Methotrexate impairs spatial memory and induces dose-associated neurodegeneration in rats

Introduction: Methotrexate (MTX) is a widely used drug in oncology and autoimmune disorders. Besides being clinically efficient, it is also associated with neurotoxic side effects, and it is reported to impair cognitive functions. MTX-related cognitive decline has been linked to oxidative stress, neuroinflammation, disruption of folate metabolism, reduced levels of neurotrophic factors, and activation of apoptotic pathways [1]. Previous studies also reported MTX-induced structural brain changes, especially in hippocampus [2].

Aim: This study aimed to investigate the dose-dependent effects of MTX on spatial learning and memory in rats. Additionally, biochemical alterations in hippocampus and histopathological investigations in various brain regions were also evaluated to assess dose-dependent neurotoxicity.

Methods: Male Wistar albino rats (n = 8 per group) received MTX (10, 20, and 40 mg/kg, i.p.) or saline. MTX was administered twice on days 5 and 9. Spatial learning and memory were assessed by the Morris Water Maze (MWM) test. Behavioral data were analyzed using one-way ANOVA followed by Tukey post-hoc test. On day six, a probe trial was executed, and rat movements were recorded for 60 seconds and then analyzed using EthoVision XT 18. For histopathological evaluation, hippocampus, cortex, and white matter were analyzed following staining with hematoxylin&eosin. ELISA analyses were performed to quantify hippocampal levels of BDNF, NGF, caspase-3, caspase-9, TNF-α, IL-1, and IL-6.

Results: No significant difference was observed in total distance moved and target quadrant entry frequency in the MWM test. But, the mean distance to the platform was increased in the 40 mg/kg MTX applied group (p<0.01), and the time spent in the target quadrant was reduced (p<0.05). Histologically, no alteration was observed in the 10 mg/kg treated group. Hippocampal neurodegenerative changes were observed in the 20 mg/kg MTX applied group, while the 40 mg/kg applied group exhibited widespread damage, including cortical and white-matter involvement with apoptotic features. The ELISA test results have shown non-significant decrease in BDNF levels. NGF levels were increased at 20 mg/kg (p<0.05), but declined at 40 mg/kg. Caspase-9 and caspase-3 levels were increased in the 40 mg/kg group (p<0.01), indicating activation of the apoptotic pathway. IL-1 levels were increased in all MTX groups. TNF-α and IL-6 levels were unchanged.

Discussion: MTX impaired spatial memory in a dose-associated manner without affecting locomotion. Histopathological findings demonstrated dose-dependent neurodegeneration, primarily in the hippocampus. ELISA results support these findings, showing activation of apoptotic pathways and selective neuroinflammatory responses. The increase in NGF at moderate doses may indicate a temporary compensatory response; its decline at higher doses may indicate failure of neuroprotection.

Conclusion: Our results confirm that MTX induces dose-associated neurotoxicity characterized by cognitive impairment, structural damage, apoptosis, and selective neuroinflammation. Moreover, neurotrophic and inflammatory pathways may be critical in MTX-induced neurotoxicity. These findings emphasize the importance of MTX dosage in clinical practice.

References

1. Chen Y.C., Sheen J.M., Wang S.C., Hsu M.H., Hsiao C.C., Chang K.A., Huang L.T., 2021. Methotrexate Neurotoxicity Is Related to Epigenetic Modification of the Myelination Process. Int J Mol Sci. Jun 23;22(13):6718.
2. Seigers R., Schagen S.B., Coppens C.M., van der Most P.J., van Dam F.S., Koolhaas J.M., Buwalda B., 2009. Methotrexate decreases hippocampal cell proliferation and induces memory deficits in rats. Behav Brain Res. 2009 Aug 12;201(2):279-84.