An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

Summary

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

Abstract

The cerebrospinal fluid (CSF) is a valuable body fluid for analysis in neuroscience research. It is one of the fluids in closest contact with the central nervous system and thus, can be used to analyze the diseased state of the brain or spinal cord without directly accessing these tissues. However, in mice it is difficult to obtain from the cisterna magna due to its closeness to blood vessels, which often contaminate samples. The area for CSF collection in mice is also difficult to dissect to and often only small samples are obtained (maximum of 5-7 µL or less). This protocol describes in detail a technique that improves on current methods of collection to minimize contamination from blood and allow for the abundant collection of CSF (on average 10-15 µL can be collected). This technique can be used with other dissection methods for tissue collection from mice, as it does not impact any tissues during CSF extraction. Thus, the brain and spinal cord are not affected with this technique and remain intact. With greater CSF sample collection and purity, more analyses can be used with this fluid to further aid neuroscience research and better understand diseases affecting the brain and spinal cord.

Introduction

The CSF is a valuable body fluid for analysis in neuroscience research. The CSF is primarily made from blood plasma, containing few cells (no red blood cells and only a few white blood cells) and is almost protein-free. It is one of the fluids in close contact with the central nervous system (CNS) and it can pass many electrolytes out from the brain and spinal cord to the peripheral system. In humans, CSF samples can be collected to aid in diagnosing disease or for research purposes in clinical trials, as a spinal tap (or lumbar puncture) is a minor, invasive procedure: the CSF fluid can reflect changes in the CNS without having to directly access these tissues. Thus,....

Protocol

All animal experiments were performed in accordance with the policies of the Society for Neuroscience (USA) and Fudan University (Shanghai, China) ethical committees. This procedure is for a non-survival surgery.

1. Setup of CSF Collection Apparatus

1. Pull the glass capillary (inner diameter 0.75 mm, outer diameter 1.0 mm) using a micropipette puller (as shown in Liu et al.¹⁰; sharpened capillary is shown in Figure 1).
2. Place a sharpened glass capillary into the capillary holder firmly mounted on a micromanipulator (Figure 1A).

Results

Using the procedure outlined here (Figure 1 and Figure 2), the CSF immediately collected in the capillary should be clear (Figure 2E), not pink or red. If there is a pink to red tinge to the fluid collected in the capillary, then there was contamination with blood.

As an example of the application of the CSF sample collected with this metho.......

Discussion

This protocol describes in detail a technique which improves on current methods¹⁰ of CSF collection to minimize contamination from blood and allow for the abundant collection of CSF (on average ~ 10-15 µL can be obtained) from mice. When breaking the capillary tip, the tip of the capillary should not be too small (as then the CSF will be extracted very slowly) or too big (will not be fine enough to collect the CSF and tissue can become lodged in the capillary). Care should be taken when .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chloral hydrate (used as anesthetic)	Sinopharm Chemicals Reagen Co. Ltd.	30037517	CAS number 302-17-0.
Dissecting scissors	66 vision technology	54002
Dissecting curved forceps	66 vision technology	53072
Dissecting straight forceps	66 vision technology	53070
Mouse adapter (with ear bars)	Made in-house.	N/A	Similar equipment available from World Precision Instruments.
Dissecting microscope	Meiji Labax	Model 15381
Micromanipulator	World Precision Instruments	M3301
Magnetic base for micromanipulator	Kanetec	MB-K
Glass capillaries	World Precision Instruments	1B100-4
Micropipette puller	Sutter Instruments	Model P-1000
Syringes (1ml)	Tansoole	02024692	For 1ml.
Microtubes (1.5ml)	Axygen	MCT-150-C
Protease inhibitor Cocktail Set III EDTA-free	Calbiochem	539134
Human Aβ42 ELISA kit	Invitrogen	KHB3441
Piping (teflon tubing)	World Precision Instruments	MMP-KIT	Obtained from a microinjection kit and attached to the capillary holder and syringe.
Mini centrifuge	Tiangen Biotech	OSE-MC8
Cotton buds	Obtained from any household store/pharmacy.	N/A