Comparative study of the effect of iloprost on neuroinflammatory changes in c8-b4 microglial cells and murine model of trypanosomiasis
- Authors: Jacobs, Ashleigh
- Date: 2024-04
- Subjects: Trypanosomiasis -- South Africa , DNA -- Methylation -- Research -- Methodology , Central nervous system -- Diseases , Nervous system -- Degeneration
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/64077 , vital:73651
- Description: Neurodegenerative conditions significantly impact well-being and quality of life in individuals with major symptoms including mood disorders, cognitive decline, and psychiatric disturbances, often resulting from neuroinflammation triggered by immune responses to bacterial or parasitic infections such as gram-negative bacteria or Human African Trypanosomiasis. Microglia play a crucial role in both neurotoxicity and cellular processes involved in restoring the neural health. Exploring the therapeutic potential of prostacyclin and its analogues in regulating microglia responses to inflammatory insult and treating Trypanosoma brucei (T.b) infection remains an unexplored area. The aim of this study was to assess the potential neuroprotective effects of Iloprost through comparative analysis of neuroinflammatory responses in both microglial cells exposed to lipopolysaccharide (LPS) and mouse brains infected with T.b brucei. In phase I of this study both resting and LPS treated C8-B4 microglial cells were exposed to varying concentrations of Iloprost. The effects of Iloprost on LPS-induced inflammation were analysed using immunofluorescence to detect microglial activation and differentiate between pro and anti-inflammatory phenotypes. Furthermore, pro and anti-inflammatory cytokine secretion was determined using an ELISA, in addition gene expression analysis was carried out using quantitative polymerase chain reaction (qPCR). Also, DNA methylation status of C8-B4 cells exposed to LPS challenge alone or in combination with various concentrations of Iloprost were determined using bisulfite sequencing technique followed by qPCR. In phase II of the study, a total of twenty-four Albino Swiss male mice (8-10 weeks old) were divided into four treatment groups with 6 mice in each group. All treatment groups except the non-infected control were inoculated with the T.b brucei parasite. One group received a single intraperitoneal injection of Diminazene aceturate (4 mg kg-1) while the remaining group received repeated intraperitoneal injections of Iloprost (200 μg kg-1). On day ten of the study, mouse brains were removed on ice using forceps. The hippocampal tissues were dissected out and processed for quantification of gene expression changes in pro and anti-inflammatory cytokines. Overall, the findings of this study indicate that LPS-induced pro-inflammatory cytokine, TNF-α and IL-1β, secretion and gene expression is down-regulated in C8-B4 microglial cells treated with Iloprost. Furthermore, there was a significant up-regulation in the expression of anti-inflammatory genes, particularly ARG-1, CD206, BDNF and CREB in response to Iloprost treatment following LPS-induced inflammation. This study is also the first to confirm M2 microglial polarization with Iloprost treatment in both resting and LPS treated cells. However, hypermethylation at CREB and BDNF promoter regions was observed 24 hours after Iloprost treatment. Additionally, Iloprost reversed hypomethylation at the BDNF promoter region that had been induced by LPS treatment. The rodent model also indicated a downregulation in the pro-inflammatory cytokine, IL-1β, expression and upregulation of BDNF transcription in T.b brucei infected mice treated with repeated doses of Iloprost. In conclusion, determining the immunomodulatory roles of Iloprost in both in vitro and in vivo models of neuroinflammation could assist in the development of alternative therapy for neurodegenerative disease. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2024
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- Date Issued: 2024-04
Establishment of a high-content neurodegenerative disease model screening platform
- Authors: Swanepoel, Bresler
- Date: 2023-12
- Subjects: Molecular neurobiology , Nervous system -- Diseases , Nervous system -- Degeneration
- Language: English
- Type: Doctorial theses , text
- Identifier: http://hdl.handle.net/10948/62644 , vital:72906
- Description: The identification of viable therapeutic targets and new treatments for central nervous system disorders, especially neurodegenerative diseases, remain major challenges in the field of drug discovery. Over the past few years there has been a steady decline in the turnaround time of current screening processes to yield viable drugs. Therefore, an increasing need exists for better technologies, protocols, and the screening of larger libraries. High-throughput screening provides the best solution to this problem and has become a key part in the drug discovery and development process. Likewise, high-content analysis has gained popularity over the past few years and is suitable for high-throughput screening. The aim of this study was to establish a comprehensive in vitro neuroprotective screening platform incorporating high throughput screening, using Parkinson’s disease as the neurodegenerative disease of interest. To evaluate the success of this platform, the neuroprotective potential of two mushrooms (Hericium erinaceus and Phlebopus sudanicus), two plants (Lippia javanica and Myrothamnus flabellifolia) and two seaweeds (Eucheuma denticulatum and Kappaphycus alvarezii) were investigated. Aqueous and ethanolic extracts of the selected natural products were evaluated across 21 parameters associated with four hallmarks of neurodegeneration: acquiring senescence, acquiring cell death, neuroinflammation and altered metabolism/cell survival. Based on the effects of these selected natural products on the 21 parameters, their potential mechanisms of action were elucidated. In addition to this, the natural products were scored under each of these therapeutic targets in an attempt to identify the most suitable animal models for future studies. The scoring system indicated that animal models investigating anti-senescence ability would be more suited for extracts of H. erinaceus, P. sudanicus and E. denticulatum whereas studies investigating the prevention of cell death would be more suited for extracts of E. denticulatum, L. javanica and K. alvarezii. Likewise, models based on neuroinflammation would be best suited for extracts of H. erinaceus, E. denticulatum and L. javanica while studies examining altered metabolism/cell survival would be best suited to extracts of H. erinaceus, E. denticulatum, K. alvarezii and M. flabellifolia. Considering the pleiotropic nature of the selected natural products, models that can combine these therapeutic targets could be of great interest. 6-OHDA proved to be capable of inducing favourable effects, in all the parameters investigated, with regard to a neurodegenerative state. However, it is known to have some disadvantages when it comes to pathological features such as the lack of the ability to induce Lewy body formation. Choosing the correct inducer remains a daunting task and no model, whether cell-based or animal-based, exists yet in which all the features of neurodegenerative diseases have been successfully replicated. The limitations of the current models, however, does not mean that they do not produce valuable insights. This is especially true if the mechanism of action for a specific compound or natural product is unknown. Animal models are still indispensable for the validation and interpretation of the results obtained from cell models with particular importance to toxicity. Therefore, this study assessed the best studied extract with the highest overall score for its toxicity using a zebrafish larvae-based model. Assessment of the toxicity of H. erinaceus revealed that both aqueous and ethanolic extracts resulted in death at the highest concentrations. This was supported by the results obtained in the in vitro cytotoxicity screening. In conclusion, this highlighted the importance of using physiologically relevant concentrations and supported the translational value of the current cell-based screening model to animal models and possibly humans. The findings of the present study suggest that a scoring system, which categorizes the different activities of selected natural products into distinct groups, can be a useful tool to improve the translatability of in vitro results to animal models. Furthermore, the current study arguably provides the most comprehensive neuroprotective screening platform in existence. Future research can look at expanding the platform through incorporation of additional parameters based on other hallmarks of neurodegeneration, not covered in this study, including protein folding and aggregation, altered epigenetics and the examination of other neuronal markers such as the involvement of astrocytes, oligodendrocytes, and microglia. In addition to this, future research can make use of more sophisticated cell models such as differentiated, human induced pluripotent stem cells and three-dimensional cultures. , Thesis (PhD) -- Faculty of Science, School of Biomolecular and Chemical Sciences, 2023
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- Date Issued: 2023-12
Evaluation and application of electroanalysis for the determination of antioxidants
- Authors: Ragubeer, Nasheen
- Date: 2007
- Subjects: Antioxidants , Nervous system -- Degeneration , Electrochemical analysis , Marine algae , Natural products , Marine metabolites , Sargassum , Legumes , Nuclear magnetic resonance
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3922 , http://hdl.handle.net/10962/d1003981 , Antioxidants , Nervous system -- Degeneration , Electrochemical analysis , Marine algae , Natural products , Marine metabolites , Sargassum , Legumes , Nuclear magnetic resonance
- Description: The role of antioxidants in the prevention of neurodegenerative diseases has been well documented. The use of synthetic antioxidants has decreased due to the ssociation of these compounds with certain cancers. Thus, the search for novel natural antioxidants has gained much focus in research. Most common methods of determining antioxidant capacity are the radical generated assays and biological assays such as lipid peroxidation and the nitroblue tetrazolium assay. Electrochemical methods have been proposed for the determination of bio-active compounds such as antioxidants. The electrochemical methods of cyclic voltammetry and square wave voltammetry were evaluated for the determination of antioxidant capacity initially examining known antioxidants and then using plant extracts of Sutherlandia frutescens as a case study. The antioxidant properties determined by electrochemical methods were validated utilising the non-biological methods of the DPPH, TEAC, ferrozine and FC assay and biological pharmacological methods. The results indicated that Sutherlandia frutescens contains potent antioxidant compounds that are able to reduce lipid peroxidation. The electrochemical techniques of square wave voltammetry and cyclic voltammetry were applied for the screening of a large number of extracts of various algae for the detection of antioxidant compounds. The results indicated that electrochemistry can be used as a preliminary method for the rapid screening of a large number of crude samples for antioxidant compounds. Electrochemical methods were also evaluated as a method for guiding the isolation and purification of antioxidant metabolites in Sargassum elegans. Solvent partitioning and fractionation of the marine alga allowed for the purification of antioxidant compounds. At each step of purification electrochemical methods were utilized to determine which fractions contained the more potent antioxidant compounds and thus guide further purification. The purified antioxidant compounds were elucidated using NMR to determine the structure of the antioxidant compounds.
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- Date Issued: 2007
An investigation of the neuroprotective effects of estrogen in a model of quinolinic acid-induced neurodegeneration
- Authors: Heron, Paula Michelle
- Date: 2002
- Subjects: Estrogen , Quinolinic acid , Nervous system -- Degeneration
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3759 , http://hdl.handle.net/10962/d1003237 , Estrogen , Quinolinic acid , Nervous system -- Degeneration
- Description: The hippocampus, located in the medial temporal lobe, is an important region of the brain responsible for the formation of memory. Thus, any agent that induces stress in this area has detrimental effects and could lead to various types of dementia. Such agents include the neurotoxin, Quinolinic acid. Quinolinic acid (QUIN) is a neurotoxic metabolite of the tryptophan-kynurenine pathway and is an endogenous glutamate agonist that selectively injures and kills vulnerable neurons via the activation of the NMDA class of excitatory amino acid receptors. Estrogen is a female hormone that is responsible for reproduction. However, in the last decade estrogen has been shown to exhibit a wide range of actions on the brain, including neuroprotection. Estrogen has been shown to exhibit intrinsic antioxidant activity and protects cultured neurons against oxidative cell death. This is achieved by estrogen’s ability to scavenge free radicals, which is dependent on the presence of the hydroxyl group at the C3 position on the A ring of the steroid molecule. Numerous studies have shown that estrogen protects neurons against various toxic substances and may play a role in delaying the onset of neurodegenerative diseases, such as Alzheimer’s disease. Neuronal damage due to oxidative stress has been implicated in several neurodegenerative disorders. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. The study aims to elucidate and further characterise the mechanism behind estrogen’s neuroprotection, using QUIN as a model of neurotoxicity. Initial studies confirm estrogen’s ability to scavenge potent free radicals. In addition, the results show that estrogen forms an interaction with iron (II) and also acts at the NMDA receptor as an agonist. Both mechanisms reduce the ability of QUIN to cause damage to neurons, since QUIN-induced toxicity is dependent on the activation of the NMDA receptor and the formation of a complex with iron (II) to induce lipid peroxidation. Heat shock proteins, especially Hsp 70 play a role in cytoprotection by capturing denatured proteins and facilitating the refolding of these proteins once the stress has been relieved. Estrogen has been shown to increase the level of expression of Hsp70, both inducible and cognate forms of the protein. This suggests that estrogen helps to protect against cellular protein damage induced by any form of stress the cell may encounter. The discovery of neuroprotective agents, such as estrogen, is becoming important as accumulating evidence indicates a protective role in vivo. Thus further research may favour the use of these agents in the treatment of several neurodegenerative disorders. Considering how devastating diseases, such as Alzheimer’s disease, are to a patient and the patient’s families, the discovery of new protective agents are a matter of urgency.
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- Date Issued: 2002