Modified rat pup cerebrospinal fluid collection method

Abstract

BackgroundCerebrospinal fluid (CSF) reflects biochemical changes in the brain due to its direct contact with brain interstitial fluid, making it a valuable tool for diagnosing and monitoring disease progression and therapeutic effectiveness in clinical practice. However, collecting CSF in animal studies, particularly from small animals like rat pups or mice, poses significant challenges. New methodAfter attempting various reported protocols, we encountered difficulties in consistently obtaining sufficient CSF from rat pups (P7-P42). Consequently, we modified these methods and developed a protocol with controllable and precise parameters for each step, enhancing reproducibility across different researchers. ResultsThe newly developed method enables rapid, single-operator, and reproducible CSF extraction while ensuring high-quality (the absorbance of the “quality control solution” at 415 nm < 0.05 AU, an indicator of oxyhemoglobin contamination for the collected CSF samples) and high-yield samples (33 ± 2.128 μL for P7 pups, 34.10 ± 2.747 μL for P8 pups, 36.67 ± 3.997 μL for P9 pups, 36.90 ± 1.946 μL for P10 pups, 35.11 ± 3.285 μL for P10 hypoxic-ischemic brain damage (HIBD) pups and 51.70 ± 5.256 μL for P42 pups, respectively). Comparison with existing methodsUnlike existing methods of CSF extraction in rat pups, our protocol has reproducible capillary pipette pulling parameters, controllable CSF quality indexes, and can be operated by a single person with high yield in a short time. ConclusionsThis paper provides a step-by-step comparison and discussion of the CSF collection process, establishing a method that enables a single operator to collect CSF rapidly, consistently, sufficiently, and with controlled quality.