Effects of Cyclophosphamide and Naproxen on Behavior, Neuroinflammation, and Neurogenesis

Cancer patients undergoing chemotherapy report changes in behavior commonly referred to as “chemo brain.” It is hypothesized that neuroinflammation contributes to the pathophysiology of “chemo-brain.” I examined the effects of naproxen, a non-steroidal anti-inflammatory drug (NSAID), on behavior, hippocampal neuroinflammation, and hippocampal neurogenesis produced by exposure to cyclophosphamide (CP), a widely used chemotherapy agent in a female mouse model. Since chemotherapy can impair estrogen synthesis, I included ovariectomized (OVX) mice in my study to examine if ovarian hormones modified the effects of CP and naproxen. An initial study analyzing 50 mg./kg. CP was used (i.p., every third day for five doses) in intact and OVX mice. CP (50 mg./kg.) significantly decreased bodyweight throughout the experimental period. There was no effect of OVX on bodyweight. There was no effect of CP (50 mg./kg.) on anxiety-like behavior, spatial working memory, or object-based attention (OBA). OVX increased the recognition index on the OBA test, likely due to increased exploration with the objects, but had no effect on anxiety-like behavior or spatial working memory. In the following study, the effects of a higher dose of CP (100 mg./kg., i.p., every third day for five doses) and naproxen were examined in intact and OVX mice. Naproxen administration began one week before CP administration and continued throughout the experiment. High-dose CP significantly decreased bodyweight. This reduction was significant to OVX mice treated with CP and was not significant in intact mice treated with CP. There was no effect of naproxen on bodyweight. Naproxen exposed mice ate significantly more food per day, compared to vehicle-treated mice. Intact mice ate significantly more food per day, compared to OVX mice. OVX mice exposed only to CP ate significantly less food per day compared to OVX mice exposed to CP and naproxen and OVX mice exposed only to naproxen. This trend was not observed in intact mice. All mice in the high-dose CP and naproxen study spent >85% of the time in the closed areas, indicating an anxiety-like behavior phenotype present across all experimental groups. CP significantly decreased protected head dips, a secondary measure of anxiety-like behavior. There were no effects of high-dose CP, naproxen, or OVX on spatial working memory. High-dose CP decreased exploratory behavior in a novel environment. This effect was specific to intact mice treated with high-dose CP and diminished in intact mice treated with both high-dose CP and naproxen. High-dose CP decreased spontaneous locomotor activity. This effect was significant in both intact and OVX mice treated with high-dose CP and diminished in mice exposed to both high-dose CP and naproxen. There was no effect of OVX on exploratory behavior in a novel environment or spontaneous locomotor activity. Lastly, there was a significant interaction between high-dose CP and time spent immobile on the tail suspension test, and between CP and naproxen on latency to immobility, measures of depression-like behavior. The combination of CP and naproxen increased the acute inflammatory marker, IL-1a, the chronic inflammatory markers, IL-2, IL-3, and IL-12, and the anti-inflammatory marker, IL-10, in the hippocampus. Post hoc comparisons confirmed that elevated inflammatory markers were seen in CP and naproxen treated mice, which may indicate an inflammatory process induced by CP, which was heightened by naproxen, contrary to our hypothesis. OVX mice had decreased levels of acute, chronic, and anti-inflammatory markers in the hippocampus compared to intact mice, regardless of CP or naproxen exposure. Analysis of neurogenesis in the dentate gyrus of the hippocampus using the S-phase marker, bromodeoxyuridine (BrdU), and the newborn neuronal marker, doublecortin (DCX), was examined using both doses of CP. CP produced significant decreases in BrdU+ cells and BrdU+DCX+ cells at 50 mg./kg. but not at 100 mg./kg. The interaction between CP (50 mg./kg.) and OVX on the number of BrdU+ cells and BrdU+DCX+ cells was significant, suggesting that 50 mg./kg. CP modified the effects of OVX on neurogenesis. A significant interaction between CP (100 mg./kg.) and naproxen on neurons that were “born” over the interval between the first BrdU administration and the time of sacrifice (percent of BrdU+DCX+ cells/total BrdU+ cells) was found, suggesting the neurogenesis was altered by the co-administration of both CP and naproxen. Collectively, our data show that chemotherapy alone, in the absence of cancer, produces behavioral impairments, which may be mediated by neuroinflammatory changes and changes in hippocampal neurogenesis. Lastly, naproxen may exacerbate cyclophosphamide-induced neuroinflammation and may not be a suitable drug for mitigating cyclophosphamide-induced adverse effects.