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

从小鼠脑和颅骨骨髓中获得单细胞悬液

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.

作者聚焦：研究脑缺血后的复杂免疫反应

从小鼠大脑和颅骨中分离免疫细胞

制备小鼠颅骨的骨髓单细胞悬液以研究对脑部疾病的免疫反应

为了从小鼠颅骨制备骨髓单细胞悬液，在解剖显微镜下，使用钝钳小心地从分离的颅骨上剥离硬脑膜。将颅骨放入含有冰上 PBS 的培养皿中，并使用 1 毫升移液器冲洗，确保去除其表面的血液残留物。将颅骨转移到一个新培养皿中，其中含有足够的新鲜冷 PBS，足以将其浸入。使用无菌剪刀在冷 PBS 中将颅骨切成大约 3 毫米 x 3 毫米的碎片。使用 18 号针头在 500 μL 微量离心管的底部戳一个孔。将此试管插入含有 20 μL 冷 PBS 的 1.5 mL微量离心管中。然后将颅骨碎片转移到 500 微升管中。将设置以 10， 000 g 在 4 摄氏度下离心 30 秒。将收集的颅骨骨髓细胞重悬于 500 μL 红细胞裂解缓冲液中，并在室温下孵育 30 秒。将悬浮液转移至含有 4 mL 冷 PBS 的 15 mL离心管中。在 4 摄氏度下以 400 g 离心 6 分钟。用 5 毫升冷 PBS 再洗涤一次。最后，在适当的缓冲液中重悬细胞上颚。

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Research

JoVE Journal

Neuroscience

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

213 次观看. 为了从小鼠颅骨制备骨髓单细胞悬液，在解剖显微镜下，使用钝钳小心地从分离的颅骨上剥离硬脑膜。将颅骨放入含有冰上 PBS 的培养皿中，并使用 1 毫升移液器冲洗，确保去除其表面的血液残留物。将颅骨转移到一个新培养皿中，其中含有足够的新鲜冷 PBS，足以将其浸入。使用无菌剪刀在冷 PBS 中将颅骨切成大约 3 毫米 x 3 毫米的碎片。使用 18 号针头在 500 μL 微量离?...

视频: 制备小鼠颅骨的骨髓单细胞悬液以研究对脑部疾病的免疫反应

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 ...