preparing single-cell suspension from mouse brain to study immune response to brain disorders

Gewinnung von Einzelzellsuspensionen aus dem Gehirn und dem Knochenmark von Mäusen

The brain, the central hub of the nervous system, is protected by the skull. Beneath the skull are three layers of connective tissue known as the meninges - the dura mater, arachnoid mater, and pia mater. Cerebrospinal fluid (CSF) circulates in the subarachnoid space between the arachnoid mater and pia mater, cushioning the brain and also removing waste via the glymphatic system1,2. Together, this unique architecture provides a secure and supportive environment that maintains the stability of the brain and shields it from potential injury.
The brain has long been considered immune-privileged. However, this notion has been partially abandoned as mounting evidence indicates that, in addition to resident microglia in the parenchyma, the borders of the brain, including the choroid plexus and meninges, host a diverse array of immune cells3. These cells play critical roles in maintaining homeostasis, surveilling brain health, and initiating the immune response to brain injury. Notably, recent findings indicate that the skull is involved in meninges immunity, and may contribute to the immune response in the brain after injury. In 2018, Herisson et al. made a seminal discovery of direct vascular channels that link the skull bone marrow to the meninges, thereby establishing an anatomical route for leukocyte migration4,5. Later, Cugurra et al. demonstrated that many myeloid cells (e.g., monocytes and neutrophils) and B cells in the meninges do not originate from the blood6. Using techniques such as calvaria bone-flap transplantation and selective irradiation regimens, the authors provided compelling evidence that the skull bone marrow serves as a local source for myeloid cells in the meninges as well as CNS parenchyma after CNS injury6. Further, another study proposed that meningeal B cells are constantly supplied by the skull bone marrow7. More recently, a novel structure, termed the arachnoid cuff exit (ACE), has been identified as a direct gateway between the dura mater and the brain for immune cell trafficking8.
These exciting findings have important implications for the origin of infiltrating immune cells into the injured brain (e.g., after ischemic stroke). A large body of evidence has indicated that after stroke, many immune cells infiltrate the brain, contributing to both acute brain damage and chronic brain recovery. The conventional notion&#160;is that these cells are circulating leukocytes in the blood that infiltrate the brain, which is largely facilitated by stroke-induced blood-brain barrier damage. However, this notion has been challenged. In one study, immune cells in the skull and the tibia of mice were labeled differently, and at 6 h after stroke, a significantly greater decrease in neutrophils and monocytes was found in the skull vs. the tibia, and more skull-derived neutrophils were present in the ischemic brain. These data suggest that in the acute stroke phase, neutrophils in the ischemic brain primarily originate from the skull bone marrow4. Interestingly, CSF may guide this migration. Indeed, two recent reports demonstrated that CSF can directly relay signaling cues from the brain into the skull bone marrow via skull channels, and instruct cell migration and hematopoiesis in the skull bone marrow after CNS injury9,10.
In light of these recent findings, it has become important to analyze changes in immune cells in both the brain and the skull bone marrow, when studying the immune response to brain disorders. In such investigations, sufficient numbers of high-quality immune cells are needed for downstream analyses such as cell sorting, flow cytometry analysis, and single-cell RNA sequencing (scRNA-seq). Here, the overall goal is to present two optimized procedures for preparing single-cell suspensions from brain tissue and skull bone marrow. It is important to note that the calvaria (frontal bone, occipital bone, and parietal bones) of the skull are typically used to extract bone marrow, and this bone marrow is specifically referred to as skull bone marrow throughout this study.

Autor im Rampenlicht: Untersuchung der komplexen Immunantwort nach Hirnischämie

Isolierung von Immunzellen aus dem Gehirn und dem Schädel von Mäusen

Our research focuses on the complex immune response following brain ischemia caused by ischemic stroke and cardiac arrest. Brain ischemia triggers the activation resident immune cells in the brain and the infiltration of immune cells. However, our understanding of this immune cells remains limited.We aim to dissect the immune state, origin, and functional roles of this brain cells after ischemia. Recent research highlights the surprising role of the skull in the brain's immune response following injury. Therefore, it is important to analyze changes in immune cells in both the brain tissue and the skull bone marrow after brain ischemia.To this end, a critical step is to obtain a sufficient number of high-quality immune cells for further analysis. Our protocols enable researchers to rapidly obtain large quantities of available immune cells from the brain and the skull bone marrow. The brain immune cell protocol uses a mechanical dissociation approach at low temperature throughout, ensuring better preservation of the transcriptional and the proteomic profiles.The skull bone marrow protocol is simple, yet effective for extracting bone marrow cells from the skull. We'll continue our comprehensive research on the impact of brain ischemia on immune cells in brain tissue and the skull. Given that neuroinflammation plays a key role in the pathophysiology of brain injury after ischemia, our research is expected to discover novel therapeutic targets for brain protection after ischemic stroke or cardiac arrest.

Herstellung einer einzelligen Suspension aus dem Gehirn von Mäusen zur Untersuchung der Immunantwort auf Hirnerkrankungen

Um eine Einzelzellsuspension aus dem isolierten Neuronengehirn zu erhalten, legen Sie das Gehirn in einen vorgekühlten 15-Milliliter-Glas-Dounce-Homogenisator, der eine angemessene Menge eiskalter Hanks'balancierter Salzlösung oder HBSS-Puffer enthält. Verwenden Sie den losen Dounce Stößel, um das Hirngewebe mit etwa 100 bis 120 Schlägen auf Eis sanft und langsam zu dissoziieren. Filtrieren Sie die resultierende Hirngewebesuspension durch ein 70-Mikron-Zellsieb in einem 50-Milliliter-Zentrifugenröhrchen.Spülen Sie das Dounce-Röhrchen mit fünf Millilitern HBSS und fahren Sie mit der Filtration fort, um die Zellentnahme zu maximieren. Die filtrierte Gewebesuspension bei 550 g sechs Minuten lang bei vier Grad Celsius zentrifugieren. Entfernen Sie den Überstand mit einem Vakuumsauger und resuspendieren Sie den Zellgaumen in einem Milliliter einer 30%igen isotonischen Dichtegradientenlösung.Übertragen Sie die Zellsuspension in ein 15-Milliliter-Zentrifugenröhrchen. Fügen Sie eine angemessene Menge einer 30%igen Dichtegradientenlösung hinzu und drehen Sie das Rohr vorsichtig um, um eine gründliche Durchmischung zu gewährleisten. Zentrifugieren Sie das Röhrchen sofort bei 845 g mit Beschleunigung und Bremse auf Stufe drei für 20 Minuten bei vier Grad Celsius.Wenn Sie fertig sind, nehmen Sie das Röhrchen vorsichtig aus der Zentrifuge, ohne es zu schütteln, und saugen Sie die obere Myelinschicht vorsichtig mit einer Ein-Milliliter-Spitze ab, die mit dem Vakuum verbunden ist. Aspirieren Sie dann den Überstand, lassen Sie ein bis zwei Milliliter zurück und resuspendieren Sie den Zellgaumen mit mindestens 10 Millilitern kaltem HBSS. Bei 550 g sechs Minuten bei vier Grad Celsius zentrifugieren.Waschen Sie noch einmal mit mindestens sieben Millilitern kaltem HBSS und zentrifugieren Sie erneut. Nachdem Sie so viel Überstand wie möglich entfernt haben, resuspendieren Sie die Zellen in 100 bis 200 Mikrolitern Durchflusszytometriepuffer für die Durchflusszytometrieanalyse.

preparing-single-cell-suspension-from-mouse-brain-to-study-immune

Research

JoVE Journal

Neuroscience

Preparing Single-Cell Suspension from Mouse Brain to Study Immune Response to Brain Disorders

Isolating Immune Cells from Mouse Brain and Skull

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only in the brain, but also in the skull. A key step toward analyzing changes in immune cell populations in both the brain and skull bone marrow after brain damage (e.g., stroke) is to obtain sufficient numbers of high-quality immune cells for downstream analyses. Here, two optimized protocols are provided for isolating immune cells from the brain and skull bone marrow. The advantages of both protocols are reflected in their simplicity, speed, and efficacy in yielding a large quantity of viable immune cells. These cells may be suitable for a range of downstream applications, such as cell sorting, flow cytometry, and transcriptomic analysis. To demonstrate the effectiveness of the protocols, immunophenotyping experiments were performed on stroke brains and normal brain skull bone marrow using flow cytometry analysis, and the results aligned with findings from published studies.

To investigate the immune response to brain disorders, one common approach is to analyze changes in immune cells. Here, two simple and effective protocols are provided for isolating immune cells from murine brain tissue and skull bone marrow.

Mounting evidence indicates that the immune response triggered by brain disorders (e.g., brain ischemia and autoimmune encephalomyelitis) occurs not only ...