| Summary: | Background and Objectives: Chemotherapy can cause long-term cognitive deficits in patients which have been termed ‘chemobrain.’ These drugs cause a reduction in hippocampal neurogenesis and behavioural deficits in animal models. Antidepressants have recently been shown to be neuroprotective against the behavioural and cellular effects of chemotherapy in animals; however the mechanism of this is unknown. This thesis used in vivo studies, in rats and mice, to determine the effects of acute and chronic 5-Fluorouracil chemotherapy on cell proliferation in the sub granular zone of the dentate gyrus immediately after treatment and after 1 week of recovery. This was done to determine if these different drug regimens affected the recovery of neurogenesis after chemotherapy which might indicate that they were affecting different stages of neurogenesis or having a different impact on the cells involved. The effect of prior treatment with the antidepressant fluoxetine on both acute and chronic chemotherapy was measured in terms of its effect on cell proliferation and markers of different stages of neurogenesis. In vitro, neural stem cells were treated with chemotherapy either on its own or in combination with fluoxetine or factors known to be upregulated by this antidepressant. This was done to determine if the actions of fluoxetine in vivo were directly on neural stem cells or required expression of downstream targets of fluoxetine, possibly from other components of the neural stem cell niche. Methods: Animals were administered either acute (single injection) or chronic (5 injections) 5- fluorouracil with or without fluoxetine pre-treatment. Cell proliferation (Ki67), early neural differentiation (doublecortin - DCX) and the numbers of cells expressing the early neurogenesis transcription factors Soxl and 2 was quantified either one day or one week after the end of chemotherapy treatment. In vitro, mouse neuroblastoma cells (N2a) or primary hippocampal neural stem cells (NSCs) treated with 5-fluorouracil with or without fluoxetine itself or associated compounds were tested using MIT assays and flow cytometry, to determine cell viability, DNA damage, apoptosis and effects on the cell cycle. Results: Treatment with a single injection of 5-fluorouracil caused an increase in cell proliferation in mice 24 hours after administration. In contrast, an acute dose of 5-fluorouracil given to rats caused a significant decrease in proliferation (quantification of Ki67 and Sox2+ cells). Prior chronic treatment with fluoxetine prevented this decline. One week after acute 5-fluorouracil treatment, cell proliferation had returned to control levels. Declines in DCX were not prevented by fluoxetine treatment, but recovered 1 week after treatment. Chronic 5-fluorouracil also reduced cell proliferation, causing significant decreases in Ki67 and Sox2+ cells, a decline which was again prevented by fluoxetine. In contrast to acute treatment however, there was no recovery 1 week after the end of treatment in animals not treated with fluoxetine. In vitro, both N2a and primary neural stem cells showed a dose dependent decline in viability after 5- fluorouracil treatment. In N2a cells this was reduced by treatment with BDNF, fluoxetine, serotonin and noradrenaline, while in NSCs, serotonin, BDNF and potassium chloride were neuroprotective. None of the compounds however prevented 5-Fluorouracil-induced DNA damage. Fluoxetine prevented the chemotherapy induced decrease in cell cycle progression found in N2a’s and BDNF prevented increases in apoptosis at 1 pM and 5 pM in N2a’s and NSCs, respectively. Conclusions: Acute 5-fluorouracil treatment in rats caused only a transient decrease in cell proliferation, possibly by apoptosis of rapidly dividing neural progenitor cells which are then replaced by division of neural stem cells. In contrast chronic 5-fluorouracil treatment caused prolonged decreases in proliferating cells, possibly due to loss of neural stem cells or by cells entering a state of quiescence due to DNA damage. Acute doses of 5-fluorouracil in mice caused an increase in proliferative cells in vivo, compared to controls, suggesting a more rapid rebound from the insult in this species. Fluoxetine prevented the decrease in cell proliferation caused by 5-fluorouracil in vivo after both acute and chronic chemotherapy. The number of Sox 1/2+ cells was also maintained when fluoxetine was given prior to chemotherapy, indicating that these possible mechanisms involved in fluoxetine protection could be by increasing serotonin and BDNF levels that prevent apoptosis and promote cell proliferation.
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