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Novel Approach to Collecting CSF from Preclinical Model Requires No Special Equipment

Key findings

  • Because of the small size of the mouse brain, methods for collecting cerebrospinal fluid in vivo typically require custom-made equipment
  • Massachusetts General Hospital researchers have devised a highly reliable method to collect cerebrospinal fluid (CSF) from mice that is free of blood and other contaminants
  • The procedure is anatomy-oriented and involves only a stereotaxic frame, scalpel, cotton swabs, Hamilton syringe, needle and desktop microscope; furthermore, only basic surgical skills are required
  • Use of the stereotaxic frame and micromanipulator enables fine movement and precise localization of the needle of the Hamilton syringe, contributing to consistent outcomes
  • 10 microliters of CSF have been collected consistently from laboratory mice with a total procedure time of 20 minutes per animal

Obtaining cerebrospinal fluid (CSF) from mice that's free of blood and other contaminants is important in neuroscience and neuro-oncology research. However, harvesting CSF from living mice is difficult because of the small size of the mouse brain, and previously published methods require customized equipment.

Kazuhide Shimizu, MD, PhD, research fellow, Hiroaki Wakimoto, MD, PhD, associate professor of neurosurgery in the Department of Neurosurgery at Massachusetts General Hospital and the Mass General Cancer Center, and colleagues developed a procedure for reproducibly collecting CSF from athymic nu/nu mice using standard laboratory tools. Their report appears in Brain Research.

The Procedure

The article describes the procedure in detail and includes color photographs, tips and troubleshooting suggestions. In brief, the steps are:

  1. Administer anesthesia and a pain reliever, then fix the mouse's head to a stereotaxic frame
  2. Using a disposable scalpel, make a skin incision in the midline from the top of the skull to the neck, then sharply dissect the superficial muscle layer to expose the base of the occipital bone
  3. With cotton swabs, dissect the deeper layers of muscle around the base of the occipital bone to expose the puncture site
  4. Connect a 10-microliter Hamilton syringe with a 26-gauge needle (2 mm bevel length) and attach them to the syringe holder of the stereotaxic frame
  5. Point the needle tip, with the bevel side upward, at the transparent dura over the cisterna magna, where there is a pool of CSF
  6. Use a desktop microscope to identify the point of puncture: the triangular-shaped structure outlined by the medial lines of bilateral gracile tubercles of the medulla oblongata and the uvula of the cerebellar vermis
  7. Puncture the cisterna magna with the needle and draw out CSF; approximately 10 microliters is typically collected in five minutes
  8. Suture the skin with a single knot and euthanize the animal

The total procedure time is usually 20 minutes.


After a few practice sessions, the researchers performed this procedure on 12 female athymic nu/nu mice weighing 25 to 31 grams:

  • CSF removed: 11 mice; in one case the syringe needle clogged
  • CSF removed uncontaminated by blood: 11 mice; in the other case the needle tip probably hit the brain parenchyma
  • More than five microliters of CSF collected: Nine mice, including four mice from which ≥10 microliters was collected (maximum, 11.1 microliters)

The researchers find five microliters of CSF adequate for pharmacokinetics and exosome extraction studies.

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