Method for collecting cerebrospinal fluid from the cisterna magna of anesthetized mice and processing single-animal samples for proteomic analysis.

Authors

Clara Ciampi, Laura Micheli, Elena Lucarini, Annapaola Andolfo, Clarissa Braccia, Lorenzo Di Cesare Mannelli, Carla Ghelardini, Cristina Lanni

Published in

Journal of neuroscience methods. Pages 110923. Oct 26, 2026. Epub Oct 26, 2026.

Abstract

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.
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.
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.
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.
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.

PMID:
42933669
Bibliographic data and abstract were imported from PubMed on 27 Oct 2026.

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