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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.
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.
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....
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
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,.......
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.......
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.
....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 |
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