1. Cognitive Impairment and Neural Pregenitor Cell Destruction (on Lister hooded rats) as a Result of Chemotherapy Morgan Earle, Department of Biological Sciences, York College of Pennsylvania Introduction It is reported that 15-50% of breast cancer patients have shown cognitive impairment following chemotherapy (Kitamura et al., 2015). Chemotherapy drugs cross the blood-brain barrier causing toxicity and leading to inflammation in the hippocampus dentate gyrus (Dietrich et al., 2015). Inflammation in this portion of the brain causes a decrease in cell proliferation resulting in a decrease of brain plasticity (Kiamura et al., 2015). COX-2 is a pro-inflammatory enzyme that increases in abundance when inflammation is present. Decreasing inflammation could help prevent cognitive impairments following chemotherapy treatment and increase the quality of life for cancer survivors Review of Literature Continued Methotrexate treatment decreases the viability of breast cancer cell lines and inhibits their growth. Treatment with this cytotoxic drug also resulted in poor behavioral tests, increased COX-2 enzymes, and a decline in immuno- positive cells (Yang et al. 2012) The negative effects of cyclophosphamide were only observed in a short time period following the last treatment. 7 days following the treatment in rats there were no effects on hippocampal cell proliferation or spatial working memory (Lyons et al. 2011) (Figure 1). Objectives Determine if methotrexate has an effect on the neurogenesis and cognitive abilities of rats. Determine if methotrexate increases the amount of the pro-inflammatory enzyme COX-2 that will indicate an increase of inflammation in the hippocampus. Determine if the inflammatory inhibitor NS- 393 can reduce the amount of cognitive impairment and increase the amount of neurogenesis following methotrexate treatment. http://www.bioseb.com/bioseb/anglais/default/item_id=2_cat_id=1_Tai l0Suspension0Test_source=news.php No Methotrexate, Tumor-Free N=8 Methotrexate, NS-393, Tumor- Bearing N=8 Intraperitoneal injection of NS- 393 (10mg/kg) Research Design No Methotrexate, Tumor-Bearing N=8 Methotrexate, Tumor-Free N=8 Methotrexate, Tumor-Bearing N=8 Intraperitoneal injection of saline (37.5mg/kg) Intraperitoneal injection of methotrexate (37.5mg/kg) Intraperitoneal injection of saline (10mg/kg) Behavioral tests: Barnes Maze, Tail-Suspension Test, and Passive Avoidance Test Immunohistochemistry Western Blot 2 week acclimation period 24 hours after last injections and 4 weeks after last injections Everyday for 4 weeks Literature Cited Briones, T., Woods, J. 2012. Dysregulation in myelination mediated by persistent neuroinflammination: Possible mechanisms in chemotherapy-related cognitive impairment. Brain, Behavior, and Immunity 35: 23- 32 Christie, L., Acharta, M., Parlhar, V., et al. 2012. Impaired Cognitive Function and Hippocampal Neurogenesis following Cancer Chemotherapy. Clinical Cancer Research 18:1954-1965 Dietrich, J., Prust M., and Kaiser, J. 2015. Chemotherapy impairments and hippocampal toxicity. Neuroscience 309: 224-232 Kitamura, Y., Hattori, S., Yoneda, S., et al. 2015. Doxorubicin and cyclophosphamide treatment produces anxiety-like behavior and spatial cognition impairment in rats: Possible involvement of hippocampal neurogenesis via brain derived neurotrophic factor and cyclin DI regulation. Behavioural Brain Research 292: 184-193 Lyons, L., EL Beltagy, M., Bennett, G., Wigmore, P. 2011. The effects of cyclophosphamide on hippocampal cell proliferation and spatial working memory in rat. Plos one 6:21445 Mustafa, S., Walker, A., Bennett, G., Wigmore, M. 2008. 5-fluorouracil chemotherapy affects spatialworking memory and newborn neurons in the adult rat hippocampus. European Journal of Neuroscience 28:323-330 Seigers, R. et al. 2015. Nerurobiological changes by cytotoxic agents in mice. Behavioural Brain Research. 299: 19-26 Yang, G., Kim, J.S., Kim, Ju., et al. 2012. Acute treatment with methotrexate induces hippocampal dysfunction on a mouse model of breast cancer. Brain Research Bulletin 89:50-56 Figure 4:Figure 1: The effect of methotrexate on learning, memory, and depressive-like behaviors. In the tail-suspension test, the time spent immobile was greatest in the methotrexate treatment groups and even greater in the tumor-bearing group. This is because previous studies show that tumor presence increases depressive-like behavior. Figure 5: Effects of methotrexate on learning, memory, and depressive-like behaviors. In the passive avoidance memory task, the control group and the methotrexate treated group with the addition of NS-393 showed the largest amount of memory formation. Figure 6:The effects of methotrexate on neurogenesis in the hippocampus. The amount of BrdU labeled proliferating cells decreased significantly in both methotrexate treatment groups and remained close to the control in the group that was injected with NS-393. Figure 7:The measurement of relative proinflammatory enzyme levels in the hippocampus. The relative OD of the methotrexate group injected with NS-393 was significantly decreased and remained at high levels in the other two methotrexate treatment groups Expected Results Continued Figure 3: Cell proliferation in the hippocampus showed to be much lower in mice treatment groups treated with doxorubicin and cyclophosphamide compared to the control group (Christie et al. 2012). http://rndb.clps.brown.edu/wp- content/uploads/2012/05/Barnes_1.jpg Expected Results Acknowledgements I would like to thank Dr. Bradley Rehnberg for all of his help and support During the tail-suspension behavioral test, the two groups treated with methotrexate will spend the largest amounts of time immobile (Figure 4). Following experimentation, the non-treatment tumor- free group and the tumor-present methotrexate with NS-393 group will have the highest cross-over latency time since cross-over latency is positively correlated with memory (Figure 5). Both methotrexate only treatment groups will have the lowest amount of new proliferating cells present in the hippocampus (Figure 7). Both methotrexate only treatment groups will have the highest abundancy of pro-inflammatory enzymes present in the hippocampus (Figure 7). In all of the results, the group that has the addition of NS-393 will be similar to those shown in the control groups. Research found that 5-Fluorouracil affected neurogenesis in the hippocampus indicated by a decrease in the levels of BDNF and DCX in the treatment group that received the cytotoxic agent. The results also showed that this specific chemotherapy drug did not affect the abundance of proliferating cells and only effected working memory slightly (Mustafa el al. 2008) When the effects of different chemotherapy drugs were compared, it showed that there was a decrease in microglia activation following short term treatment using cyclophosphamide, doxotaxel, 5-fluorouracil, and topotecan (Seiger et al 2015). Cyclophosphamide and doxorubicin, individually or in combination, induced anxiety-like behavior, decreased cell proliferation in the hippocampus, decreased cell survival in dentate gyrus, reduced the amount of red blood cells, and decreased BDNF levels. All of these effects lead to the destruction of neuroplasticity in the brain and caused cognitive impairments (Kitamura et al. 2015) Review of Literature Figure 1: Lyons et al. 2011 Figure 2: Briones and Woods (2014) results showed that NS-398, an inflammatory inhibitor, improved how rats performed in behavioral tests following treatment with CMF.