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Updated: Oct 28, 2026
An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice
Published on: March 19, 2018
Method for collecting cerebrospinal fluid from the cisterna magna of anesthetized mice and processing single-animal
Clara Ciampi1, Laura Micheli1, Elena Lucarini1
1Department of Neuroscience, Psychology, Drug Research and Child Health-NEUROFARBA, Pharmacology and Toxicology Section, University of Florence, 50139 Florence, Italy.
Background:
CSF proteomics in preclinical mouse models holds great potential for biomarker discovery and CNS disease characterization, but low sample volume and protein content limit the applicability of existing protocols.
New Method:
We describe a workflow for cisternal CSF collection and single-animal proteomic analysis in mice, combining: continuous subcutaneous ketamine/xylazine infusion for extended anaesthesia; semi-automated microinjector-based withdrawal at a constant aspiration rate; syringe preloading with protease inhibitors and cold storage to prevent degradation; and bead-based protein enrichment (ENRICH-iST, PreOmics) for dynamic range reduction without sample pooling.
Results:
The procedure yields 20µl of CSF per animal and was validated in healthy mice and a DSS-induced colitis model, enabling identification and quantification of 502 proteins from single-animal samples. The coefficient of variation in quantified proteins was below 10%, demonstrating adequate reproducibility for comparative proteomic analyses.
Comparison With Existing Methods:
Bead-based enrichment provided higher protein recovery than immunodepletion without pooling, preserving inter-individual variability. Semi-automated withdrawal reduces operator-dependent variability compared to manual aspiration. The protocol relies exclusively on commercially available equipment, unlike methods requiring custom-built tools.
Conclusions:
This workflow enables reliable single-animal CSF proteomics in mice, including debilitated animals, applicable to both basic research and preclinical pharmacological settings. Reproducibility is supported by a step-by-step protocol and troubleshooting guide. The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE repository (PXD058129) and are publicly available.
