Agmatine improves methotrexate-induced neurotoxicity and spatial memory impairment in rats

Introduction

Methotrexate (MTX) is known to impair cognitive function, particularly at higher doses [1]. Agmatine has been suggested to produce neuroprotective effects by modulating alpha-2 receptors, NMDA receptors, nitric oxide pathways, and neuroinflammatory processes. Although many studies report its beneficial effects against memory impairment [2], no studies have yet investigated agmatine’s protective effects against MTX-induced memory impairment.

Aims

This study aimed to investigate whether agmatine reduces MTX-induced spatial memory impairment and neurotoxicity in rats. Moreover, locomotor activity and anxiety-related behavior were analyzed.

Methods

Wistar albino rats (n= 8 per group) were treated intraperitoneally with saline, MTX (40 mg/kg, applied on days 5 and 9), and MTX combined with agmatine (25, 50, or 100 mg/kg/day). Hippocampal tissues were formalin-fixed, paraffin-embedded, sectioned, and stained with H&E for histopathological evaluation under light microscopy. Locomotor activity and anxiety was assessed in the open field (OF) test, spatial memory in the modified elevated plus maze (mEPM) and the Morris Water Maze (MWM) test. In the MWM test, the probe trial was recorded and analyzed using EthoVision XT 18. Data were analyzed using one-way ANOVA followed by Tukey post-hoc test.

Results

MTX induced hippocampal damage characterized by pyknosis, vascular congestion, and edema, and combination of agmatine 50 and 100 mg/kg reduced these changes. In the OF test, the total distance moved, time spent in the center, and center entry frequency were similar. In the mEPM test, day two, the MTX applied group exerted higher transfer latency versus vehicle group (p<0.05). This increase was reversed in the 50 mg/kg agmatine combination applied group (p<0.05). In the MWM test probe trial, no significant difference was observed between groups in total distance moved, target quadrant entry frequency, and mean distance to the platform. Yet, time spent in the target quadrant was reduced in the MTX applied group (p<0.05) and was reversed in the agmatine 50 mg/kg combination applied group (p<0.05). Interestingly, agmatine combinations at 25 and 100 mg/kg did not significantly alter results in either memory test.

Discussion

Histological results have shown neuroprotective effect of agmatine in hippocampus, especially in higher doses indicated dose-associated protection. MTX led to spatial memory impairments, but it did not affect anxiety and locomotor activity. In the mEPM test, agmatine combinations producing a protective effect at 50 mg/kg dose suggest it is a modulatory molecule in a dose-associated manner. Similarly, in the MWM test, agmatine produced a dose-associated protective effect, with significance observed only at 50 mg/kg. This effect pattern supports a non-linear, dose-associated protective profile of agmatine. The lack of efficacy at low and high doses may also be influenced by reduced sample sizes, which limited statistical significance.

Conclusions

Our results confirm that agmatine reduces MTX-induced neurotoxicity and spatial memory impairment without altering anxiety and locomotor activity. These effects appear selective and dose-associated, supporting a potential neuroprotective role of agmatine in neurotoxicity-related memory impairment.

Acknowledgments: This work was supported by TÜBİTAK (Project No. 324S314)

References

1. Berlin C., Lange K., Lekaye H.C., Hopland K., Phillips S., Piao J., Tabar V., 2020. Long-term clinically relevant rodent model of methotrexate-induced cognitive impairment. Neuro Oncol. Aug 17;22(8):1126-1137.
2. Aglawe M.M., Kale M.B., Rahangdale S.R., Kotagale N.R., Umekar M.J., Taksande B.G., 2021. Agmatine improves the behavioral and cognitive impairments associated with chronic gestational ethanol exposure in rats. Brain Res Bull. Feb;167:37-47.