SACM - United Kingdom
Permanent URI for this collectionhttps://drepo.sdl.edu.sa/handle/20.500.14154/9667
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Item Restricted In vitro and in vivo models to study the effect of biofilms and antimicrobial wound dressings on wound healing(The University of Manchester, 2024-06-25) Baqader, Sajwa; Thomason, Helen; Humphrey, Gavin; McBain, AndrewBackground: Biofilms have been strongly implicated in delayed wound healing. Reduced oxygen and low microbial growth rates within wound biofilms are a recognized driver of tolerance towards antimicrobial agents, and poor treatment outcomes. There is however uncertainty about the contribution of biofilm to delayed healing and the mechanisms involved. This emphasises the need to examine the differences in the wound healing process between the planktonic and biofilm-infected wounds through preclinical models. Improved knowledge in this area may lead to the development of improved treatment strategies. Antimicrobial chemicals, especially silver-based ones, are frequently employed in wound dressings, but little is known about their efficacy and there are few reliably predictive preclinical wound models. This doctoral thesis focuses on visualising the variations in metabolic activity in bacterial cells during biofilm development and studying the differences in the wound healing process between planktonic and biofilm-infected wounds. This thesis also evaluates the antibacterial efficacy of innovative wound dressings that incorporate silver oxynitrate, which produces highly reactive Ag2+ and Ag3+ ions, using in vitro and in vivo models. Methods: Initially, a 96 well plate-based assay was used to determine the bactericidal activity of dressings containing silver oxynitrate and untreated controls against planktonic S. aureus in both log- and stationary phases. A colony biofilm model was then used to assess the efficacy of dressings containing silver oxynitrate in eradicating biofilms over 1, 3, and 7 days. Next, an in vivo excisional wound model either uninfected or infected with 104 S. aureus in planktonic form, or as preformed biofilms was established, and used to assess effects of dressings containing silver oxynitrate compared to non-antimicrobial controls and uninfected wound controls. Bromodeoxyuridine (BrdU), a nucleotide analogue that incorporates into nascent DNA in actively dividing cells was used to visualise proliferating. Biofilms were exposed to BrdU for 4 h before harvesting at 1, 3 and 7 days. Wound healing was characterised by quantification of wound width, area, and re-epithelialisation from histological sections. Inflammation was determined based on neutrophil/macrophage marker immunohistochemistry. Wound biofilms were characterised by anti- BrdU immunofluorescence, fluorescence in-situ hybridisation specific prob, viability mapping, tissue Gram stain, quantitative bacterial culture, and scanning electron microscopy. Results: Strong BrdU staining was observed corresponding to active growth of 1 day S. aureus biofilm and slow metabolic rates occurred following 3 days old biofilms. No BrdU signal was detected in the 7 days old biofilms of S. aureus. Over 7 days, wound width and areas increased significantly in wounds infected with preformed biofilm and in planktonic infected wounds compared to uninfected wounds. A significant reduction in the reepithelialisation percentage was observed in wounds infected with preformed biofilms and infected planktonic wounds. In contrast, uninfected wounds exhibited a small wound width, area, and comparatively high reepithelialisation percentage. The number of neutrophils and macrophages was significantly higher in wounds infected with preformed biofilm than wounds that infected with planktonic bacteria. S. aureus abundance on surface of both planktonic and biofilm infected wounds was markedly increased after seven days. Dressings containing silver oxynitrate significantly reduced planktonic bacterial cell counts more during log phase than stationary phase. Furthermore, this dressing effectively decreased number of bacterial cells in colony biofilm following a 7-day exposure. The dressings containing silver oxynitrate also significantly reduced S. aureus viable counts, wound width, area, inflammation, and increased reepithelialisation more in planktonic infected wounds than biofilm infected wounds over 7 days. SEM imaging revealed that infected wounds treated with silver oxynitrate-incorporating dressings contained fewer bacteria and biofilm extracellular polymeric substance was disrupted. Conclusion: The in vitro colony biofilm method of labelling metabolically active S. aureus with BrdU over 7 days demonstrated the bacterial cells dormancy. The murine wound model provides evidence of the impact of S. aureus in planktonic and biofilm form in delaying healing process. The use of dressings containing silver oxynitrate demonstrated a greater impact on the treatment of infected murine wounds in both states, with greater efficacy against planktonic infected wounds. The in vitro and in vivo models mentioned above can be used to evaluate effectiveness of other wound treatments obtain better wound management and patient outcomes.21 0Item Restricted Anti-atherogenic actions of hydroxytyrosol(Saudi Digital Library, 2023-11-02) Alotibi, Reem Mansour; Ramji, DipakIntroduction: Atherosclerosis is a chronic inflammatory disorder characterised by the accumulation of lipids in the arterial wall, and is considered to be a major contributor to cardiovascular disease (CVD). Worldwide, CVD is responsible for a third of all deaths. Current pharmacological therapeutic agents for CVD, such as statin therapy, are not fully effective and have considerable residual risk for the disease. Alternative agents for the prevention and treatment of the disease are therefore required. Hydroxytyrosol (HT) is a polyphenol compound found mainly in olive oil. HT has been reported to prevent CVD predominantly due to its antioxidant effects. HT is the only polyphenol recognised by the European Food Safety Authority as a protector against low-density lipoprotein-mediated oxidative damage. Previous investigations carried out in the host laboratory showed that HT altered multiple atherosclerosis-associated risk factors in wild-type mice fed a high-fat diet (HFD) for 3 weeks, as well as various anti-inflammatory and anti-atherogenic actions on human monocytes/macrophages in vitro. Unfortunately, the actions of HT in atherosclerosis in vivo are poorly understood. The aim of this study therefore was to elucidate its effects on atherosclerosis progression and regression in a mouse model system. Methods: In order to study the development of atherosclerosis in vivo, 8-week-old male or female low-density lipoprotein receptor-deficient (ldlr-/-) mice were given HFD alone or in combination with 10 mg/kg/day of HT for 12 weeks. For regression studies, the mice were fed a HFD for 12 weeks to promote the formation of established lesions, and then switched to normal chow diet (NCD) alone or in combination with HT. The two procedures were then followed up by an in-depth investigations of atherosclerosis- associated risk factors and the plaques that formed in the aortic root. RNA-sequencing and bioinformatic analyses was used to assess changes in gene expression and associated pathways in the thoracic aorta. The liver was also analysed in relation to non-alcoholic fatty liver disease (NAFLD) that is often associated with atherosclerosis and extended via the use of an in vitro hepatoma HepG2 cell culture model system. Results: As part of the progression study, female ldlr-/- mice that had received HT supplemented HFD for 12 weeks had attenuated weight gain, plaque size in the aortic root and neutrophil content in the peripheral blood, while male ldlr-/- mice had attenuated occlusion and T cells in the peripheral blood. In both cases, there was reduced plaque inflammation and improved plasma lipid profile and plaque stability. As part of the regression studies on male ldlr-/- mice, intervention with HT combined with NCD reversed hepatic injury and enhanced plaque stability (to a greater extent than NCD intervention alone). Conclusions: These findings provide support for the anti-atherogenic actions of HT as well as its possible use as an alternative nutraceutical agent for preventing the development of atherosclerosis. This is potentially possible due to the lack of adverse effects as well as the relatively low cost in comparison to that of typical pharmacological treatments. Additional research is necessary in order to determine the mechanisms that are responsible for these favourable anti-atherogenic and other beneficial changes.16 0