; ALC = Alcohol; ALC+SIM5 = 5 mg simvastatin and alcohol; ALC+SIM15 = 15 mg simvastatin and alcohol. NS = Not significant at P>0.05. 39 3.2.3 Measurement of collagen distribution In the females, the collagen distribution (%) was highest in the ALC and was lowest in the NT (Table 3.2). A Kruskal-Wallis test revealed that the collagen distribution was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that collagen distribution in any paired groups was significantly different (P<0.000) but was similar in NT vs ALC+SIM15 (P=0.639) or SIM vs ALC+SIM5 (P=0.799) (Fig. 3.7). In addition, collagen distribution was significantly higher in the ALC than in the NT (P=0.000), SIM (P=0.002), ALC+SIM5 (P=0.001) or ALC+SIM15 (P=0.000) but it was significantly lower in the NT than in the SIM (P=0.000) as well as SIM was significantly lower than in the ALC+SIM15 (P=0.000). Similar to the cardiomyocyte hypertrophy, the significant reduction in collagen distribution in the NT than in the ALC also validates an alcohol-induced myocardial fibrosis. Similarly, both concentrations of simvastatin were effective against alcohol-induced myocardial fibrosis. In the males, the collagen distribution (%) was also highest in the ALC but was lowest in the ALC+SIM5 (Table 3.2). A Kruskal-Wallis test revealed that the collagen distribution was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the collagen distribution was also significantly higher in the ALC than in the NT (P=0.000), SIM (P=0.000) or ALC+SIM5 (P=0.000). Collagen distribution was also significantly higher in the SIM than in the ALC+SIM5 (P=0.013) but significantly lower than in the ALC+SIM15 (P=0.000). However, collagen distribution was similar in NT vs SIM (P=0.409) or ALC vs ALC+SIM15 (P=0.708) (Fig. 3.7). The significant reduction in collagen distribution in the NT than in the ALC also validates an alcohol-induced myocardial fibrosis. Surprisingly, 5 mg simvastatin was effective against alcohol-induced myocardial fibrosis to levels that are significantly lower than NT or SIM, 40 but 15 mg simvastatin had no benefit against alcohol-induced damage in the males unlike in the females. Figure 3.7: Box plots of the percentage collagen distribution across the different experimental groups and for both sexes. In both sexes, collagen distribution was significantly higher in the ALC than in the other groups thus confirming alcohol-induced myocardial fibrosis. Low and high simvastatin concentrations significantly reduced alcohol-induced myocardial fibrosis in the females but only the low simvastatin concentration was effective in the males. This seems to indicate a sex-specific differences in the effects of simvastatin against alcohol-induced myocardial fibrosis. NT = Non-treatment; SIM = Simvastatin; ALC = Alcohol; ALC+SIM5 = 5 mg simvastatin and alcohol; ALC+SIM15 = 15 mg simvastatin and alcohol. NS = Not significant at P>0.05. 41 3.2.4 Measurement of TNF-α distribution In the females, the TNF-α distribution was highest in the ALC but was lowest in the NT (Table 3.2). A Kruskal-Wallis test revealed that the TNF-α distribution was significantly different across the experimental groups (P=0.000) while a Dunn’s post hoc revealed that the TNF-α distribution in any paired groups was significantly different except for NT vs SIM (P=0.751), SIM vs ALC+SIM5 (P=0.718) or NT vs ALC+SIM5 (P=0.538) (Fig. 3.8). The significant reduction in TNF-α distribution in the NT than in the ALC also validates an alcohol-induced myocardial inflammation. Both concentrations of simvastatin were effective against alcohol-induced myocardial inflammation, but the low simvastatin concentration was more effective than the high simvastatin concentration against alcohol-induced myocardial inflammation. In the males, the TNF-α distribution was highest in the ALC but was lowest in the NT like in the females (Table 3.2). A Kruskal-Wallis test revealed that the TNF-α distribution was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the TNF-α distribution in any paired groups was significantly different except NT vs SIM (P=0.267) or ALC+SIM5 vs ALC+SIM15 (P=0.049) (Fig. 3.8). The significant reduction in TNF- α distribution in the NT than in the ALC also validates an alcohol-induced myocardial inflammation. Both concentrations of simvastatin were effective against alcohol-induced myocardial inflammation unlike in the females. 42 Figure 3.8: Box plots of the percentage TNF-α distribution across the different experimental groups and for both sexes. In both sexes, TNF-α distribution was significantly higher in the ALC than in the other groups thus confirming alcohol toxicity on the cardiomyocytes. TNF-α distribution was improved in the ALC+SIM5 in the females to the level of NT but not in the ALC+SIM15. However, either ALC+SIM5 or ALC+SIM15 did not significantly lower TNF-α distribution to the level of the NT in the males. This is an indication of sex-specific differences in the effect of simvastatin again alcohol effects in the cardiomyocytes. NT = Non-treatment; SIM = Simvastatin; ALC = Alcohol; ALC+SIM5 = 5 mg simvastatin and alcohol; ALC+SIM15 = 15 mg simvastatin and alcohol. NS = Not significant at P>0.05. 43 3.3 The Kidney 3.3.1 General morphology of the renal corpuscles and tubules The morphology of the tubules and renal corpuscles from the NT group was normal i.e., the tubular epithelial brush border was intact, and the glomeruli were neither swollen nor retracted. The tubular epithelia from the ALC group showed loss of brush border and the glomeruli were swollen. The NT and SIM groups had no interstitial fibrosis, while most of the sections from the ALC group showed tubulointerstitial fibrosis. The NT group showed little to no TNF-α expression in the renal corpuscles and tubules while the renal corpuscles and tubules in the ALC showed intense TNF-α expression. 44 Figure 3.9: Representative micrograph of the cross-section of the kidney demonstrating the cortex and medulla regions. C= cortex region, M= medulla region. 45 Figure 3.10: Representative micrographs of the kidney demonstrating normal renal morphology and renal tissue with pathology. a and b. Kidneys from NT showing normal morphology, c. Kidney from ALC showing a loss of brush border, d. Kidney from ALC showing a swollen glomerulus and a loss of brush border. NT= non-treatment group, ALC= alcohol only group, BB- brush border. 46 Figure 3.11: Representative micrograph of the kidney demonstrating how the measurements of the glomerulus and renal corpuscle was obtained, and a demonstration of tubular morphology. a. Tracing of renal corpuscle and glomerular area measurements, b. Renal tubule morphology. RC= renal corpuscle area, GL= glomerular area, RT= renal tubules. 47 Figure 3.12: Representative micrographs of the kidney demonstrating the differences in collagen distribution in normal tissue and alcohol-treated tissue. a. Kidney from the NT showing no collagen deposition in the tubules and the interstitium, b and b.1. Kidneys from the ALC showing increased collagen deposition in the tubules and interstitium i.e., tubulointerstitial fibrosis, c. Kidney from ALC+SIM5 showing minimal collagen deposition in the tubules, d. Kidney from ALC+SIM15 showing minimal collagen deposition in the tubules and no collagen deposition in the interstitium, e. Kidney from SIM showing no collagen deposition in the tubules and the interstitium. NT= non-treatment, ALC= alcohol only group. 48 Figure 3.13: Representative micrographs of the kidney demonstrating the levels of inflammation in the renal tissues from different treatment groups. a. NT kidney showing minimal TNF-α expression in the tubules and corpuscles, b. ALC kidney showing intense TNF-α expression in the tubules and corpuscles, c. ALC+SIM5 kidney showing moderate TNF-α expression in the tubules and corpuscles, d. ALC+SIM15 kidney showing minimal TNF-α expression in the tubules and corpuscles. NT= non-treatment, ALC= alcohol only group, ALC+SIM5= alcohol and simvastatin 5 mg, ALC+SIM15= alcohol and simvastatin 15 mg. 49 3.3.2 Morphometry of the area of renal corpuscle In the females, the area of renal corpuscle was highest in the ALC and was lowest in the ALC+SIM5 (Table 3.3). A Kruskal-Wallis test revealed that the area of renal corpuscle was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the area of renal corpuscle was significantly higher in the ALC than in the NT (P=0.001) or the ALC+SIM5 (P=0.000). However, the area of renal corpuscle was similar in the NT vs SIM (P=0.155), the NT vs ALC+SIM5 (P=0.132), SIM vs ALC (P=0.073) or ALC vs ALC+SIM15 (P=0.140) (Fig. 3.14). The significant difference between the ALC vs NT validates the toxicity of alcohol on the size of the renal corpuscle. 5 mg simvastatin significantly reduced the effect of alcohol on the size of the renal corpuscle but not 15 mg simvastatin. In contrary to the females, the area of renal corpuscle was highest in the ALC+SIM15 and was lowest in the ALC+SIM5 in the males (Table 3.3). A Kruskal-Wallis test revealed that the renal corpuscle area was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the area of the renal corpuscle in any paired groups was significantly different except the SIM vs ALC+SIM5 (P=0.058) or ALC vs ALC+SIM15 (P=0.117) (Fig. 3.14). The significant difference between the ALC vs NT (P=0.012) also validates the toxicity of alcohol on the area of renal corpuscle. Similar to the females, 5 mg simvastatin significantly reduced the effect of alcohol on the size of the renal corpuscle but not 15 mg simvastatin. 3.3.3 Morphometry of the glomerular area In the females, the glomerular area was highest in the ALC and was lowest in the ALC+SIM5 (Table 3.3). A Kruskal-Wallis test revealed that the glomerular area was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the 50 glomerular area in any paired groups was significantly different except the NT vs SIM (P=0.557) or the NT vs ALC+SIM5 (P=0.188) or SIM vs ALC+SIM5 (P=0.057) (Fig. 3.15). In addition, the glomerular area was significantly higher in the ALC than in the NT (P=0.000), SIM (P=0.000), ALC+SIM5 (P=0.000) or ALC+SIM15 (P=0.001). The significant difference between ALC vs NT also validates the toxicity of alcohol on the glomerular area. Both concentrations of simvastatin were effective against the alcohol effect on glomerular area however, 5 mg simvastatin seems to be more effective than 15 mg simvastatin. In the males, the glomerular area was highest in the ALC+SIM15 and was lowest in the ALC+SIM5 (Table 3.3). A Kruskal-Wallis test revealed that the glomerular area was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the area of the glomerulus in any paired groups was significantly different except for SIM vs ALC+SIM5 (P=0.856) or ALC vs ALC+SIM15 (P=0.139) (Fig. 3.15). The significant difference between ALC vs NT also validates the toxicity of alcohol on the glomerular area. In contrary to the females, only the lower concentration of simvastatin (5 mg) was effective against the alcohol effect on the glomerular area. 3.3.4 Morphometry of the area of the urinary space In the females, the area of urinary space was highest in the SIM and was lowest in the ALC (Table 3.3). A Kruskal-Wallis test revealed that the area of urinary space was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the area of the urinary space was significantly lower in the ALC than in the NT (P=0.000), SIM (P=0.000), ALC+SIM5 (P=0.000) or ALC+SIM15 (P=0.001). However, there was no significant difference in the area of urinary space in the NT vs SIM (P=0.085) or the NT vs ALC+SIM5 (P=0.101) (Fig. 3.16). The significant difference between ALC vs NT validates the toxicity of alcohol on the area of urinary 51 space. Both concentrations of simvastatin were effective against the alcohol effect on the area of urinary space however, 5 mg simvastatin seems to be more effective than 15 mg simvastatin. In the males, the area of urinary space was highest in the SIM and was lowest in the ALC+SM15 (Table 3.3). A Kruskal-Wallis test revealed that the urinary space area was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the area of urinary space was significantly lower in the ALC than in the NT (P=0.000), SIM (P=0.000) or ALC+SIM5 (P=0.005) but not in the NT vs SIM (P=0.687) or the ALC vs ALC+SIM15 (P=0.449) (Fig. 3.16). The significant difference between the ALC vs NT also validates the toxicity of alcohol on the area of urinary space. In contrary to the females, only the lower concentration of simvastatin (5 mg) was effective against the alcohol effect on the area of urinary space in the males. 52 Table 3.3: Summary of the morphometries of the renal corpuscle, glomerular and urinary space No of animals No of images assessed Renal corpuscle area Glomerular area Urinary Space area Mean (± SD) (μm) Median (μm) Mean (± SD) (μm) Median (μm) Mean (± SD) (μm) Median (μm) Female NT 5 269 845.47 (±380.61) 799.91 577.55 (±260.94) 560.64 267.92 (±163.05) 228.66 SIM 5 269 857.75 (±337.09) 828.23 574.32 (±230.79) 569.94 283.42 (±148.15) 247.99 ALC 5 268 902.97 (±306.31) 856.60 710.14 (±242.61) 670.43 192.84 (±113.00) 166.11 ALC+SIM5 5 270 786.936 (±291.34) 766.83 545.28 (±218.28) 516.37 243.17 219.11 (±121.72) ALC+SIM15 5 262 880.54 (±330.33) 820.07 642.62 (±240.89) 615.23 237.92 (±160.28) 189.13. Male NT 5 259 857.30 (±306.82) 791.63 601.46 (±236.43) 568.06 255.84 (±116.24) 237.58 SIM 5 260 808.62 (±414.51) 762.49 528.72 (±262.39) 524.96 279.90 (±184.92) 240.31 ALC 5 265 917.64 (±288.23) 885.31 726.15 (±246.20) 704.54 191.49 (±103.41) 179.08 ALC+SIM5 5 269 754.59 (±351.60) 692.95 525.04 (±254.04) 487.06 229.55 (±144.06) 199.47 ALC+SIM15 5 264 966.82 (±314.27) 922.38 755.08 (±242.84) 734.53 211.74 (±144.11) 173.81 53 Figure 3.14: Box plots of the area of renal corpuscle across the different experimental groups and for both sexes. In both sexes, the area of renal corpuscle was significantly higher in the ALC than in the NT thus confirming alcohol-induced renal damage. Low simvastatin concentration (5 mg) significantly reduced the effect of alcohol on the area of renal corpuscle but not the high concentration of simvastatin (15 mg) in both sexes. NT = Non-treatment; SIM = Simvastatin; ALC = Alcohol; ALC+SIM5 = 5 mg simvastatin and alcohol; ALC+SIM15 = 15 mg simvastatin and alcohol. NS = Not significant at P>0.05. 54 Figure 3.15: Box plots of the glomerular area across the different experimental groups and for both sexes. In both sexes, the glomerular area was significantly higher in the ALC than in the NT thus confirming alcohol-induced renal damage. In the females, both concentrations of simvastatin were effective against alcohol effect on glomerular area however only the lower concentration of simvastatin (5 mg) was effective in the males. This is an indication of a sex- specific differences in the effect of simvastatin against alcohol toxicity on the renal tissues. NT = Non-treatment; SIM = Simvastatin; ALC = Alcohol; ALC+SIM5 = 5 mg simvastatin and alcohol; ALC+SIM15 = 15 mg simvastatin and alcohol. NS = Not significant at P>0.05. 55 Figure 3.16: Box plots of the area of urinary space across the different experimental groups and for both sexes. In both sexes, the glomerular area was significantly lower in the ALC than in the NT thus confirming alcohol-induced renal damage. In the females, both concentrations of simvastatin were effective against alcohol effect on the area of urinary space however only the lower concentration of simvastatin (5 mg) was effective in the males. This is an indication of a sex-specific differences in the effect of simvastatin against alcohol toxicity on the renal tissues. NT = Non-treatment; SIM = Simvastatin; ALC = Alcohol; ALC+SIM5 = 5 mg simvastatin and alcohol; ALC+SIM15 = 15 mg simvastatin and alcohol. NS = Not significant at P>0.05. 56 3.3.5 Measurement of collagen distribution In the females, the percentage collagen distribution in the kidney was highest in the ALC and was lowest in the NT (Table 3.4). A Kruskal-Wallis test revealed that the collagen distribution was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the collagen distribution was significantly higher in the ALC than in the NT (P=0.000), SIM (P=0.000), ALC+SIM5 (P=0.000) or ALC+SIM15 (P=0.000) (Fig 3.17). The significant difference between ALC vs NT validates alcohol-induced fibrosis in the kidney. Both concentrations of simvastatin were significantly effective against alcohol-induced renal fibrosis, but the higher simvastatin concentration seems to be more effective. In the males, the percentage collagen distribution in the kidney was highest in the ALC and was lowest in the NT (Table 3.4). A Kruskal-Wallis test revealed that the collagen distribution was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the collagen distribution was significantly higher in the ALC than in the NT (P=0.000), SIM (P=0.000), ALC+SIM5 (P=0.000) or ALC+SIM15 (P=0.000) except in the SIM vs ALC+SIM15 (P=0.163) (Fig. 3.17). The significant difference between the ALC vs NT also validates the alcohol-induced renal fibrosis. Similar to the females, both concentrations of simvastatin were significantly effective against alcohol-induced renal fibrosis in the males. 3.3.6 Measurement of TNF-α distribution The pattern of TNF-α distribution was identical in the renal tubule and in the renal corpuscle for both sexes. The TNF-α distribution in the renal tubules or in the renal corpuscle was highest in the ALC and was lowest in the NT for both sexes (Table 3.4). In the females, a Kruskal- Wallis test revealed that the TNF-α distribution was significantly different across the experimental groups (P=0.000) while a Dunn’s post hoc revealed that the TNF-α distribution in any paired groups was significantly different except in the SIM vs ALC+SIM15 for the renal 57 tubule (P=0.453) or in the SIM vs ALC+SIM5 (P=0.200) for the renal corpuscle (Fig. 3.18 & 3.19). The significant difference between the ALC vs NT also validates alcohol-induced inflammation in the renal tissue. Both concentrations of simvastatin were effective in reducing alcohol-induced inflammation in the renal tissue. In the males, a Kruskal-Wallis test also revealed that the TNF-α distribution was significantly different across the experimental groups (P=0.000), while a Dunn’s post hoc revealed that the TNF-α distribution in any paired groups for the renal tubule or corpuscle was significantly different except in the ALC+SIM5 vs ALC+SIM15 (P=0.733) for the renal tubule (Fig. 3.18 & 3.19). The significant difference between the ALC vs NT also confirms alcohol-induced inflammation in the renal tissue and both concentrations of simvastatin were effective in reducing alcohol-induced inflammation in the renal tissue. 58 Table 3.4: Summary of the collagen and TNF-α distributions in the kidney No of animals Collagen Distribution TNF-α Distribution No of images assessed Mean (±SD) (%) Median (%) Renal tubules Renal corpuscles No of ROIs assessed Mean (±SD) (%) Median (%) Mean (±SD) (%) Median (%) Female NT 5 260 0.51 (±0.14) 0.48 225 1.03 (±0.15) 1.04 0.70 (±0.15) 0.68 SIM 5 260 1.48 (±1.22) 1.14 215 1.93 (±0.84) 1.79 2.39 (±1.09) 2.13 ALC 5 231 6.52 (±2.60) 6.35 260 11.04 (±1.54) 11.43 11.13 (±2.71) 10.61 ALC+SIM5 5 259 3.18 (±1.73) 2.78 260 1.69 (±0.78) 1.74 2.56 (±1.60) 2.35 ALC+SIM15 5 257 1.35 (±1.44) 0.76 259 1.67 (±0.20) 1.67 1.41 (±0.33) 1.43 Male NT 5 260 0.67 (±0.21) 0.65 217 1.00 (±0.21) 0.97 0.79 (±0.20) 0.77 SIM 5 259 1.81 (±1.66) 1.14 191 1.56 (±0.54) 1.52 1.84 (±0.66) 1.77 ALC 5 258 6.13 (±5.87) 5.87 260 9.51 (±2.02) 9.40 8.94 (±2.92) 8.99 ALC+SIM5 5 255 2.78 (±1.41) 2.38 261 1.85 (±0.30) 1.88 2.48 (±1.05) 2.26 ALC+SIM15 5 261 2.13 (±1.49) 2.05 259 1.90 (±0.37) 1.84 1.58 (±0.57) 1.63 59 Figure 3.17: Box plots of the collagen distribution across the different experimental groups and for both sexes. In both sexes, the collagen distribution was significantly higher in the ALC than in the NT thus confirming alcohol-induced renal fibrosis. Both concentrations of simvastatin were effective against alcohol-induced renal fibrosis. NT = Non-treatment; SIM = Simvastatin; ALC = Alcohol; ALC+SIM5 = 5 mg simvastatin and alcohol; ALC+SIM15 = 15 mg simvastatin and alcohol. NS = Not significant at P>0.05. 60 Figure 3.18: Box plots of the TNF-α distribution in the renal tubules across the different experimental groups and for both sexes. In both sexes, the TNF-α distribution was significantly higher in the ALC than in the NT thus confirming alcohol-induced renal inflammation. Both concentrations of simvastatin were effective against alcohol-induced renal inflammation in the tubules. NT = Non-treatment; SIM = Simvastatin; ALC = Alcohol; ALC+SIM5 = 5 mg simvastatin and alcohol; ALC+SIM15 = 15 mg simvastatin and alcohol. NS = Not significant at P>0.05. 61 Figure 3.19: Box plots of the TNF-α distribution in the renal corpuscles across the different experimental groups and for both sexes. In both sexes, the TNF-α distribution was significantly higher in the ALC than in the NT thus confirming alcohol-induced renal inflammation. Both concentrations of simvastatin were effective against alcohol-induced renal inflammation in the corpuscles. NT = Non-treatment; SIM = Simvastatin; ALC = Alcohol; ALC+SIM5 = 5 mg simvastatin and alcohol; ALC+SIM15 = 15 mg simvastatin and alcohol. NS = Not significant at P>0.05. 62 3.4 The liver 3.4.1 General morphology of the liver The morphology of the liver in the NT showed normal hepatic parenchyma which consists of normally arranged hepatocytes in plates radiating outward from the central vein. The hepatocytes were normally shaped with centrally placed nuclei. The plates of hepatocytes were separated by sinusoids, the lumens of the sinusoids were clear of red blood cells. There was no aggregation of Kupffer cells in the parenchyma in the NT. In the ALC, the hepatocytes in the parenchyma were normally shaped with centrally placed nuclei and there was aggregation of Kupffer cells along the length of the parenchyma. The parenchyma in the ALC+SIM5 and ALC+SIM15 had a few hepatocytes which were not normally shaped, and the radial arrangements of the parenchyma was disrupted. The hepatocytes showed ballooning degeneration also called hydropic swelling, which is characterised by a pale and distended cytoplasm. The sinusoids were filled with red blood cells. There was an aggregation of Kupffer cells along the length of the parenchyma. The livers from the ALC showed the early stages of perisinusoidal fibrosis. The parenchyma from the ALC showed intense TNF-α expression. 63 Figure 3.20: Representative micrographs of the liver demonstrating hepatic morphology from liver tissues from different treatment groups. a. Liver from NT showing normal hepatocyte and endothelial morphology, b. Liver from ALC showing normal hepatocyte morphology and an aggregation of Kupffer cells, c. ALC+SIM15 liver showing hepatocytes with ballooning degeneration and the presence of red blood cells in the sinusoids. NT= non-treatment, ALC= alcohol only group, ALC+SIM15= alcohol and simvastatin 15 mg, KC= Kupffer cells, BD= ballooning degeneration. 64 Figure 3.21: Representative micrographs of the liver demonstrating collagen distribution in hepatic tissues from different treatment and a demonstration of perisinusoidal fibrosis. a. Liver from NT showing no collagen deposition, b. Liver from ALC showing the early stages of perisinusoidal fibrosis indicated by the collagen deposition along the walls of the sinuoids, c. ALC+SIM5 liver showing extensive collagen deposition. NT= non-treatment group, ALC= alcohol only group, ALC+SIM5= alcohol and simvastatin 5 mg, F= fibrosis. 65 Figure 3.22: Representative micrographs of the liver demonstrating the levels of inflammation in hepatic tissues from different treatment groups. a. Liver from NT showing minimal TNF-α expression, b. Liver from ALC showing areas of intense T NF-α expression, c. Liver from ALC+SIM5 showing moderate TNF-α expression, d. Liver from ALC+SIM15 showing moderate TNF-α expression. NT= non-treatment group, ALC= alcohol only group, ALC+SIM5= alcohol and simvastatin 5 mg, ALC+SIM15= alcohol and simvastatin 15 mg. 66 3.4.2 Density of hepatocytes In the females, the hepatocyte density was highest in the ALC and was lowest in the NT (Table 3.5). A Kruskal-Wallis test revealed that the hepatocyte cell density was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the hepatocyte cell density in any paired groups was significantly different except in the ALC vs ALC+SIM5 (P=0.300), ALC vs ALC+SIM15 (P=0.641) or ALC+SIM5 vs ALC+SIM15 (P=0.568) (Fig. 3.23). From these observations, all the experimental groups that received alcohol produced a significantly higher cell densities than the NT or the SIM while the alcohol effect on the hepatocyte density was similar in the ALC, ALC+SIM5 or ALC+SIM15. This seems to show that alcohol stimulated a compensatory mechanism which may have been caused by alcohol-induced cell death of hepatocytes. Surprisingly, simvastatin did not seem to have prevented alcohol-induced cell death because the hepatocyte density in groups that were administered alcohol was significantly different from the NT (or the SIM). In the males, the hepatocyte density was highest in the ALC and was lowest in the NT (Table 3.5). A Kruskal-Wallis test revealed that the hepatocyte density was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the hepatocyte density in any paired groups was significantly different (Fig. 3.23). The cell density of hepatocytes was significantly higher in the ALC than in the NT (P=0.000), SIM (P=0.000) or ALC+SIM5 (P=0.000) but was significantly lower than in ALC+SIM15 (P=0.000). Like the observations in the females, all the experimental groups that received alcohol produced a significantly higher cell densities than the NT or the SIM. This seems to show that alcohol stimulated a compensatory mechanism which may have been caused by alcohol-induced cell death of hepatocytes. 15 mg simvastatin also showed a better improvement on hepatocyte density than 5 mg simvastatin. 67 3.4.3 Collagen distribution analyses In the females, the collagen distribution in the liver was highest in the ALC+SIM5 and was lowest in the NT (Table 3.5). A Kruskal-Wallis test revealed that the collagen distribution was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the collagen distribution was significantly higher in the ALC than in the NT (P=0.000) or ALC+SIM15 (P=0.000) but was significantly lower than in the ALC+SIM5 (P=0.000). Collagen distribution was however similar in the NT vs ALC+SIM15 (P=0.410) or in the SIM vs ALC (P=0.601) (Fig. 3.24). The significant difference between ALC vs NT validates the toxicity of alcohol on the hepatocytes i.e., fibrosis. Alcohol effect on collagen distribution was significantly reduced by the administration of 15 mg simvastatin but not the 5 mg simvastatin. In the males, the collagen distribution in the liver was highest in the ALC+SIM5 and was lowest in the NT (Table 3.5). A Kruskal-Wallis test revealed that the collagen distribution was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the collagen distribution was significantly higher in the ALC than in the NT (P=0.000), SIM (P=0.000) or ALC+SIM15 (P=0.000) but was significantly lower than in the ALC+SIM5 (P=0.000) except. On the other hand, collagen distribution was similar in the NT vs SIM (P=0.240) (Fig. 3.24). The significant difference between ALC vs NT also validates the toxicity of alcohol on the hepatocytes i.e., fibrosis. Similar to the observations in the females, alcohol effect on collagen distribution was improved by a higher concentration of simvastatin but not the lower concentration. 3.4.4 Measurement of TNF-α distribution In the females, the TNF-α distribution in the liver was highest in the ALC+SIM5 and was lowest in the SIM (Table 3.5). A Kruskal-Wallis test revealed that the TNF-α distribution 68 was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the TNF-α distribution in any paired groups was significantly different except in the NT vs ALC (P=0.099), NT vs ALC+SIM15 (P=0.600) or ALC vs ALC+SIM15 (P=0.218) (Fig. 3.25). The similarity in the TNF-α distribution in the NT vs ALC did not reveal alcohol toxicity. At the same time, both concentrations of simvastatin following chronic alcohol did not perform better (with respect to the expression of TNF-α) than in the ALC. In the males, the TNF-α distribution in the liver was highest in the ALC+SIM15 and was the lowest in the SIM (Table 3.5). A Kruskal-Wallis test revealed that the TNF-α distribution was significantly different across the experimental groups (P=0.000). A Dunn’s post hoc revealed that the TNF-α distribution in any paired groups was significantly different except in the NT vs SIM (P=0.224) or ALC+SIM5 vs ALC+SIM15 (P=0.418) (Fig. 3.25). In contrary to the females, chronic alcohol induced inflammation in the liver, an indication of alcohol toxicity in the liver but both concentrations of simvastatin did not prevent inflammation following chronic alcohol. 69 Table 3.5: Summary of hepatocyte morphometries, collagen and TNF-α distributions No of animals Hepatocyte cell density Collagen distribution TNF-α distribution No of grids assessed Mean count per grid (± SD) Median No of images assessed Collagen distribution (Mean ± SD) (%) Median No of images assessed TNF-α distribution (Mean ± SD) (%) Median Female NT 5 300 39.81 (±14.61) 50.5 100 3.10 (±1.36) 3.59 86 12.73 (±11.25) 8.73 SIM 5 300 57.18 (±13.22) 63 96 5.79