Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells

Summary

This paper presents two optimized protocols for examining resident and peripherally derived immune cells within the central nervous system, including the brain, spinal cord, and meninges. Each of these protocols helps to ascertain the function and composition of the cells occupying these compartments under steady state and inflammatory conditions.

Abstract

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

Introduction

The central nervous system (CNS) is an immunologically specialized site. The CNS parenchyma, excluding the CSF space, the meninges, and the vasculature, is classically viewed as an immune-privileged site^1,2,3,4,5 and is relatively devoid of immune cells during homeostatic conditions^2,6,7. In contrast, the meninges, comprised of the dura, arachnoid, and pia layers, are crucial components of the CNS compartment, acti....

Protocol

All animal work utilizes protocols reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Geisel School of Medicine at Dartmouth.

1. Processing brain and spinal cord samples for decalcification

1. Isolating brain and spinal cord samples
   1. Euthanize the mouse via CO₂ inhalation. Ensure that the CO₂ flow rate displaces 10%–30% of the cage volume per minute.
   2. Using forceps, lift the xiphoid proc.......

Discussion

Methods for evaluating the cellular composition in the CNS compartment during homeostasis and disease are essential for understanding the physiological and pathological states of the CNS. However, despite serving as an important barrier in the CNS and housing a diverse array of immune cells, the meninges are often omitted from analysis because many conventional tissue extraction methods for the brain and spinal cord do not allow for the collection of these membranes. This omission is a critical limitation in the advancem.......

Materials

Name	Company	Catalog Number	Comments
Aluminum foil	any	N/A
Bovine Serum Albumin	ThermoFisher Scientific	37002D
Centrifuge	Beckman Coulter	Allegra X-12R centrifuge
Collagenase I	Worthington	LS004196
Conical tube, 15 mL	VWR	525-1069
Conical tube, 50 mL	VWR	89039-658
Cover glass	Hauser Scientific	5000
Cryomold	VWR	18000-128
Curved forceps	Fine Science Tools	11003-14
Disposable polystyrene tube, 14 mL	Fisher Scientific	14-959-1B
Disposable Scalpel	Fisher Scientific	NC0595256
DNAse I	Worthington	LS002139
Dry ice	Airgas	N/A
Durmont #7Forceps	Fine Science Tools	11271-30
EDTA disodium salt dihydrate	Amresco	0105-500g
Ethanol, 100%	any	N/A
Fetal Bovine Serum (FBS)	Hyclone	SH30910.03
Filter top tube, 5 mL	VWR	352235
Fixable viability stain 780	Becton Dickinson	565388
Flow cytometer	Beckman Coulter	Gallios
Glucose	Fisher Chemical	D16-500
Goat anti-mouse IgG (488 conjugate)	Jackson immunoresearch	115-546-146
Goat anti-mouse IgG (594 conjugate)	Jackson immunoresearch	115-586-146
Goat anti-rabbit 488	Jackson immunoresearch	111-545-144
Goat anti-rat 594	Jackson immunoresearch	112-585-167
Goat anti-rat 650	Jackson immunoresearch	112-605-167
Hank's Balnced Salt Solution (HBSS)	Corning	21-020-CV
Hemacytometer	Andwin Scientific	02-671-51B
Hemostat	Fine Science Tools	13004-14
HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid)	ThermoFisher Scientific	15630080
KCl	Fisher chemical	BP366-500
KH2PO4 (anhydrous)	Sigma Aldrich	P5655-100G
Liquid Nitrogen	Airgas	N/A
Mouse FC block (CD16/32)	Becton Dickinson	553141
Na2HP04 (anhydrous)	Fisher Chemical	S374-500
NaCl	Fisher chemical	S671-500
Needle, 25 gauge	Becton Dickinson	305122
Normal mouse serum	ThermoFisher Scientific	31881
Nylon mesh strainer	VWR	352350
OCT	Sakura	4583
Paraformaldehyde, 20%	Electron Microscopy Sciences	15713-S	Diluted to 4% using 1 x PBS
Pasteur pipette, 9 inch, unplugged	Fisher Scientific	13-678-20C
PBS (1x)	Corning	21-040-CV
PE Rat Anti-Mouse CD4	Becton Dickinson	553730
PE-CF594 Rat Anti-Mouse CD19	Becton Dickinson	562329
Percoll density gradient media	GE healthcare	17-0891-01
PerCP-Cy5.5 Rat Anti-Mouse CD45	Becton Dickinson	550994
Petri dish, 100 mm	VWR	353003
pH meter	Fisher Scientific	13-636-AB150
Pipet-Aid	Drummond Scientific Corporation	4-000-101
Pipette 200 µl	Gilson	FA10005M
Pipette tips, 1 mL	USA Scientific	1111-2831
Pipette tips, 200 µl	USA Scientific	1111-1816
Pipette, 1 mL	Gilson	FA10006M
Prolong Diamond mountant with DAPI	ThermoFisher Scientific	P36962
Purified Rat Anti-Mouse CD16/CD32	Becton Dickinson	553141
Rabbit anti-mouse CD3 (SP7 clone)	Abcam	ab16669
Rabbit anti-mouse laminin	Abcam	ab11575
Rat anti-mouse ERT-R7	Abcam	ab51824
RPMI 1640	Corning	10-040-CV
Serological pipet, 1 mL	VWR	357521
Serological pipet, 10 mL	VWR	357551
Serological pipet, 5 mL	VWR	357543
Sodium hydroxide	Fisher Scientific	S318-100
Sucrose	Fisher chemical	S5-500
Surgical scissors	Fine Science Tools	14001-16
Surgical scissors, extra fine	Roboz	RS-5882
Syringe, 10 mL	Becton Dickinson	302995
Syringe, 5 mL	Becton Dickinson	309646
Trypan blue	Gibco	15250-061
Vacuum filter system	Millipore	20207749
Vacuum flask	Thomas Scientific	5340-2L
Vacuum in-line filter	Pall Corporation	4402
Vacuum line	Cole Palmer	EW-06414-20
Water bath	ThermoFisher Scientific	Versa bath