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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 site1,2,3,4,5 and is relatively devoid of immune cells during homeostatic conditions2,6,7. 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.......

Representative Results

This representative experiment was aimed at quantifying B and T cells and describing B and T cell localization in the meningeal and parenchymal CNS compartments in homeostatic conditions as well as in a murine progressive MS model (i.e., TMEV-IDD). TMEV-IDD was induced in 5-week-old female SJL mice by intracranial infection with 5 x 106 plaque forming units (PFU) of TMEV BeAn as previously described29.

The present study assessed B and T cells in the meninges,.......

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.......

Acknowledgements

The authors thank the staff of the Center for Comparative Medicine and Research (CCMR) at Dartmouth for their expert care of the mice used for these studies. The Bornstein Research Fund funded this research.

....

Materials

NameCompanyCatalog NumberComments
Aluminum foilanyN/A
Bovine Serum AlbuminThermoFisher Scientific37002D
CentrifugeBeckman CoulterAllegra X-12R centrifuge
Collagenase IWorthingtonLS004196
Conical tube, 15 mLVWR525-1069
Conical tube, 50 mLVWR89039-658
Cover glassHauser Scientific5000
CryomoldVWR18000-128
Curved forcepsFine Science Tools11003-14
Disposable polystyrene tube, 14 mLFisher Scientific14-959-1B
Disposable ScalpelFisher ScientificNC0595256
DNAse IWorthingtonLS002139
Dry iceAirgasN/A
Durmont #7ForcepsFine Science Tools11271-30
EDTA disodium salt dihydrateAmresco0105-500g
Ethanol, 100%anyN/A
Fetal Bovine Serum (FBS)HycloneSH30910.03
Filter top tube, 5 mLVWR352235
Fixable viability stain 780Becton Dickinson565388
Flow cytometerBeckman CoulterGallios
GlucoseFisher ChemicalD16-500
Goat anti-mouse IgG (488 conjugate)Jackson immunoresearch115-546-146
Goat anti-mouse IgG (594 conjugate)Jackson immunoresearch115-586-146
Goat anti-rabbit 488Jackson immunoresearch111-545-144
Goat anti-rat 594Jackson immunoresearch112-585-167
Goat anti-rat 650Jackson immunoresearch112-605-167
Hank's Balnced Salt Solution (HBSS)Corning21-020-CV
HemacytometerAndwin Scientific02-671-51B
HemostatFine Science Tools13004-14
HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid)ThermoFisher Scientific15630080
KClFisher chemicalBP366-500
KH2PO4 (anhydrous)Sigma AldrichP5655-100G
Liquid NitrogenAirgasN/A
Mouse FC block (CD16/32)Becton Dickinson553141
Na2HP04 (anhydrous)Fisher ChemicalS374-500
NaClFisher chemicalS671-500
Needle, 25 gaugeBecton Dickinson305122
Normal mouse serumThermoFisher Scientific31881
Nylon mesh strainerVWR352350
OCTSakura4583
Paraformaldehyde, 20%Electron Microscopy Sciences15713-SDiluted to 4% using 1 x PBS
Pasteur pipette, 9 inch, unpluggedFisher Scientific13-678-20C
PBS (1x)Corning21-040-CV
PE Rat Anti-Mouse CD4Becton Dickinson553730
PE-CF594 Rat Anti-Mouse CD19Becton Dickinson562329
Percoll density gradient mediaGE healthcare17-0891-01
PerCP-Cy5.5 Rat Anti-Mouse CD45Becton Dickinson550994
Petri dish, 100 mmVWR353003
pH meterFisher Scientific13-636-AB150
Pipet-AidDrummond Scientific Corporation4-000-101
Pipette 200 µlGilsonFA10005M
Pipette tips, 1 mLUSA Scientific1111-2831
Pipette tips, 200 µlUSA Scientific1111-1816
Pipette, 1 mLGilsonFA10006M
Prolong Diamond mountant with DAPIThermoFisher ScientificP36962
Purified Rat Anti-Mouse CD16/CD32Becton Dickinson553141
Rabbit anti-mouse CD3 (SP7 clone)Abcamab16669
Rabbit anti-mouse lamininAbcamab11575
Rat anti-mouse ERT-R7Abcamab51824
RPMI 1640Corning10-040-CV
Serological pipet, 1 mLVWR357521
Serological pipet, 10 mLVWR357551
Serological pipet, 5 mLVWR357543
Sodium hydroxideFisher ScientificS318-100
SucroseFisher chemicalS5-500
Surgical scissorsFine Science Tools14001-16
Surgical scissors, extra fineRobozRS-5882
Syringe, 10 mLBecton Dickinson302995
Syringe, 5 mLBecton Dickinson309646
Trypan blueGibco15250-061
Vacuum filter systemMillipore20207749
Vacuum flaskThomas Scientific5340-2L
Vacuum in-line filterPall Corporation4402
Vacuum lineCole PalmerEW-06414-20
Water bathThermoFisher ScientificVersa bath

References

  1. Mastorakos, P., McGavern, D. The anatomy and immunology of vasculature in the central nervous system. Science Immunology. 4 (37), 0492 (2019).
  2. Carson, M. J., Doose, J. M., Melchior, B., Schmid, C. D., Ploix, C. C. CNS immune privilege: h....

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