Effect of 5-fluorouracil chemotherapy and the potential protective effect of the SSRI antidepressant fluoxetine on memory and neurogenesis in the adult hippocampus

*Please note: The abstract, acknowledgments, list of publications, table of contents and abbreviations in this PDF file appear in a different order to that of the print version of this thesis. Cancer patients, treated with systemic adjuvant chemotherapy, have described experiencing persistent deteriorations in cognition. The nature of these effects is unclear, and although a wide range of theories have been advanced, there is currently no treatment. This thesis uses an animal model to investigate the effects of a commonly prescribed chemotherapeutic agent, 5-fluorouracil (5-FU). The cognitive effects of 5-FU were examined using two behavioural tests, the object location recognition test (OLR) and the conditioned emotional response test (CER) both of which require input from the hippocampus, a brain region associated with memory. Memory consolidation by the hippocampus requires the continual production of new neurons (adult neurogenesis) from progenitor cells in the sub granular zone (SGZ) of the dentate gyrus. As an anti mitotic agent, 5-FU could be reducing the cell proliferation required for neurogenesis and this could be a cause of the cognitive deterioration. This hypothesis was tested by quantifying the numbers of proliferating cells (Ki67+) in the SGZ in sections together with the levels of doublecortin (DCX), a neurofilament expressed in developing neurons and brain- derived neurotrophic factor (BDNF), a factor required for new neuron survival and synaptic plasticity, by Western blotting. After developing the methodology (chapter 2); adult male Lister Hooded rats were given five i.v injections of 5-FU (25mg/kg) over a two week period and their behaviour and cellular aspects of the hippocampus compared with saline injected controls (chapter 3). 5-FU treated animals showed significant impairments in their performance of both the OLR and CER behavioural tests. Animals were sacrificed after the behavioural tests were performed and analysis showed they had significantly reduced numbers of dividing cells in the SGZ and non significant reductions in the levels of BDNF and DCX within the hippocampus. These results demonstrate that 5-FU treatment can produce cognitive impairments in this animal model which are similar in nature to those described by patients after chemotherapy. These behavioural changes are correlated with a reduction in the cell proliferation required for hippocampal neurogenesis providing support for the hypothesis that chemotherapy drugs are affecting this aspect of hippocampal function. In order to develop a treatment for the cognitive effects of chemotherapy the antidepressant fluoxetine was co-administered with 5-FU (chapter 4). This approach was based on recent evidence that fluoxetine can increase neurogenesis and protect neurons after damage. As with the experiment described above, performance in the CER test was impaired by five injections of 5-FU (25 mg/kg) as compared with saline treated controls. Similarly, animals treated with six injections of 5-FU (20mg/kg) were unable to discriminate between objects in novel and familiar locations in the OLR task. However co-administration of fluoxetine in drinking water (10mg/kg/day) for three weeks, starting a week before 5-FU treatment, prevented the impaired performance of this task found in the 5-FU only group. 5-FU chemotherapy caused a significant reduction in the number of proliferating cells in the SGZ compared to controls but this reduction was eliminated in the group co administered with fluoxetine. Fluoxetine on its own had no effect on proliferating cell number or behaviour. Moreover hippocampal BDNF or DCX protein levels in the co-treated group (5-FU+fluoxetine) were significantly increased compared to the 5-FU only treated group. These findings suggest that while 5-FU can negatively affect cell proliferation and hippocampal dependent memory, these deficits can be reversed by co- administration of fluoxetine. To understand the long term effects of chemotherapy, the cellular effects of 5-FU treatment were quantified one day, 2 and 6 weeks after the end of two weeks of 5-FU (20mg/kg) treatment (chapter 5). The results showed that 2 weeks of 5-FU treatment did not significantly reduce cell proliferation in the SGZ when quantified one day after the end of treatment. However proliferating cell numbers were significantly reduced compared to controls two and six weeks after the end of treatment. This suggests that 5-FU has a delayed effect on cell proliferation with its maximum effect two weeks after the end of treatment. Cell survival was quantified by BrdU labelling cells immediately prior to 5-FU treatment, and quantifying the numbers of BrdU positive cells at the different time points. BrdU+ cell numbers were significantly reduced at the end of treatment and continued to decline at 2 weeks but stabilised by 6 weeks. These results demonstrate that 5-FU has prolonged effects on neurogenesis after the end of chemotherapy treatment. The effects of 5-FU on cognition and neurogenesis are discussed and correlated with chemotherapy treated patient reports of continued cognitive impairment for months or years after completion of chemotherapy treatment.