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.

isolating-central-nervous-system-tissues-associated-meninges-for

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 cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

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 pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

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 cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

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) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

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), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

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 towards the immune synapse to assure a stable binding and signal exchange. Classical confocal, TIRF, or super-resolution microscopy have been used to study the immune synapse. Since these methods require manual image acquisition and time-consuming quantification, the imaging of rare events is challenging. Here, we describe a workflow that enables the morphological analysis of tens of thousands of cells. Immune synapses are induced between primary human T cells in pan-leukocyte preparations and Staphylococcus aureus enterotoxin B (SEB)-loaded Raji cells as APCs. Image acquisition is performed with imaging flow cytometry, also called In-Flow microscopy, which combines features of a flow cytometer and a fluorescence microscope. A complete gating strategy for identifying T cell/APC couples and analyzing the immune synapses is provided. As this workflow allows the analysis of immune synapses in unpurified pan-leukocyte preparations and hence requires only a small volume of blood (i.e., 1 mL), it can be applied to samples from patients. Importantly, several samples can be prepared, measured, and analyzed in parallel.

Here, we describe a complete workflow for the qualitative and quantitative analysis of immune synapses between primary human T cells and antigen-presenting cells. The method is based on imaging flow cytometry, which allows the acquisition and evaluation of several thousand cell images within a relatively short period of time.

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 macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

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 dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the 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 central nervous system (CNS). Recent studies demonstrate the use of a peptide, RVG9R, incorporating the minimal receptor-binding domain of the Rabies virus glycoprotein, in eliciting the delivery of siRNA into neurons in the brain. In this protocol, the peptide-siRNA formulation is delivered intranasally with a pipette in the dominant hand, while the anesthetized mouse is restrained by the scruff with the nondominant hand in a "head down-and-forward position" to avoid drainage into the lung and stomach upon inhalation. This precise gripping of mice can be learned but is not easy and requires practice and skill to result in effective CNS uptake. Furthermore, the process is long-drawn, requiring about 45 min for the administration of a total volume of ~20-30 &#181;L of solution in 1-2 &#181;L droplet volumes per inhalation, with 3-4 min rest periods between each inhalation. The objective of this study is to disclose a mouse positioning device that enables the appropriate placement of mice for efficient IN administration of the peptide-siRNA formulation. Multiple features are incorporated into the design of the device, such as four or eight positioning chairs with adjustable height and tilt to restrain anesthetized mice in the head down-and-forward position, enabling easy visualization of the mice's nares and a built-in heating pad to maintain the mice's body temperatures during the procedure. Importantly, the ability to treat four or eight mice simultaneously with RVG9R-siRNA complexes in this manner enables studies on a much quicker time scale, for the testing of an IN therapeutic siRNA approach. In conclusion, this device allows for appropriate and controlled mouse head positioning for IN application of RVG9R-siRNA and other therapeutic molecules, such as nanoparticles or antibodies, for CNS delivery.

Here, we present a protocol using a mouse positioning device that enables the appropriate placement of mice for the intranasal administration of a brain-targeting peptide-siRNA formulation enabling effective gene silencing in 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 genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

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