Hypoxia adversely affects cells and tissues, and neuronal cells in particular have been shown to be more susceptible to the injurious effects of hypoxia i.e. they may begin to die when oxygen supply is reduced or completely eliminated. Cannabinoid (CB1) receptor and opioid (μ, δ and κ) receptor agonists have been shown to elicit several central nervous system (CNS) effects, mediated via G protein-coupled receptors. The aim of the research presented in this thesis was to study the effect of hypoxia on neuronal cell signalling and the consequent neuroprotectant effects of cannabinoid and opioid receptor agonists against hypoxia in the rat cortical neuronal cell line (B50) in culture. The B50 cells cultured in hypoxic conditions were treated and concurrently cultured with cannabinoid and opioid receptor agonists to determine the effects of these drugs on hypoxia-induced changes using downstream signalling activities such as cellular morphogenesis, growth, proliferation, differentiation, lactate dehydrogenase (LDH) leakage, second messenger (cAMP) and extracellular signalregulated kinases (ERK1/2) quantification, to assess the level of cellular damage and injury, repair and protection. Cortical B50 cells were cultured in either a normal incubator (21%O2; 5% CO2) as the normoxic control group, or a hypoxic incubator (5%O2; 5% CO2) as the experimental group. Three cannabinoid agonists [Win55,212- 2 mesylate (Win), anandamide or arachidonoylethanolamide (AEA), and 2- arachidonylglycerol (2-AG)] and three opioid agonists [DAMGO (μ), DSLET (δ) and ICI-199,441 hydrochloride (κ)], were selected and administered to the cells as treatment group for 48 hours after 48 hours of initial culture for a total of 96 hours of culture and pre-treatment group treated at 0 hour for a total of 96 hours in hypoxic conditions at concentrations of 10nM, 50nM and 100nM for cannabinoid agonists, and 10μM, 50μM and 100μM for opioid agonists. Neuronal viability, proliferation, differentiation and second messenger activity were assessed using morphological same-field assessment, LDH leakage, cellular proliferation assay, second messenger (cAMP) assay, and phospho-ERK1 & 2 assay and dibutyryl cyclic adenosine monophosphate (DbcAMP) induced differentiation method. Levels of G-protein coupled receptor (cannabinoid, CB1 and mu opioid, MOR) mRNAs were assessed using the RT-PCR method. The results showed that hypoxia induced a 4-fold increase in LDH leakage from B50 cells cultured in hypoxia when compared to the cells xxviii cultured in normoxic conditions (440% versus 100%, respectively; p<0.05). Cannabinoid receptor agonist treatment was able to reduce the LDH release in hypoxic cells to between 2-to 4-folds: 100nM AEA (69%), 100nM 2-AG (103%) and 10nM Win (217%), when compared to untreated hypoxic B50 cells (440% versus cannabinoid treated; p<0.05). The results of opioid administration showed a 3-fold decrease in the level of LDH leakage in B50 cells cultured in hypoxia when compared to untreated hypoxic cells (587%). The results of hypoxic treated B50 cells with opioid agonists are 100μM ICI-199,441 (318%); 50μM DSLET (339%) and 50μM DAMGO (352%) (p<0.05; untreated hypoxia versus opioid treated). The result of cAMP quantification in B50 cells in culture showed a reduction in cAMP concentration in untreated hypoxic B50 cells when compared to normoxic cells (0.7 pmol/ml versus 3.0 pmol/ml; p<0.05). Cannabinoid treated hypoxic cells showed increases in cAMP concentration: 2-AG 10nM (3.5 pmol/ml), 50nM (3.1 pmol/ml) and 100nM (0.9 pmol/ml), (p<0.05; Cannabinoid treated versus hypoxia untreated). The cAMP concentration in B50 cells treated in hypoxia with opioid agonist, ICI 199,441 hydrochloride, was significantly increased when compared to untreated hypoxic B50 cells (0.7 pmol/ml). The treatment with ICI 199,441 hydrochloride are 10μM (10.0 pmol/ml), 50μM (3.15 pmol/ml) and 100μM (1.15 pmol/ml), (p<0.05; opioid treated versus hypoxia untreated). The result of phospho-ERK1&2 assay in B50 cells showed decrease in phospho-ERK1&2 in untreated hypoxic cells when compared to normoxic untreated cells (6.0 units/ml versus 87.0 units/ml; p<0.05). The result of cannabinoid treated hypoxic cells showed increases in phospho-ERK1&2 when compared with the hypoxic untreated B50 cells: Win 10nM (98 units/ml), Win 100nM (27 units/ml), AEA 10nM (62 units/ml), AEA 100nM (60.5 units/ml), 2-AG 10nM (45 units/ml) and 2-AG 100nM (68 units/ml) (cannabinoid treated versus untreated hypoxia; p<0.05). The phospho-ERK1&2 in hypoxic B50 cells treated with opioid showed increase with DAMGO 10μM (22 units/ml), DSLET 10μM (16 units/ml) and ICI 199,441 hydrochloride 10μM (23.5 units/ml) (P<0.05; opioid treated versus hypoxia untreated). The result showed a decrease in cellular proliferation in untreated hypoxic cells when compared to the normoxic cells (7x106 cells/ml versus 20x106 cells/ml; p<0.05), while cannabinoid and opioid treatments was able to increase cell proliferation in hypoxic treated cells with: Win 10nM (11x106 cells/ml), AEA 100nM (12x106 cells/ml) and 2-AG 100nM (13.8x106 cells/ml), DAMGO 10μM (16x106 cells/ml), DSLET 10μM (20x106 cells/ml) and ICI xxix 199,441 100μM (21.5x106 cells/ml) when compared to hypoxic untreated cells (7x106 cells/ml) (hypoxia untreated versus hypoxia treated; p<0.05). Some of these changes were shown to be concentration-dependent between the normal and hypoxic B50 neurons, and between treated and untreated hypoxic B50 cells in culture, while the CB1 and MOR mRNA levels showed no appreciable change. The results show that B50 neuronal cells are susceptible to damage and injurious effects of hypoxia, as are most brain cells, while the results of the administration of cannabinoid and opioid agonists suggest that these agents have some potential therapeutic and protective benefits in the treatment and prevention of hypoxia-induced toxicity in neuronal B50 cells in culture. This could be of potential benefit in the treatment and protection against hypoxia-related neurodegenerative diseases and disorders such as stroke, dementias, ageing, Alzheimer’s and Parkinson’s diseases.

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Statistics for The effects of Hypoxia on neuronal cell signalling

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