Abstract
BACKGROUND: Dynamic contrast-enhanced MRI has become an invaluable tool for mapping cerebrospinal fluid (CSF) flow and delineating solute transport pathways in the brain. However, the influence of experimental parameters, including infusion location, infusion rate, and anesthetic regimen, on tracer transport kinetics and distribution patterns remains incompletely characterized, potentially confounding interpretation of results. METHODS: We investigated the impact of infusion parameters on tracer transport in mice using Gd-DTPA as a CSF tracer. The effect of infusion rate was assessed by administering Gd-DTPA into the cisterna magna (ICM) at two different rates under isoflurane anesthesia. The influence of anesthesia was evaluated by comparing tracer transport patterns under isoflurane versus ketamine/xylazine and low-dose isoflurane at the slower infusion rate. To examine the impact of infusion site, Gd-DTPA was also delivered into the lateral ventricle (ICV) for comparison with ICM delivery. Region-specific signal time courses were analyzed using cross-correlation and hierarchical clustering to characterize tracer transport pathways. RESULTS: Tracer transport within the brain was significantly influenced by infusion location, rate, and anesthetic regimen. ICV infusion produced rapid, extensive transport into deep brain structures, while ICM infusion promoted transport toward ventral regions. Cross-correlation analysis revealed that ICM infusion primarily facilitated tracer transport along periarterial spaces, whereas ICV infusion favored transport across the ventricular-parenchymal interface. Hierarchical clustering of region-specific signal time courses further demonstrated distinct transport patterns associated with different infusion sites. CONCLUSION: Experimental conditions substantially impact tracer transport kinetics and its spatial distribution in CSF dynamics studies. While infusion site determines primarily transport pathways, i.e., via periarterial spaces versus across ventricular-parenchymal interface, infusion rate and anesthesia modulate transport efficiency. These findings underscore the importance of methodological considerations in the design and interpretation of CSF tracer studies and emphasize the need for standardized protocols to facilitate meaningful comparisons across research groups. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12987-026-00758-w.