Proteomic Profiling of Cerebrospinal Fluid in Adult Rats with Compromised Lymphatic Drainage Using DIA-PASEF

The glymphatic system’s outflow of “dirty” brain fluids collects in the meningeal lymphatic vessels, which drain via lymphatic collectors primarily to the deep cervical lymph nodes (dcLN). It is unclear, however, whether lymphatic dcLN drainage is a prerequisite for normal cerebral homeostasis. Here, we applied proteomic profiling of cerebrospinal fluid (CSF) using Data-Dependent Acquisition (DDA) and Data-Independent Acquisition Parallel Accumulation–Serial Fragmentation (DIA PASEF) approaches to characterize the CSF obtained from 3-month-old young rats. The study included two sets of samples: Sham (n=5) and Cauterized dcLN (n=4). For spectral library generation, 20% of each digested sample was pooled and analyzed using timsTOF Pro2 in DDA PASEF mode. Following bottom-up analysis, enrichment analysis using Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) identified several pathways indicative of a damaging phenotype and cellular stress-related neurodegeneration. The CSF proteomic analysis revealed cellular stress in the form of low-grade inflammation and up-regulation of pathways associated with neurodegeneration in young rats with lymphatic drainage impairment. Our findings illuminate the brain’s age-dependent adaptive mechanisms to chronic mechanical stress and underscore the urgency of developing targeted therapies for lymphatic dysfunction.