Isolation and Flow Cytometric Assessment of Neuroimmune Interactions in a Mini-Stroke Murine Model

Summary

Here, we present a protocol to assess immune cell function following ischemic stroke using flow cytometric analysis in a mini-stroke model. This method enables detailed examination of neuroimmune interactions and can be adapted for studying other neurodegenerative diseases, enhancing our understanding of immune responses in stroke pathology.

Abstract

Stroke is a leading cause of death and long-term disability worldwide, with ischemic stroke representing the majority of cases. Following an ischemic stroke, resident and infiltrating immune cells become activated, contributing to further neuronal damage. However, the roles of the immune system in the pathology of ischemic stroke are not fully understood, largely due to the complex and dynamic regulation of immune responses in reaction to changes in the microenvironment during neuroinflammation. Therefore, it is essential to monitor and analyze the activation of resident and infiltrating immune cells over time after an ischemic stroke. In this study, we present a protocol for assessing the function of these immune cells following ischemic stroke using flow cytometric analysis in a mini-stroke model. We microdissect the infarcted brain tissue at specific time points and then dissociate it into a single-cell suspension using both mechanical and enzymatic methods. The cells are passed through a 70 µm cell strainer and labeled with fluorescently tagged antibody cocktails before being quantified by flow cytometric analysis. While this assay was specifically developed to investigate neuroimmune interactions after ischemic stroke, it can also be easily adapted to study neuroimmune mechanisms in other neurodegenerative diseases, such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease.

Introduction

Stroke is a predominant cause of mortality and long-term disability worldwide, with ischemic stroke accounting for the vast majority of cases^1,2,3. The overall goal of this method is to enhance our understanding of the neuroimmune interactions that occur following an ischemic stroke by providing a refined approach for assessing the activation of both resident and infiltrating immune cells in a mini-stroke murine model^4,5. The intricate interplay between these immune cells and the neuronal microenvironment is crucial for understanding the pathophysiology of ischemic stroke. However, the precise mechanisms underlying these neuroimmune interactions remain poorly elucidated, primarily due to the dynamic and multifaceted nature of immune responses triggered by neuroinflammation⁶. This technique aims to elucidate the complex dynamics of immune responses in the context of neuroinflammation and their subsequent impact on neuronal damage and recovery outcomes.

The rationale behind the development of this technique stems from the critical role that immune cells play in exacerbating neuronal damage and influencing recovery following an ischemic event. Traditional methods of studying these interactions often lack the resolution necessary to capture the temporal and spatial nuances of immune cell activation. By employing flow cytometric analysis on single-cell suspensions derived from microdissected infarcted brain tissue, this protocol offers a more detailed and dynamic assessment of immune cell profiles over time^7,8. This approach enables the identification of specific immune cell populations and their functional states, which are pivotal for understanding the pathophysiology of ischemic stroke.

Compared to alternative techniques such as histological staining or bulk RNA sequencing, which may provide limited insights into the individual contributions of immune cell types, this flow cytometric method presents several advantages. For instance, previous studies have shown that flow cytometry allows for high-throughput analysis of multiple markers simultaneously, facilitating a more comprehensive characterization of immune cell phenotypes and functions^{9,10,11,12,13}. Additionally, this method's ability to adapt to a variety of neurodegenerative conditions -- such as multiple sclerosis, Parkinson's disease, and Alzheimer's disease -- demonstrates its versatility and relevance in the wider body of literature addressing neuroimmune interactions.

For researchers considering the application of this method, evaluating the specific aims of their study is essential. This technique is particularly suitable for investigations requiring detailed temporal analysis of immune responses in the context of ischemic stroke or related neurodegenerative diseases. It is recommended that users possess a foundational understanding of flow cytometry and experience with murine models to effectively implement this protocol and interpret the resulting data. By providing a robust framework for studying neuroimmune interactions, this method contributes significantly to the ongoing efforts to delineate the complexities of immune involvement in stroke pathology.

Protocol

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Beijing Friendship Hospital, Capital Medical University. We recommend that all solutions be subjected to sterile filtration and that all tubes utilized be sterile.

1. Preparing reagents and buffer

1. Digestion enzyme mix: Dissolve collagenase IV to obtain a concentration of 0.25% and dilute DNase I to reach a final concentration of 2 U/mL.
2. Magnetic activated cell sorting (MACS) buffer: Dissolve bovine serum albumin (BSA) and Ethylenediaminetetraacetic acid (EDTA) to reach concentrations of 0.5% and 2 mM in 1x PBS, respectively.

2. Focal ischemic stroke model

NOTE: C57BL/6 J mice (8-10-week-old)were employed to establish a focal ischemic stroke model, as previously described¹⁴. Any alternative pharmaceutical-grade injectable anesthetics that are well-documented for rodents may be utilized.

1. Induce anesthesia in the mouse using 3% isoflurane delivered at a flow rate of 0.8-1.0 L/min of oxygen, and subsequently maintain anesthesia with 1.5% isoflurane. Maintain the body temperature at 37 °C throughout the entire procedure using a heating pad.
   NOTE: This study emphasizes the importance of verifying that anesthetized animals have reached a surgical plane of anesthesia prior to incision. This is accomplished by assessing the paw withdrawal reflex by performing a firm pinch of the hindlimb digits to confirm an absence of response. If none are observed, the animal is adequately anesthetized.
2. Trim the fur in the region between the right eye and right ear using an electric razor. Ensure the skin is sterile by applying alternating rounds of chlorhexidine and alcohol in a circular motion for a minimum of three applications. Subsequently, administer 5 mg/kg of meloxicam and 3.25 mg/kg of sustained-release buprenorphine subcutaneously before making the incision. Prior to the incision, perform an intradermal injection of up to 4 mg/kg of lidocaine, diluted in saline, along the incision line. Then apply ophthalmic lubricant on the surface of the eyes to prevent corneal desiccation and create a 0.5 cm vertical incision in the designated area adjacent to the eyes under a dissection microscope.
3. Gently separate all tissues to expose the skull and identify the distal branch of the right middle cerebral artery (MCA), characterized by a vertical "Y"-shaped vascular structure. Utilize an electric drill to penetrate the skull, taking care to remove it cautiously.
4. Delicately excise the meninges with micro-forceps.
5. Expose the distal branch of the right middle cerebral artery (MCA), and then perform permanent ligation of the distal branch of the right MCA using a 10-0 non-absorbable polypropylene suture, securing it with a double knot succeeded by a reverse knot. Subsequently, remove the excess ends of the suture.
6. Reposition the tissue to its original state and close the facial skin incision with 4-0 nylon suture.
7. Position the mouse in a supine orientation and trim the fur in the neck area with an electric razor. Ensure the skin is sterile by applying alternating rounds of chlorhexidine and alcohol in a circular motion for a minimum of three applications.
8. Create a horizontal incision in the neck region and gently pull apart the submandibular glands. Briefly, a transverse incision is skillfully made through the skin with a scalpel, facilitating the visualization of the submandibular glands on either side of the midline. The submandibular gland is then carefully subjected to blunt dissection with the aid of forceps and blunt-ended scissors, revealing the underlying carotid artery.
9. Carefully dissect the carotid sheath with micro-forceps, identifying the common carotid artery and separating it from the vagus nerve.
10. Thread a 4-0 nylon suture through the common carotid artery and secure it with a slipknot.
11. Repeat step 10 on the contralateral common carotid artery. Temporarily occlude the bilateral common carotid arteries (CCAs) for 7 min.
12. Replace the submandibular gland tissue to its original position and close the neck incision with tissue adhesive.
    NOTE: The sham operation group underwent procedures that were identical to those of the stroke group, with the singular exception that the distal MCA and CCA remained unligated.
13. Monitor the mouse throughout and following the surgical procedure, including its respiration and body temperature. Place the mouse in a cage devoid of bedding, positioned halfway on the heating pad, and maintain this setup until it is fully awake. To mitigate discomfort, administer 0.05 mg/kg of buprenorphine during the initial 48 hours post-surgery.

3. Brain perfusion and dissection

1. Prepare a 50 mL syringe and attach a 26 G needle to its end. Fill the syringe with saline solution.
2. Deeply anesthetize the mouse using 3% isoflurane, followed by euthanasia via carbon dioxide asphyxiation.
   NOTE: This study emphasizes the critical necessity of ensuring that anesthetized animals have attained a surgical plane of anesthesia before any incisions are made. This verification is achieved by evaluating the paw withdrawal reflex through a firm pinch of the digits on the hindlimb, thereby confirming the absence of any response. If no reaction is observed, it can be concluded that the animal is sufficiently anesthetized.
3. Position the mouse in a supine orientation on a foam board, extending its forelimbs and hindlimbs, securing them in place with 26 G needles.
4. Once the mouse is thoroughly anesthetized, grasp the abdominal skin with serrated thumb forceps and make a lateral incision to reveal the liver. Proceed to cut along both sides of the ribs to expose the heart.
5. Insert the 26 G needle into the left ventricle and sever the right auricle, then depress the syringe to allow the blood to flow out.
6. Continue the perfusion process until the blood draining from the auricle appears clear.
7. Decapitate the mouse by severing the cervical spine using straight surgical scissors.
8. Create a posterior-anterior incision in the skin of the head to uncover the skull. Make two lateral incisions at the junction of the lateral walls and the base of the skull.
9. Cut through the skull along the sagittal suture and make an incision between the orbits.
10. Utilize forceps to remove the skull covering each hemisphere, thereby exposing the brain, and transfer it to a mouse steel brain matrix. Subsequently, section the brain into consecutive 2-mm-thick coronal slices.

4. Digestion of cortical tissue into single-cell suspension and antibody staining for flow cytometry

1. Introduce 500 µL of digestion enzyme mix into each well of a 12-well plate.
2. Delicately separate the infarct tissue and transfer it into the digestion enzyme mix using micro-forceps (Figure 1).
3. Minutely fragment the tissue into the smallest possible pieces with an ophthalmic scissor and incubate at 37 °C for 20 min.
4. Employing a P1000 pipette, gently aspirate and dispense the solution 10 times to dissociate the tissue into individual cells, then pass the solution through a 70 µm cell strainer. Continuously rinse both the 12-well plate and the cell strainer with ice-cold HBSS containing calcium and magnesium.
5. Centrifuge at 500 × g at 4 °C for 5 min, then carefully discard the supernatant.
   NOTE: Given that the pellets produced are quite loose and easily dislodged, please refrain from disturbing them to prevent any loss of cells.
6. Resuspend the pellet in 1 mL of 1x PBS and repeat step 5. Subsequently, resuspend the pellet in 150 µL of 1x PBS and transfer the suspension into a 96-well plate with a round bottom. Then centrifuge again at 500 × g at 4 °C for 5 min.
7. Firmly grasp the 96-well plate and swiftly invert it to discard the supernatant.
8. Dilute cell viability fluorescence dye at a ratio of 1:100 in 1x PBS, then resuspend the pellet in a 150 µL aliquot. Incubate for 10 min at room temperature (RT), shielded from light.
9. Centrifuge at 500 × g at 4 °C for 5 min and discard the supernatant. Dilute the cell surface antibody cocktail in MACS buffer. Without washing the cells, add the cell surface antibody cocktail and incubate for an additional 15-20 min at 4 °C.
10. Centrifuge at 500 × g at 4 °C for 5 min and discard the supernatant. Resuspend the pellet in 150 µL of MACS buffer.
11. Repeat step 4.10 and transfer the suspension to 1.5 mL centrifuge tubes or 5 mL round bottom polystyrene test tubes.

5. Flow cytometry analysis and sorting

1. Design a multicolor flow cytometry panel as per the instrument configuration, utilizing either manual techniques or an online design tool, to optimize the selection of fluorophores conjugated to antibodies and minimize spectral overlap.
   NOTE: Several pivotal considerations must be taken into account when designing a multicolor flow cytometry panel. Firstly, it is essential to familiarize oneself with the configuration of the instrument in use, including the lasers and filters, prior to commencement. Secondly, employ bright fluorophore labels on antibodies for low-abundance antigens, while opting for dim fluorophore labels on antibodies targeting highly expressed antigens. Finally, it is imperative to include a cell viability dye in the panel to effectively exclude non-viable cells and debris from the data. Notably, to ensure appropriate gating of low abundance or poorly characterized antigens, fluorescence minus one (FMO) controls should be utilized¹⁵. FMO controls encompass all lineage markers except for the one of interest, facilitating precise delineation between positively and negatively stained cells¹⁶.
2. Adjust the settings of the forward scatter (FSC) and side scatter (SSC) detectors to ensure that the cells of interest are appropriately displayed on the scale and can be gated as required.
3. Meticulously fine-tune FSC and SSC to eliminate debris or extraneous noise. Differentiate doublets from single cells within a dot plot that illustrates both the area and height of FSC. Isolate viable cells based on the cell viability fluorescence dye-negative signal.
   NOTE: The settings for various instruments may exhibit slight variations. For instance, some cell sorters employ the width and height of FSC to distinguish doublets from single cells, whereas others utilize the area and height of FSC for the same purpose.
4. Adjust compensation for each channel.
   NOTE: Both cellular samples and commercially available compensation beads may be employed for the purpose of fluorescent spillover compensation.
   1. Compare the auto-fluorescence control (unstained cells) with stained cell positive controls to ensure the stained cells are appropriately scaled for each parameter. Additionally, fine-tune the compensation by analyzing multi-color cell samples, monitoring 2-color dot plots, and adjusting compensation settings upward or downward to ensure that the cell populations are aligned either vertically or horizontally with one another.
   2. Utilize CD45 and CD11b signals to distinguish between lymphocytes (CD11b^-CD45^high), microglia (CD11b⁺CD45^int), myeloid cells (CD11b⁺CD45^high), and astrocytes (CD11b^-CD45^- GLAST⁺)^17,18.
   3. Employ sham brain tissue as an auto-fluorescence control and a reference for discriminating lymphocytes. If a specific target is under examination, utilize an FMO control with post-stroke brain tissue.
5. Begin the collection of control and sample data and ensure it is saved.
   NOTE: Given that the mini-stroke model described here results in a small infarct, typically fewer cells are collected than the middle cerebral artery occlusion (MCAO) model. Generally, collecting 5 × 10⁵ cells yields approximately 5 × 10³ microglia and 1 × 10⁵ astrocytes, respectively.
6. Select and sort the desired cell populations into 5 mL round-bottom polystyrene test tubes or 15 mL conical centrifuge tubes.
   NOTE: Add 500 µL of PBS and meticulously rinse the tube to ensure the viability of the cells and aid in the formation of the cell pellet.

6. Analysis of gene expression in selected cell populations

NOTE: Total RNA extraction may alternatively be conducted using commercial kits following the manufacturer's guidelines.

1. Centrifuge the cells from step 5.6 at 500 × g at 4 °C for 3 min, then discard the supernatant. Resuspend the cells in 1 mL of TRIzol reagent.
   NOTE: The number of sorted cells is considerably lower compared to cultured cells or tissue. Consequently, a higher centrifugation speed may aid in forming a more compact cell pellet. The cells obtained from this step may be utilized for subsequent applications, such as cell culture or single-cell RNA sequencing pipelines.
2. Allow the sample to stand at RT for 5 min to facilitate the dissociation of nucleoprotein complexes.
3. Add 0.2 mL of chloroform to the sample and secure the tube. Shake the tubes vigorously for 15 s, then incubate for 2 min at RT.
4. Centrifuge the samples at 10,000 g for 15 min at 4 °C. The RNA will remain solely in the upper aqueous phase.
5. Cautiously transfer the upper aqueous phase to a clean tube, taking care not to disturb the interface.
   NOTE: The volume of the aqueous phase should be approximately 500 µL.
6. Precipitate the RNA from the aqueous phase by introducing 0.5 mL of isopropanol and gently mixing by inverting the tube several times. Incubate the samples at RT for 10 min.
7. Centrifuge the samples at 10,000 g for 10 min at 4 °C to collect the RNA.
8. Decant the supernatant and thoroughly remove any residual liquid by aspiration.
9. Wash the RNA pellet once with 75% ethanol, adding a minimum of 1 mL of 75% ethanol. Mix the sample thoroughly, then centrifuge at 7500 × g for 5 min at 4 °C.
10. Eliminate all traces of ethanol. Air-dry the RNA pellet for 5 min.
11. Dissolve the RNA in 10 µL of RNase-free water by pipetting up and down.
12. Proceed to conduct real-time PCR according to the manufacturer's instructions.

Results

In this study, we present a refined protocol for evaluating the functionality of immune cells following ischemic stroke, employing flow cytometric analysis within a mini-stroke model. The mini-stroke was induced by the permanent ligation of the distal branch of the right middle cerebral artery (MCA), succeeded by a 7 min occlusion of the bilateral common carotid arteries (CCAs). This methodology yields a mild stroke characterized by small, consistent lesion volumes and a remarkably low mortality rate. Moreover, the model...

Discussion

In this study, we present a detailed protocol for assessing the function of immune cells following ischemic stroke, utilizing flow cytometric analysis in a mini-stroke model. The critical steps of this protocol include the establishment of a focal ischemic stroke via the permanent ligation of the distal branch of the right middle cerebral artery (MCA) alongside a seven-minute occlusion of the bilateral common carotid arteries (CCAs). This surgical approach yields a mild stroke that is characterized by consistent lesion v...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL Microcentrifuge Tube	NEST	615601	Lab material
1× PBS	Corning	21-040-CM	Reagent
96-well Clear Round Bottom TC-treated Microplate	Corning	3799	Lab material
APC anti-mouse CD19	Biolegend	152410	Antibody
APC/Cyanine7 anti-mouse CD45	Biolegend	103116	Antibody
Avertin (1.25%)	Nanjing Aibei Biotechnology	M2920	Reagent
BD FACSAria II	BD	642510	Equipment
Brilliant Violet 421 anti-mouse CD4	Biolegend	100437	Antibody
Brilliant Violet 605 anti-mouse / human CD11b	Biolegend	101237	Antibody
BSA	Sigma-Aldrich	A1933-1G	Chemical
Collagenase IV	Sigma-Aldrich	C4-28-100MG	Enzyme
DNase I	New England Biolabs	M0303L	Enzyme
EDTA	Sigma-Aldrich	EDS-100G	Chemical
Falcon 5 mL Round Bottom Polystyrene Test Tubes	Corning	352052	Lab material
Falcon 15 mL Conical Centrifuge Tubes	Corning	352095	Lab material
FlowJo V10	BD		Software
GLAST (ACSA-1) Antibody, PE	Miltenyi Biotec	130-118-344	Antibody
Isoflurane	RWD Life Science	R510-22-10	Reagent
PE/Cyanine7 anti-mouse CD3	Biolegend	100220	Antibody
PE/Dazzle 594 anti-mouse NK-1.1	Biolegend	156518	Antibody
TRIzol Reagent	Thermo Fisher Scientific Inc.	15596026	Reagent
UltraPure DNase/RNase-Free Distilled Water	Thermo Fisher Scientific Inc.	10977015	Reagent
Vetbond Tissue Adhesives	3M	1469SB	Lab material
Zombie Aqua Fixable Viability Kit	Biolegend	423102	Amine-reactive fluorescent dye