Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

Summary

We describe an intracisternal injection that employs a needle bent at the tip that can be stabilized to the skull, thus eliminating the risk of damage to the underlying parenchyma. The approach can be used for genetic fate mapping and manipulations of leptomeningeal cells and for tracking cerebrospinal fluid movement.

Abstract

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the underlying parenchyma. Previously published protocols recommend using straight needles that should be lowered to a maximum of 1-2 mm from the dural surface. The sudden drop in resistance once the dural membrane has been punctured makes it difficult to maintain the needle in a steady position. Our method, instead, employs a needle bent at the tip that can be stabilized against the occipital bone of the skull, thus preventing the syringe from penetrating into the tissue after perforation of the dural membrane. The procedure is straightforward, reproducible, and does not cause long-lasting discomfort in the operated animals. We describe the intracisternal injection strategy in the context of genetic fate mapping of vascular leptomeningeal cells. The same technique can, furthermore, be utilized to address a wide range of research questions, such as probing the role of leptomeninges in neurodevelopment and the spreading of bacterial meningitis, through genetic ablation of genes putatively implicated in these phenomena. Additionally, the procedure can be combined with an automatized infusion system for a constant delivery and used for tracking cerebrospinal fluid movement via injection of fluorescently labelled molecules.

Introduction

Leptomeningeal cells are a fibroblast-like population of cells organized in a thin layer overlaying the brain and expressing genes implicated in collagen crosslinking (e.g., Dcn and Lum), and in the establishment of a brain-meningeal barrier (e.g., Cldn11)^1,2. Leptomeningeal cells are implicated in a wide range of physiological functions, from strict control over the cerebrospinal fluid drainage³ to guidance of neural progenitors in the developing brain^4,5. A recent study has also proposed that leptome....

Protocol

The surgical procedures hereby presented were approved by Stockholms Norra Djurförsöksetiska Nämnd and carried out in agreement with specifications provided by the research institute (Karolinska Institute, Sweden).

NOTE: Intracisternal injection can be flexibly adapted for multiple research purposes. We present below a procedure developed to efficiently label leptomeningeal cells for fate mapping based on injection of endoxifen in a transgenic mouse line carrying R26R-tdTomato¹² and CreER, the latter under the Cx30 promoter¹³. Labelling of this population of cel....

Results

Intracisternal injection of endoxifen in transgenic mice expressing CreER under the Cx30 promoter¹³ and an inducible fluorescent reporter allows for specific recombination of leptomeningeal cells without labelling the neighboring Cx30-expressing surface and parenchymal astrocytes in the cortex (Figure 1). In order to gain access to the cisterna magna, the anesthetized animal is positioned with its body and its head at an angle of appro.......

Discussion

The protocol outlined here presents a straightforward and reproducible procedure to label leptomeningeal cells for fate mapping. We use intracisternal injection of endoxifen, an active metabolite of Tamoxifen, to induce expression of tdTomato fluorescent reporter in Cx30-CreER; R26R-tdTomato mice^12,13.

Compared to other protocols used for gaining access to the cerebrospinal fluid through the cisterna magna⁹, our.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anesthesia unit	Univentor 410	8323102	Complete of vaporizer, chamber, and tubing that connects to chamber and mouse head holder
Anesthesia (Isoflurane)	Baxter Medical AB	000890
Betadine	Sigma-Aldrich	PVP1
Carprofen	Orion Pharma AB	014920	Commercial name Rymadil
Cyanoacrylate glue	Carl Roth	0258.1	Use silk 5-0 sutures, in alternative
Medbond Tissue Glue	Stoelting	50479
DMSO	Sigma-Aldrich	D2650
Endoxifen	Sigma-Aldrich	E8284
Ethanol 70%	Histolab	01370
Hamilton syringe (30 G beveled needle)	Hamilton	80300
Lidocaine	Aspen Nordic	520455
Mouse head holder	Narishige International	SGM-4	With mouth piece for inhalational anhestetics. Alternatively, use a stereotactic frame
Scissors	Fine Science Tools	15009-08
Shaver	Aesculap	GT420
Sterile absorption spears	Fine Science Tools	18105-01	Sterile cotton swabs are also a good option
Surgical separator	World Precision Instrument	501897
Tweezers	Dumont	11251-35
Viscotears	Bausch&Lomb Nordic AB	541760