Effects of early chemotherapeutic treatment on learning in adolescent mice: implications for cognitive impairment and remediation in childhood cancer survivors

Abstract

Purpose: Among children diagnosed with acute lymphoblastic leukemia (ALL) and given chemotherapy-only treatment, 40% to 70% of survivors experience neurocognitive impairment. The present study used a preclinical mouse model to investigate the effects of early exposure to common ALL chemotherapeutics methotrexate (MTX) and cytarabine (Ara-C) on learning and memory.

Experimental design: Preweanling mouse pups were treated on postnatal day (PND) 14, 15, and 16 with saline, MTX, Ara-C, or a combination of MTX and Ara-C. Nineteen days after treatment (PND 35), behavioral tasks measuring different aspects of learning and memory were administered.

Results: Significant impairment in acquisition and retention over both short (1 hour) and long (24 hours) intervals, as measured by autoshaping and novel object recognition tasks, was found following treatment with MTX and Ara-C. Similarly, a novel conditional discrimination task revealed impairment in acquisition for chemotherapy-treated mice. No significant group differences were found following the extensive training component of this task, with impairment following the rapid training component occurring only for the highest MTX and Ara-C combination group.

Conclusions: Findings are consistent with those from clinical studies suggesting that childhood cancer survivors are slower at learning new information and primarily exhibit deficits in memory years after successful completion of chemotherapy. The occurrence of mild deficits on a novel conditional discrimination task suggests that chemotherapy-induced cognitive impairment may be ameliorated through extensive training or practice.

Figure 1

Effects of MTX and Ara-C on body weight in pups injected on PND 14, 15, and 16. Overall, no significant differences in body weights were observed at PND 35 prior to behavioral testing. Values are Mean ± SEM. Ordinate: Weight in grams. Abscissa: Postnatal day.

Figure 2

Effects of early treatment of MTX and Ara-C on mean-adjusted latency (A), reinforced dipper response rate (B), and non-reinforced response rate (C) on day 1 acquisition sessions (open bars) and day 2 retention sessions (filled bars) in an autoshaping task on PND 35. (A) Amount of time to obtain the tenth reinforcer was compared between mice treated with saline, MTX, Ara-C, or a MTX and Ara-C combination on day 1 and day 2. Mice treated with 2 MTX or a combination of MTX and Ara-C displayed a significantly slower acquisition on day 1, compared to saline controls. ^p < 0.01, ^^p < 0.001. Mice treated with MTX or the higher MTX and Ara-C combination showed impaired retention on day 2. *p < 0.05. Ordinate: Latency to the tenth reinforcer minus the latency to the first reinforcer, in seconds. (B) Total reinforced response rates in the dipper nose-poke hole were compared between mice treated with saline, MTX, Ara-C, or a MTX and Ara-C combination on day 1 and day 2. All groups except for the lower MTX and ARA-C combination displayed a significantly increased dipper response rate on day 2 compared to day 1. *p < 0.05, **p < 0.01, ***p < 0.001. Ordinate: Rate of nose-poke responses per second in the dipper well. (C) Total non-reinforced response rates in the left and right nose-poke holes were compared between mice treated with saline, MTX, Ara-C, or a MTX and Ara-C combination on day 1 and day 2. All groups displayed a significantly decreased non-reinforced response rate on day 2 compared to day 1. *p < 0.05, **p < 0.01, ***p < 0.001. Ordinate: Rate of nose-pokes in either the left or right holes per second as a measure of general activity. All Abscissa: Saline or chemotherapy treatment group (dose in milligrams per kilogram, administered i.p.). Values are Mean ± SEM.

Figure 3

Effects of early treatment of MTX and Ara-C on novel object recognition at PND 35: (A) Discrimination index and (B) Total exploration time. (A) Discrimination of the novel object compared to the familiar object following a 1h delay was compared between mice treated with saline, MTX, Ara-C, or a MTX and Ara-C combination. Mice treated with the higher dose of MTX (2 mg/kg), Ara-C (20 mg/kg), or MTX and Ara-C combination (2 mg/kg MTX and 20 mg/kg Ara-C) spent significantly less time exploring the novel object during the recognition trial, compared to saline controls. *p < 0.05. Ordinate: (Time spent exploring the novel object/time spent exploring the familiar object) × 100. (B) Total exploration time of both objects during the recognition trial was compared between mice treated with saline, MTX, Ara-C, or a MTX and Ara-C combination. There were no significant differences observed between treatment groups. Ordinate: Time spent exploring the novel object + familiar object, in seconds. All Abscissa: Saline or chemotherapy treatment group (dose in milligrams per kilogram, administered i.p.). Values are Mean ± SEM.

Figure 4

Effects of early treatment of MTX and Ara-C on conditional discrimination at PND 35: (A) Acquisition and (B) Discrimination ratio following extensive and rapid training. (A) Number of sessions to reach criteria for Phase 1 of the conditional discrimination procedure was compared between mice treated with saline, MTX, Ara-C, or a MTX and Ara-C combination. There was a significant effect of pre-weanling treatment on acquisition, although only mice treated with 2 mg/kg MTX or 10 mg/kg Ara-C required a significantly higher number of sessions to reach criteria compared to saline controls. *p < 0.05. Ordinate: Number of sessions required to reach Phase 1 criteria. Abscissa: Saline or chemotherapy treatment group (dose in milligrams per kilogram, administered i.p.). (B) Accuracy in responding was compared between mice treated with saline, MTX, Ara-C, or a MTX and Ara-C combination following extensive and rapid training. There was no significant difference in discrimination ratio between groups following extensive training. There was a significantly lower discrimination ratio in mice treated with the higher combination (2 mg/kg MTX and 20 mg/kg Ara-C), relative to saline controls, following rapid training. *p < 0.05. Ordinate: [correct responding/(correct + incorrect responding)]*100. Abscissa: Training type. Values are Mean ± SEM.