Central nervous system resilience to cyclophosphamide: preserved behavior, astrocytes and LGALS3 expression in immunosuppressed mice.

Background: Cyclophosphamide is widely used as an immunosuppressive agent in experimental models. However, its potential effect on central nervous system (CNS) function and behavior may confound studies investigating neuroimmune interactions, interpretation of results, and translational relevance. Objectives: To characterize longitudinal behavioral outcomes and CNS-related molecular and histological changes following repeated cyclophosphamide-induced immunosuppression in mice. Methods: Adult male C57BL/6 mice received an intraperitoneal injection of cyclophosphamide (100 mg/kg) for three or four consecutive weeks. Peripheral immunosuppression was assessed by body weight monitoring and leukocyte differential counts. Behavioral performance was evaluated longitudinally using the SHIRPA protocol, open-field test, and tail suspension test (TST). CNS integrity was analyzed through histopathology, GFAP immunohistochemistry, and quantitative PCR analysis of Lgals3 in brain tissue. Results: Cyclophosphamide administration induced a sustained reduction in body weight gain and significant alterations in leukocyte profile, confirming immunosuppression. Behavioral assessment did not demonstrate significant changes, only mild and transient ones, mainly related to muscle strength and subtle sickness-like behavior. No histopathological alterations or astrocytic activation were detected in brain tissue, as GFAP immunoreactivity remained unchanged across experimental groups. Lgals3 gene expression was significantly upregulated in the brains of immunosuppressed animals following both three and four cycles. Conclusion: Repeated low-dose cyclophosphamide administration induces effective peripheral immunosuppression without causing overt neuroinflammation or major impairment, indicating CNS resilience under these experimental conditions. The upregulated Lgals3 expression suggests an adaptive neuroimmune response that may contribute to maintaining CNS homeostasis during systemic immune dysregulation. These findings support the use of this immunosuppressive protocol in experimental models targeting CNS infections or neuroimmune mechanisms while minimizing confounding neurotoxic effects.