Name: isolating central nervous system tissues and associated meninges for the downstream analysis of immune cells
Uploaded: 2020-05-19T14:45:00
Description: Watch this Scientific Journal Video about Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells at JoVE.com

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.

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
<ol>
	<li>Isolating brain and spinal cord samples
	<ol>
		<li>Euthanize the mouse via CO2 inhalation. Ensure that the CO2 flow rate displaces 10%&#8211;30% of the cage volume per minute.</li>
		<li>Using forceps, lift the xiphoid proc...

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 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, actively participating in homeostatic immune surveillance and inflammatory processes during disease pathogenesis3,6,7,8. During steady state conditions, the meninges support numerous immune sentinel cells, including innate lymphoid cells (ILC), macrophages, dendritic cells (DC), mast cells, T cells, and to a lesser extent, B cells9,10,11.
The meninges are highly vascularized structures and contain lymphatic vessels that provide a lymphatic connection between the CNS and its periphery8,12,13,14. In inflammatory conditions induced by CNS injury, infections, autoimmunity, or even neurodegeneration, peripherally derived immune cells infiltrate the parenchyma and alter the immune landscape within the meninges. Following cell infiltration, the meninges may represent a functional niche for peripherally derived immune cells, promoting immune cell aggregation, local immune cell activation, and long-term survival in the CNS compartment. Prominent meningeal inflammation is observed in multiple diseases affecting the CNS, including multiple sclerosis (MS)15,16,17,18,19, stroke20,21, sterile injury22,23 (i.e., spinal cord injury and traumatic brain injury), migraines24, and microbial infection25,26,27,28,29. Thus, the characterization of resident cells and peripherally derived immune cells in the meningeal compartment is essential for understanding the role of these cells during steady state conditions and disease pathogenesis.
The extraction of the brain, spinal cord, and meninges from the cranium and vertebral bodies is technically challenging and time-consuming. There are currently no techniques available for the rapid extraction of the brain with all three meningeal layers intact. While laminectomy yields excellent spinal cord tissue morphology and preserves the meningeal layers, it is both extremely time-consuming and complicated30,31. Conversely, more conventional extraction methods such as the removal of the brain from the cranium and the hydraulic extrusion of the spinal cord facilitate the quick extraction of the CNS tissue, but both the arachnoid and dural meninges are lost with these techniques30,31. The omission of dura and arachnoid layers during conventional isolation of brain and spinal cord tissues results in an incomplete analysis of the cells within the CNS compartment. Thus, the identification of new techniques focused on the quick extraction of CNS tissues with intact meninges is crucial for the optimal analysis of the CNS compartment.
This manuscript presents two methods for the rapid extraction of the brain, spinal cord, and meninges from mice, facilitating the downstream analysis of resident cells and peripherally derived immune cells in the CNS parenchyma and meninges. These optimized protocols focus on 1) isolating single-cell suspensions for downstream analysis and 2) preparing tissue for histological processing. Obtaining single-cell suspensions from the brain, spinal cord tissue, and dural and arachnoid meninges32 allows for the simultaneous analysis of cells residing in both the parenchymal and meningeal compartments. Single-cell suspensions can be used in different applications, including cell culture assays to perform in vitro stimulation33, enzyme-linked immunospot (ELISpot)28,34,35, flow cytometry36,33, and single-cell37 or bulk transcriptomics. Additionally, the optimized protocol for decalcification of whole brains and spinal cords with intact skulls or vertebral columns, respectively, allows for the gentle decalcification of the surrounding bone, leaving the meninges intact and preserving the tissue morphology. This method allows for the selective identification of proteins or RNA using immunohistochemistry (IHC) or in situ hybridization (ISH) techniques within both the parenchymal and meningeal spaces. The characterization of the phenotype, activation state, and localization of resident cells and peripherally derived immune cells within the CNS may provide information essential to understanding how individual cell types in the CNS compartment contribute to homeostasis and disease pathogenesis.

<table>
	<tbody>
		<tr>
			<td>Aluminum foil</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>ThermoFisher Scientific</td>
			<td>37002D</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra X-12R centrifuge</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Worthington</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube, 15 mL</td>
			<td>VWR</td>
			<td>525-1069</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical tube, 50 mL</td>
			<td>VWR</td>
			<td>89039-658</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Hauser Scientific</td>
			<td>5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryomold</td>
			<td>VWR</td>
			<td>18000-128</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Fine Science Tools</td>
			<td>11003-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable polystyrene tube, 14 mL</td>
			<td>Fisher Scientific</td>
			<td>14-959-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel</td>
			<td>Fisher Scientific</td>
			<td>NC0595256</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Worthington</td>
			<td>LS002139</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td>Airgas</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Durmont #7Forceps</td>
			<td>Fine Science Tools</td>
			<td>11271-30</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA disodium salt dihydrate</td>
			<td>Amresco</td>
			<td>0105-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 100%</td>
			<td>any</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter top tube, 5 mL</td>
			<td>VWR</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixable viability stain 780</td>
			<td>Becton Dickinson</td>
			<td>565388</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>Gallios</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Chemical</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (488 conjugate)</td>
			<td>Jackson immunoresearch</td>
			<td>115-546-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG (594 conjugate)</td>
			<td>Jackson immunoresearch</td>
			<td>115-586-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit 488</td>
			<td>Jackson immunoresearch</td>
			<td>111-545-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rat 594</td>
			<td>Jackson immunoresearch</td>
			<td>112-585-167</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rat 650</td>
			<td>Jackson immunoresearch</td>
			<td>112-605-167</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balnced Salt Solution (HBSS)</td>
			<td>Corning</td>
			<td>21-020-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Andwin Scientific</td>
			<td>02-671-51B</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>Fine Science Tools</td>
			<td>13004-14</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid)</td>
			<td>ThermoFisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher chemical</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4 (anhydrous)</td>
			<td>Sigma Aldrich</td>
			<td>P5655-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid Nitrogen</td>
			<td>Airgas</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse FC block (CD16/32)</td>
			<td>Becton Dickinson</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HP04 (anhydrous)</td>
			<td>Fisher Chemical</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher chemical</td>
			<td>S671-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle, 25 gauge</td>
			<td>Becton Dickinson</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal mouse serum</td>
			<td>ThermoFisher Scientific</td>
			<td>31881</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh strainer</td>
			<td>VWR</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 20%</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713-S</td>
			<td>Diluted to 4% using 1 x PBS</td>
		</tr>
		<tr>
			<td>Pasteur pipette, 9 inch, unplugged</td>
			<td>Fisher Scientific</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1x)</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PE Rat Anti-Mouse CD4</td>
			<td>Becton Dickinson</td>
			<td>553730</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-CF594 Rat Anti-Mouse CD19</td>
			<td>Becton Dickinson</td>
			<td>562329</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll density gradient media</td>
			<td>GE healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP-Cy5.5 Rat Anti-Mouse CD45</td>
			<td>Becton Dickinson</td>
			<td>550994</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 100 mm</td>
			<td>VWR</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Fisher Scientific</td>
			<td>13-636-AB150</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-Aid</td>
			<td>Drummond Scientific Corporation</td>
			<td>4-000-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 200 &#181;l</td>
			<td>Gilson</td>
			<td>FA10005M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips, 1 mL</td>
			<td>USA Scientific</td>
			<td>1111-2831</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips, 200 &#181;l</td>
			<td>USA Scientific</td>
			<td>1111-1816</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette, 1 mL</td>
			<td>Gilson</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Diamond mountant with DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>P36962</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse CD16/CD32</td>
			<td>Becton Dickinson</td>
			<td>553141</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse CD3 (SP7 clone)</td>
			<td>Abcam</td>
			<td>ab16669</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse laminin</td>
			<td>Abcam</td>
			<td>ab11575</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse ERT-R7</td>
			<td>Abcam</td>
			<td>ab51824</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipet, 1 mL</td>
			<td>VWR</td>
			<td>357521</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipet, 10 mL</td>
			<td>VWR</td>
			<td>357551</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipet, 5 mL</td>
			<td>VWR</td>
			<td>357543</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher chemical</td>
			<td>S5-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>14001-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors, extra fine</td>
			<td>Roboz</td>
			<td>RS-5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 10 mL</td>
			<td>Becton Dickinson</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe, 5 mL</td>
			<td>Becton Dickinson</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum filter system</td>
			<td>Millipore</td>
			<td>20207749</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum flask</td>
			<td>Thomas Scientific</td>
			<td>5340-2L</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum in-line filter</td>
			<td>Pall Corporation</td>
			<td>4402</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum line</td>
			<td>Cole Palmer</td>
			<td>EW-06414-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>ThermoFisher Scientific</td>
			<td>Versa bath</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating central nervous system tissues and associated meninges for the downstream analysis of immune cells

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.

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,...

Watch this Scientific Journal Video about Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells at JoVE.com

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

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 ...

Rapid isolation of CNS tissues including the meninges allows for a comprehensive analysis of immune cell phenotype, function, and localization in homeostatic and pathogenic conditions. The two demonstrated methods allow for the quick extraction of CNS tissues with the meninges, ideal for downstream analysis using single cell techniques and histological methods. Although the representative results focus on the analysis of immune cells, these two methods can be used to analyze CNS resident cells, including microglia, astrocytes, pericytes, endothelial cells, stromal cells, and neurons.Begin by isolating brain and spinal cord samples from euthanized mice. After removing the head, make a midline incision in the skin and flip it over the eyes to free the skull. Cut at the nasal bone to release the mandible from the skull.Then remove the mandible, tongue, and eyes. Cut along the lateral aspects of the skull to release the tissue along the external auditory meatus and trim it. To collect the spinal cord sample, separate the rib cage from the spinal column by cutting parallel to the spine with sharp scissors.Make a small cut at the lower lumbar region to isolate the spinal column, then trim and remove any remaining muscle along the spine to expose the vertebrae. When performing decalcification, check the bone daily to determine if it is soft and pliable. Remove the tissue from the EDTA solution with forceps, place it on a Petri dish, and gently test the bone softness with a 25 gauge needle.If the needle easily penetrates the bone, the decalcification process is complete. To extract the skull cap in the brain, make a midline incision in the skin and flip the skin over the eyes. Place the scissors within the foramen magna and begin cutting the skull laterally along the cortices towards the olfactory bulb, keeping the incisions above the external auditory meatus and mandible.Perform the same cuts on the opposite side with cuts meeting at the olfactory bulb to free the skull cap from the brain. Using forceps, peel back the skull cap and place it into a 15 milliliter conical tube containing five milliliters of cold RPMI medium supplemented with 25 millimolar HEPES. Place curved forceps below the base of the brain and lift them to free the brain from the skull cap.Extract the vertebrae column and spinal cord tissue by cutting parallel to the spine to separate the rib cage from the spinal column. Then make a small cut at the lower lumbar region to isolate the vertebrae column. Trim and remove any remaining muscle along the spine to expose the vertebrae.Next, place the extra fine surgical scissors within the vertebral column and cut along the lateral edge of the column. Cut the opposite lateral edge completely to divide the vertebral column into an anterior and posterior portion. Use forceps to peel away the spinal cord slowly and carefully from the vertebral column and place it in a 15 milliliter conical tube containing five milliliters of cold RPMI with 25 millimolar HEPES.Then transfer the anterior and posterior portions of the spinal column to another tube. To remove the meninges from the skull cap, take the skull cap out of the RPMI media and use sharp forceps to score around the outer edge. Peel the meninges away from the edge of the skull cap, scraping to remove the dural and arachnoid meninges and place the meninges on a Petri dish.To remove the meninges from the vertebral column, take the column out of the media and score around the edges of the vertebral column with sharp forceps to free the meninges. Use curved forceps to peel away the meninges from the edge of the vertebrae and place the meninges on a Petri dish. Place a nylon mesh strainer in a 50 milliliter conical tube.To create a cell suspension, move the meninges into a strainer and add three milliliters of HEPES supplemented RPMI, then use a plunger from a five milliliter syringe to grind the tissue and media through the strainer. Pour the brain or spinal cord tissue with the media into a 100 millimeter Petri dish and use forceps to move the tissue to the bottom. Mince the brain tissue with a sterile razor blade and gather the tissue at the bottom of the plate.Add three milliliters of RPMI supplemented with 10%FCS to the Petri dish, then use a 10 milliliter serological pipette to resuspend the tissue in the media and transfer it to a 15 milliliter conical tube. Add collagenase type 1 and DNase 1 to the tissue and incubate the tubes in a 37 degree Celsius water bath for 40 minutes. Invert the tubes every 15 minutes to thoroughly mix the tissue with the enzymes.After incubation, add EDTA to each tube for a final concentration of 0.01 molar and incubate the tubes for an additional five minutes to inactivate the collagenase. Add nine milliliters of RPMI supplemented with 10%FCS to each tube, then centrifuge the tubes at 450 G for five minutes at four degrees Celsius. Use a Pasteur pipette with a vacuum to aspirate the supernatant, taking care not to touch the cell pellet.Add three milliliters of 100%stock isotonic density gradient solution to the cell pellet, then add additional RPMI 10%FCS media to bring the final volume to 10 milliliters and resuspend the cell pellet. Invert and mix the tube well, then insert a serological pipette with one milliliter of 70%stock isotonic density gradient solution into the bottom of the tube. Slowly underlay the solution, being careful not to make bubbles.Remove the serological pipette from the tube, making sure not to disturb the gradient. Centrifuge the tube at 800 G for 30 minutes at four degrees Celsius, then aspirate the supernatant, including the myelin debris layer, until two to three milliliters remain in the tube. Use a one milliliter pipette to harvest the cell layer between the 30%and 70%density gradient and transfer it to a new 15 milliliter tube.Add RPMI 10%FCS media to bring the final volume to 15 milliliters, then centrifuge the cells at 450 G for five minutes at four degrees Celsius. This protocol was used to assess B and T cells in the meninges, brain, and spinal cord during progressive multiple sclerosis. Gating was conducted to distinguish CD45 high peripherally derived infiltrating immune cells from CD45 low microglia and CD45 negative neurons, astrocytes, and oligodendrocytes.Few CD45 high cells were present in the spinal cord and brain tissue of sham-treated mice. The same gating cutoff for CD45 high expression was used in the meninges to identify CD45 high immune cells and exclude non-immune cells. In all TMEV-IDD CNS tissues, increased percentages of CD45 high immune cells were observed compared to sham treated mice.During chronic TMEV-IDD, the percentage of B cells among CD45 high immune cells in the brain and spinal cord was higher compared to the meningeal compartment. Decalcified brains and spinal cords were imaged to identify the localization of B and T cells within the CNS compartment. Conventional extraction of the brain from the skull cap resulted in an intact pia layer, but the remaining meningeal layers were excluded.In both decalcified brains and spinal cords, all meningeal layers were intact. Isotype switched B cells and T cells were localized with IgG and CD3 expression, respectively. Costaining IgG with ER-TR7 revealed that B cells were present in the CNS parenchyma and the meninges, while cellular aggregates within ER-TR7 positive meninges contained multiple IgG positive B cells and CD3 positive T cells.A critical step in these protocols is the careful extraction of the CNS tissues, essential for improving the purity of single cell suspensions and for obtaining histology tissue with high quality morphology. The described techniques allow for the comprehensive analysis of cells occupying the CNS compartment, ideal for examining cell phenotype, function, and localization under homeostasis and during disease pathogenesis.

Research

JoVE Journal

Immunology and Infection

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Qualitative and Quantitative Analysis of the Immune Synapse in the Human System Using Imaging Flow Cytometry

The immune synapse is the area of communication between T cells and antigen-presenting cells (APCs). T cells polarize surface receptors and proteins ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

A Positioning Device for the Placement of Mice During Intranasal siRNA Delivery to the Central Nervous System

Intranasal (IN) drug delivery to the brain has emerged as a promising method to bypass the blood-brain barrier (BBB) for the delivery of drugs into the ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...