Integrated Protocol for Assessing Meningeal Lymphatics: Cisterna Magna Injection, Lymph Node Imaging, Meningeal Dissection, and Whole-Mount Staining

Summary

Here, a protocol is presented to assess mouse meningeal lymphatic vessels (MLVs), which play an important role in multiple neurologic diseases. The goal of this protocol is to provide an integrated method that enables both functional evaluation of tracer drainage and detailed morphological characterization of the dorsal MLVs network.

Abstract

Meningeal lymphatic vessels (MLVs) have emerged as critical regulators of central nervous system (CNS) homeostasis, and MLVs dysfunction has been implicated in various neurological disorders. A comprehensive assessment of MLVs' morphology and function is therefore essential for understanding their role in CNS physiology and disease. This protocol describes an integrated method for evaluating MLVs in mice using four key techniques: Intra-cisterna-magna (i.c.m.) injection of fluorescent tracers, live imaging of cervical lymph nodes (CLNs), meningeal dissection, and whole-mount staining. First, the 30 G needle is connected to the syringe and infusion pump via a polyethylene (PE) tube. After the mouse is anesthetized, the dura mater is exposed by blunt dissection of the neck muscles, and the injection needle is inserted into the cisterna magna (CM) to deliver the tracer into the cerebrospinal fluid (CSF) with precise control. Tracer drainage to the CLNs can be visualized in real time, while subsequent meningeal dissection and whole-mount staining enable detailed morphological analysis of the dorsal MLVs. The entire procedure can be completed within 3 h, encompassing i.c.m. injection, tissue perfusion, skull isolation, and placement in fixative solution. This protocol can also be adapted for intracranial drug delivery or combined with molecular analyses, as the meningeal dissection procedure is compatible with fresh, unfixed tissues suitable for RNA and protein extraction.

Introduction

MLVs are located within the dural sinuses and are able to carry CSF components and immune cells from the CNS to the CLNs^1,2. The discovery of MLVs has challenged the traditional concept of central nervous system "immune privilege" and has emerged as a research hotspot in recent years³. Studies have shown that impaired MLVs function is closely associated with various diseases, including Alzheimer's disease, age-related cognitive decline, brain tumors, and CNS infections^4,5,6,

Protocol

The mouse procedures described in this protocol were performed in compliance with the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai University (Approval No. ECSHU 2023-099). 

1. Preparation of surgical instruments and buffers

CAUTION: Operate in a chemical fume hood and wear gloves to minimize the potential risks when working with PFA, a moderately toxic and carcinogenic substance. All operations involving Isoamyl alcohol must be carried out in a chemical fume hood away from ignition sources, while wearing gloves and a mask, to reduce exposure risks.<....

Results

Following i.c.m. injection of OVA-647, in vivo imaging of small animals revealed distinct oval-shaped fluorescent signals distributed bilaterally in the cervical region (Figure 6A). In successful injections, the tracer was clearly visible in the CLNs after injection, with fluorescence intensity peaking at approximately 60 min (Figure 6B). In addition, fluorescent signals were observed in the isolated superficial and deep cervical lymph nodes (

Discussion

This protocol provides a comprehensive method for evaluating MLVs function and morphology by integrating i.c.m. injection, in vivo imaging of CLNs, meningeal dissection, and whole-mount staining. A key advantage of this integrated approach over conventional methods is that it enables simultaneous assessment of lymphatic drainage function and vessel morphology within the same animal, eliminating the need for separate cohorts and thereby reducing experimental variability while improving data comparability. Further.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ACSF (Artificial Cerebrospinal Fluid)	Tocris	Cat# 3525
Alexa Fluor 488 donkey anti-rat IgG (1:1000 dilution)	Invitrogen	Cat# A-21208
Antifade Mounting Medium	Beyotime	Cat# P0126
Blunt-tipped forceps	RWD Life Science	Cat# F12011-10
BSA	Beyotime	Cat# ST023
Coverslips	CITOTEST	Cat# 80312-3161
Curved sharp forceps	RWD Life Science	Cat# F11003-11
DAPI	Sigma-Aldrich	Cat# D9542
Depilatory cream	Veet	N/A
Dish-10 cm	Thermo Fisher Scientific	Cat# 150464
Disposable intravenous (IV) infusion needle	Zhejiang Kindly Medical Devices	N/A
Ethanol-75%	Sinopharm Chemical Reagent	Cat# 80176961
Fetal Bovine Serum	Excell	Cat# FSD500
Filter-0.22 µm	Merck Millipore	Cat# SLGPR33RB
Glass slides	CITOTEST	Cat# 80340-3610
Isoamyl alcohol	Sinopharm Chemical Reagent	Cat# 10003218
IVIS Spectrum	PerkinElmer	N/A
Micro forceps	Shinva Medical Instrument	Cat# ZD372RB
Micro scissors	Shinva Medical Instrument	Cat# ZO13056R
Microinjection needle (25 µL)	Shanghai Gaoge Industry & Trade	N/A
Microinjection pump	Voyage Power	Cat# QHZS-001A
Mouse: C57BL/6 wild-type, 8 weeks old, male	Cavens	N/A
Needle-30G	Zhejiang Kindly Medical Devices	N/A
Normal Saline	Beyotime	Cat# ST341
Ovalbumin, Alexa Fluor™ 647 Conjugate	Thermo Fisher Scientific	Cat# O34784
Paraformaldehyde Fix Solution-4%	Beyotime	Cat# P0099
PE-10 tubing	Shanghai Qiujing Biochemical Reagent and Instrument	N/A
Phosphate-buffered saline (1x PBS)	Beyotime	Cat# ST447
Rat anti-LYVE-1 (1:200 dilution)	Santa Cruz	Cat# sc-65647
Stereomicroscope	Jonec	Cat# JSZ6
Stereotaxic frame	RWD Life Science	Cat# 68030
Straight scissors	RWD Life Science	Cat# S12007-10
Syringe-1-mL	Zhejiang Kindly Medical Devices	N/A
Tissue Adhesive I	3M Vetbond	Cat# 1469SB
Tribromoethanol	Sigma-Aldrich	Cat# T48402
Triton X-100	Sigma-Aldrich	Cat# T8787
VS120 High-throughput fluorescence imaging system	Olympus	N/A