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

Obtaining Single-Cell Suspensions from Murine Brain and Skull Bone Marrow

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.

Author Spotlight: Investigating the Complex Immune Response Following Brain Ischemia

Isolamento delle cellule immunitarie dal cervello e dal cranio del topo

Preparazione di una sospensione unicellulare dal cervello di topo per studiare la risposta immunitaria ai disturbi cerebrali

Per ottenere la sospensione di una singola cellula dal cervello del neurone isolato, posizionare il cervello in un omogeneizzatore Dounce di vetro da 15 millilitri pre-raffreddato contenente una quantità appropriata di soluzione salina bilanciata di Hanks ghiacciata, o tampone HBSS. Usa il pestello Dounce sciolto per dissociare delicatamente e lentamente il tessuto cerebrale con circa 100-120 colpi di ghiaccio. Filtrare la sospensione di tessuto cerebrale risultante attraverso un filtro cellulare da 70 micron in una provetta da centrifuga da 50 millilitri.Sciacquare la provetta Dounce con cinque millilitri di HBSS e procedere con la filtrazione per massimizzare la raccolta cellulare. Centrifugare la sospensione tissutale filtrata a 550 g per sei minuti a quattro gradi Celsius. Rimuovere il surnatante utilizzando un aspiratore sottovuoto e risospendere il palato cellulare in un millilitro di soluzione isotonica a gradiente di densità al 30%.Trasferire la sospensione cellulare in una provetta da centrifuga da 15 millilitri. Aggiungere una quantità appropriata di soluzione con gradiente di densità al 30% e capovolgere delicatamente la provetta per garantire una miscelazione accurata. Centrifugare immediatamente la provetta a 845 g con accelerazione e freno impostati al livello tre per 20 minuti a quattro gradi Celsius.Una volta fatto, rimuovere delicatamente la provetta dalla centrifuga senza agitare e aspirare con cura lo strato superiore di mielina utilizzando una punta da un millilitro collegata al vuoto. Quindi aspirare il surnatante, lasciando da uno a due millilitri, e risospendere il palato cellulare con un minimo di 10 millilitri di HBSS freddo. Centrifugare a 550 g per sei minuti a quattro gradi Celsius.Lavare ancora una volta con un minimo di sette millilitri di HBSS freddo e centrifugare nuovamente. Dopo aver rimosso la maggior parte possibile del surnatante, risospendere le cellule in 100-200 microlitri di tampone per citometria a flusso per l'analisi della citometria a flusso.

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 ...

Ricerca

Neuroscienze

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

Preparazione della sospensione unicellulare del midollo osseo da calvaria di topo per studiare la risposta immunitaria ai disturbi cerebrali