Neuroscience

This paper presents a refined, user-friendly protocol for using boron-dipyrromethene (BODIPY) to assess and quantify foam cells and lipid droplet–accumulating microglia (LDAM) in mouse brain tissue. The protocol aims to enhance existing methodologies by offering precise and efficient evaluation of foam cells and LDAM burden in various neuropathological conditions linked to lipid metabolism and neuroinflammation. A notable challenge in analyzing tissue from mouse models of these neurodegenerative disorders is the interference caused by the autofluorescent molecule lipofuscin. Our protocol addresses this issue with specific steps that effectively distinguish BODIPY fluorescence from lipofuscin autofluorescence, using advanced imaging techniques and filter settings to ensure accurate and reliable analysis. By providing a straightforward and accessible method, this research aims to facilitate the broader adoption of BODIPY-based techniques for detailed foam cell and LDAM analysis in mouse brain tissue, potentially enhancing diagnostic capabilities and deepening our understanding of how these cells contribute to neurodegenerative disease mechanisms.

Microglia, the brain's primary resident immune cell, exists in various phenotypic states depending on intrinsic and extrinsic signaling. Distinguishing between these phenotypes can offer valuable biological insights into neurodevelopmental and neurodegenerative processes. Recent advances in single-cell transcriptomic profiling have allowed for increased granularity and better separation of distinct microglial states. While techniques such as immunofluorescence and single-cell RNA sequencing (scRNA-seq) are available to differentiate microglial phenotypes and functions, these methods present notable limitations, including challenging quantification methods, high cost, and advanced analytical techniques. This protocol addresses these limitations by presenting an optimized cell preparation procedure that prevents ex vivo activation and a flow cytometry panel to distinguish four distinct microglial states from murine brain tissue. Following cell preparation, fluorescent antibodies were applied to label 1) homeostatic, 2) disease-associated (DAM), 3) interferon response (IRM), and 4) lipid-droplet accumulating (LDAM) microglia, based on gene markers identified in previous scRNA-seq studies. Stained cells were analyzed by flow cytometry to assess phenotypic distribution as a function of age and sex. A key advantage of this procedure is its adaptability, allowing the panel provided to be enhanced using additional markers with an appropriate cell analyzer (i.e., Cytek Aurora 5 laser spectral flow cytometer) and interrogating different brain regions or disease models. Additionally, this protocol does not require microglial cell sorting, resulting in a relatively quick and straightforward experiment. Ultimately, this protocol can compare the distribution of microglial phenotypic states between various experimental groups, such as disease state or age, with a lower cost and higher throughput than scRNA-seq.

Myeloid cells, specifically microglia and macrophages, are activated in retinal diseases and can improve or worsen retinopathy outcomes based on their inflammatory phenotype. However, assessing the myeloid cell response after retinal injury in mice remains challenging due to the small tissue size and the challenges of distinguishing microglia from infiltrating macrophages. In this protocol paper, we describe a flow cytometry–based protocol to assess retinal microglia/macrophage and their inflammatory phenotype after injury. The protocol is amenable to the incorporation of other markers of interest to other researchers.

Key features

• This protocol describes a flow cytometry–based method to analyze the myeloid cell response in retinopathy mouse models.

• The protocol can distinguish between microglia- and monocyte-derived macrophages.

• It can be modified to incorporate markers of interest.

We show representative results from three different retinopathy models, namely ischemia-reperfusion injury, endotoxin-induced uveitis, and oxygen-induced retinopathy.

Microglia are a unique type of tissue-resident innate immune cell found within the brain, spinal cord, and retina. In the healthy nervous system, their main functions are to defend the tissue against infectious microbes, support neuronal networks through synapse remodeling, and clear extracellular debris and dying cells through phagocytosis. Many existing microglia isolation protocols require the use of enzymatic tissue digestion or magnetic bead-based isolation steps, which increase both the time and cost of these procedures and introduce variability to the experiment. Here, we report a protocol to generate single-cell suspensions from freshly harvested murine brains or spinal cords, which efficiently dissociates tissue and removes myelin debris through simple mechanical dissociation and density centrifugation and can be applied to rat and non-human primate tissues. We further describe the importance of including empty channels in downstream flow cytometry analyses of microglia single-cell suspensions to accurately assess the expression of protein targets in this highly autofluorescent cell type. This methodology ensures that observed fluorescence signals are not incorrectly attributed to the protein target of interest by appropriately taking into account the unique autofluorescence of this cell type, a phenomenon already present in young animals and that increases with aging to levels that are comparable to those observed with antibodies against highly abundant antigens.

Immunofluorescence is a reliable method for identifying specific proteins in neuronal and glial cell populations of the hypothalamus. Several immunofluorescence protocols are available to detect protein markers and neuropeptides in the hypothalamus; however, published methods may vary in subtle details that can potentially impact the final outcome of the procedure. Here, we provide a detailed protocol suitable for thin cryostat sections, which has been successful for specific antibodies directed against key markers of hypothalamic neurons and glial cells. We include every detail concerning brain tissue collection, processing, sectioning, and labeling with optimal dilutions of antibodies with the aim of reducing non-specific background. Our background-optimized immunostaining protocol has been routinely used in the lab and allows efficient detection of specific neuropeptides, glial cells, and markers of inflammation and endoplasmic reticulum stress in the hypothalamus.

Microglia are the resident immune cells of the central nervous system (CNS). In the last year, the improvements in the transgenic mouse technologies and imaging techniques have shed light on microglia functions under physiological conditions. Microglia continuously scan the brain parenchyma with their highly motile processes, maintaining tissue homeostasis and participating in neuronal circuits refinement. Here, we describe a protocol that enables us to perform time-lapse imaging of microglial cells in acute hippocampal slices, making image acquisition possible on an electrophysiology rig equipped with a standard imaging system. Using this ex vivo approach, we investigated microglial processes scanning abilities under physiological condition in hippocampus.

Microglia reside in the central nervous system (CNS) and are involved in the maintenance of the physiologic state. They constantly survey their environment for pathologic alterations associated with injury or diseases. For decades, researchers have investigated the role of microglia under different pathologic conditions, using approaches aiming to inhibit or eliminate these phagocytic cells. However, until recently, methods have failed to achieve complete depletion. Moreover, treatments often affected other cells, making unequivocal conclusions from these studies difficult. Recently, we have shown that inhibition of colony stimulating factor 1 receptor (CSF1R) by oral treatment with PLX5622 containing chow enables complete depletion of retinal microglia and almost complete microglia depletion in the optic nerve without affecting peripheral macrophages or other cells. Using this approach, we investigated the role of microglia in neuroprotection in the retina and axon regeneration in the injured optic nerve under different conditions. Thus, this efficient, reliable and easy to use protocol presented here will enable researchers to unequivocally study the contribution of microglia on neurodegeneration and axon regeneration. This protocol can be also easily expanded to other paradigms of acute and chronic injury or diseases in the visual system.