Extended Abstract
Background and Purpose
Introduction
Childhood infections represent one of the most significant and pervasive health challenges globally, with consequences extending far beyond the immediate acute phase of illness. A growing body of evidence suggests that systemic infections and the subsequent activation of the peripheral immune system during critical periods of postnatal brain development can lead to enduring alterations in neurobiology and behavior. These early-life immune challenges are increasingly implicated in the etiology and exacerbation of various neurodevelopmental disorders, including Autism Spectrum Disorder (ASD) and schizophrenia. The central mechanism underlying these long-term behavioral sequelae is postulated to be the induction of neuroinflammation. The developing brain is uniquely vulnerable to inflammatory insults. A systemic infection triggers a cascade of events, notably the production and release of pro-inflammatory cytokines such as Interleukin-6 (IL-6), Interleukin-1 beta (IL-1β), and Tumor Necrosis Factor-alpha (TNF-α). These peripheral signals can traverse the blood-brain barrier (BBB) or signal to the brain via vagal afferents and circumventricular organs, leading to the activation of resident immune cells—microglia and astrocytes—within the central nervous system (CNS). Activated microglia enter a state of chronic surveillance, resulting in a persistent, low-grade inflammatory environment within key brain regions. This sustained neuroinflammation disrupts essential developmental processes, including synaptic pruning, neurogenesis, and myelination, ultimately remodeling the neural circuitry responsible for complex behaviors. In this study, the LPS administration model was utilized in neonatal mice (Postnatal Day 5, PND 5) to mimic a robust, yet non-lethal, systemic infection that induces a lasting inflammatory phenotype and associated behavioral deficits. The focus of this research was on two critical brain regions: the Hippocampus (HPC) and the Prefrontal Cortex (PFC). The PFC is paramount for executive function, social cognition, and decision-making, while the HPC is central to learning, memory, and affective processing. Disruption in the functional and structural integrity of both regions has been consistently linked to social withdrawal and impaired cognitive flexibility, characteristic features observed in animal models following early-life immune activation. Specifically, elevated levels of the pro-inflammatory cytokine Interleukin-6 (IL-6) were chosen as the primary biochemical marker of neuroinflammation due to its central role in mediating communication between the immune system and the brain, and its established correlation with behavioral abnormalities. Given the significant public health implications of post-infectious neurobehavioral deficits, identifying effective, non-invasive, and accessible therapeutic strategies is imperative. The present investigation explores two distinct, yet potentially synergistic, intervention modalities: Minocycline and Aerobic Exercise (Swimming). Minocycline, a semi-synthetic tetracycline antibiotic, possesses potent anti-inflammatory and neuroprotective properties independent of its antimicrobial effects [5, 60]. Mechanistically, minocycline has been shown to suppress microglial activation, inhibit the release of pro-inflammatory cytokines, prevent the induction of key inflammatory enzymes (like iNOS and COX-2), and reduce excitotoxicity. Its ability to cross the BBB makes it a promising pharmacological agent for targeting CNS inflammation. Conversely, physical exercise, particularly aerobic training, is a powerful non-pharmacological intervention known to exert profound neurobiological benefits. Exercise is recognized to enhance neurogenesis, increase the expression of neurotrophic factors (e.g., Brain-Derived Neurotrophic Factor, BDNF), and significantly modulate the inflammatory status of the CNS. The routine of swimming was selected as a form of moderate-intensity aerobic exercise, suitable for the post-weaning developmental stage of the mice, and known for its stress-reducing properties. The primary objective of this study was, therefore, to rigorously assess the efficacy of post-infectious treatment with either swimming exercise, minocycline administration, or a combination of both, in mitigating the social behavioral impairments and the associated increase in IL-6 levels within the hippocampus and prefrontal cortex of NMRI mice subjected to a neonatal BCG challenge. This comprehensive approach aimed to provide critical evidence supporting the use of these interventions as potential translational strategies for managing the long-term sequelae of early-life immune activation.
Materials and Methods
Fifty newborn male NMRI mice were randomly assigned to five groups (n = 10): control (saline), LPS-only, LPS + swimming, LPS + minocycline, and LPS + swimming + minocycline. Systemic inflammation was induced via intraperitoneal injection of LPS (50 µg/kg) on postnatal days 3 and 5 [8]. Interventions began during adolescence (days 28–56). The swimming protocol consisted of four weeks of forced swimming, five sessions per week, with duration gradually increasing from 10 to 30 minutes per session. Water temperature was maintained at 32 ± 1°C, and mild water resistance was created using wave generators to prevent floating and enhance physical load. Minocycline (20 mg/kg/day) was administered intraperitoneally daily from days 28 to 42. At adulthood (day 115), social behavior was assessed using a 7-minute social interaction test in a square arena (80 × 80 × 20 cm), where time spent in direct contact (sniffing, chasing, touching, climbing over) with an unfamiliar mouse was recorded [11]. Following behavioral testing, animals were deeply anesthetized, transcardially perfused with saline, and brains rapidly extracted. Hippocampal and prefrontal cortical tissues were dissected, homogenized in TBS buffer containing Triton X-100, EDTA, PMSF, and protease inhibitors, and centrifuged at 15,000 g for 15 min at 4°C. Total protein concentration was determined using a BCA assay (Sigma), and IL-6 levels were measured via ELISA (Abcam, UK) according to manufacturer instructions. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test (P < 0.05) in SPSS v24.
Findings
The LPS-only group showed significantly reduced social interaction time compared to controls (P = 0.003), confirming lasting social deficits following early immune activation. Neither minocycline nor swimming alone significantly improved social behavior (P > 0.05); however, the combination of swimming and minocycline resulted in a marked improvement in social interaction (P = 0.030 vs. LPS group). Regarding neuroinflammation, IL-6 levels were significantly elevated in both the hippocampus (P = 0.016) and prefrontal cortex (P = 0.001) of the LPS group compared to controls. While individual interventions had minimal impact, the combined treatment led to a significant reduction in IL-6 in both regions: hippocampus (P = 0.016) and prefrontal cortex (P = 0.002). Notably, no significant change in IL-6 was observed with minocycline monotherapy, suggesting its limited efficacy without concomitant physical activity.
Conclusion
This study demonstrates that while minocycline or swimming alone may exert only modest effects after early-life infection, their combination effectively attenuates neuroinflammation and improves social deficits in adult mice. The synergistic effect likely arises from complementary mechanisms: minocycline suppresses microglial activation and pro-inflammatory signaling, while swimming enhances neuroplasticity, increases anti-inflammatory mediators, and promotes a shift from M1 to M2 microglial phenotype. These findings underscore the potential of multimodal interventions in addressing the long-term neurological consequences of childhood infections and support further exploration of combined pharmacological and lifestyle strategies in neurodevelopmental disorders.

Article Message
Early-life immune activation can cause persistent neuroinflammation and social deficits. This study examined the effects of swimming exercise, minocycline, and their combination in LPS-exposed mice. While either intervention alone showed limited benefits, combined swimming and minocycline significantly improved social behavior and reduced IL-6 levels in the hippocampus and prefrontal cortex. These findings highlight the potential of combined exercise and pharmacological approaches to reduce long-term neurological consequences of childhood infections.
Funding
This research received no external funding.
Authors' Contributions
All authors contributed to the study design, data interpretation, and manuscript revision. The first author conducted experiments, performed statistical analysis, and drafted the manuscript. Co-authors supervised procedures and provided critical feedback. All approved the final version.
Conflicts of Interest
The authors declare no competing interests.
Acknowledgement
We thank the Salari laboratory for providing animal facilities and technical support. We also acknowledge the laboratory staff for their assistance in sample processing.