Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

Summary

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

Abstract

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

Introduction

The CSF is a complex fluid that bathes the developing neuroepithelium ^1-6 and provides essential pressure ⁷ and growth promoting cues for the developing brain ^8-12. To study the CSF over the course of brain development, we developed techniques to isolate ventricular CSF from developing rat or mouse embryos during various stages of development ^6,9. Previous methods of isolation included using a glass micro-needle and isolating the CSF using a micro-injector ^1,2. Our method utilizes a glass micro-capillary pipette whose tip has been pulled to create an ultrafine point for improved tissue penetration. The glass micro....

Protocol

1. Embryo Isolation/Preparation

This technique can be used for mouse or rat. In this protocol we demonstrate the CSF collection technique and cerebral cortical explant dissection with mouse embryonic brain. We will comment on any important differences for rats versus mice that exist within the general techniques. For the embryonic age staging system, E1 is classified as the day of the plug for rats, and E0.5 is classified as the day of the plug for mice.

1. Prepare a micro-dissection dish with Sylgard Silicone Elastomer. Sylgard 184 Silicone Elastomer is supplied as a two part liquid component, Part A and Part B. Part A and Part B ....

Results

The CSF collection should yield a clear, transparent fluid. There should be no evidence of contamination from blood, as demonstrated by a red or yellow tinged fluid in the aspirate and in the Eppendorf tube. There should also be no evidence of tissue in the aspirate and Eppendorf tube. When the CSF is centrifuged, one can also assess the CSF microscopically to ensure that there is no contamination. If there are signs of contamination, the CSF should be discarded. From one E14.5 mouse litter, one can anticipate co.......

Discussion

The described method for ventricular CSF collection has yielded relatively pure samples of embryonic CSF with stable protein composition and consistent activity in a number of cellular assays ⁹ . With a good collection technique and litter size of ten E14.5 mice, one can expect to collect 10-15 μl of CSF, and from a litter of E16 rats, one can expect to collect about 50-90 μl of CSF. This collection technique minimizes contamination from blood and tissue, by careful observation, centrifugation, and.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Wiretrop II capillary needles	Drummond Scientific	#5-000-2020
Sylgard	Ellsworth Adhesives	#184 SIL ELAST kit
Aspirator tube assembly	Sigma	#A5177-5EA
Disposable filter	Venturi or local pharmacy	not available	Standard cigarette filter
Roundstock opthalmic knife (15 degree stab knife)	World Precision Instruments, Inc.	#500250
35mm glass bottomed culture dish	MatTek Corp.	#P35G-1.5-14-C
Platinum-iridium wire	Tritech Research	#PT-9010-010-3FT
Nuclepore Track-Etch Membrane	Whatman	#09-300-57
Hanks Balanced Salt Solution	Fisher Scientific	#SH30031.FS
Iridectomy Scissors	Fine Science Tools	#15000-02