往期刊物2021
卷册: 11, 期号: 22
生物物理学
Macroscopic Structural and Connectome Mapping of the Mouse Brain Using Diffusion Magnetic Resonance Imaging
利用弥散磁共振成像绘制小鼠大脑的宏观结构和连接组图
Translational work in rodents elucidates basic mechanisms that drive complex behaviors relevant to psychiatric and neurological conditions. Nonetheless, numerous promising studies in rodents later fail in clinical trials, highlighting the need for improving the translational utility of preclinical studies in rodents. Imaging of small rodents provides an important strategy to address this challenge, as it enables a whole-brain unbiased search for structural and dynamic changes that can be directly compared to human imaging. The functional significance of structural changes identified using imaging can then be further investigated using molecular and genetic tools available for the mouse. Here, we describe a pipeline for unbiased search and characterization of structural changes and network properties, based on diffusion MRI data covering the entire mouse brain at an isotropic resolution of 100 µm. We first used unbiased whole-brain voxel-based analyses to identify volumetric and microstructural alterations in the brain of adult mice exposed to unpredictable postnatal stress (UPS), which is a mouse model of complex early life stress (ELS). Brain regions showing structural abnormalities were used as nodes to generate a grid for assessing structural connectivity and network properties based on graph theory. The technique described here can be broadly applied to understand brain connectivity in other mouse models of human disorders, as well as in genetically modified mouse strains.Graphic abstract: Pipeline for characterizing structural connectome in the mouse brain using diffusion magnetic resonance imaging. Scale bar = 1 mm.
Cortical Laminar Recording of Multi-unit Response to Distal Forelimb Electrical Stimulation in Rats
大鼠前肢远端电刺激的多单位反应的皮层层析记录
Severe traumatic brain injury (sTBI) survivors experience permanent functional disabilities due to significant volume loss and the brain’s poor capacity to regenerate. Chondroitin sulfate glycosaminoglycans (CS-GAGs) are key regulators of growth factor signaling and neural stem cell homeostasis in the brain. In this protocol, we describe how to perform recordings to quantify the neuroprotective and regenerative effect of implanted engineered CS-GAG hydrogel (eCS) on brain tissue. This experiment was performed in rats under three conditions: healthy without injury (Sham), controlled cortical impact (CCI) injury on the rostral forelimb area (RFA), and CCI-RFA with eCS implants. This protocol describes the procedure used to perform the craniotomy, the positioning of the cortical recording electrode, the positioning of the stimulation electrode (contralateral paw), and the recording procedure. In addition, a description of the exact electrical setup is provided. This protocol details the recordings in the brain of injured animals while preserving most of the uninjured tissue intact, with additional considerations for intralesional and laminar recordings of multi-unit response.Graphic abstract: Sensorimotor response to paw stimulation using cortical laminar recordings.
癌症生物学
Measurement of Bone Metastatic Tumor Growth by a Tibial Tumorigenesis Assay
用胫骨肿瘤发生法测定骨转移瘤的生长
Bone metastasis is a frequent and lethal complication of many cancer types (i.e., prostate cancer, breast cancer, and multiple myeloma), and a cure for bone metastasis remains elusive. To recapitulate the process of bone metastasis and understand how cancer cells metastasize to bone, intracardiac injection and intracaudal arterial animal models were developed. The intratibial injection animal model was established to investigate the communication between cancer cells and the bone microenvironment and to mimic the setting of prostate cancer patients with bone metastasis. Given that detailed protocols of intratibial injection and its quantitative analysis are still insufficient, in this protocol, we provide hands-on procedures for how to prepare cells, perform the tibial injection, monitor tibial tumor growth, and quantitatively evaluate the tibial tumors in pathological samples. This manuscript provides a ready-to-use experimental protocol for investigating cancer cell behaviors in bone and developing novel therapeutic strategies for bone metastatic cancer patients.
Measurement of DNA Damage Using the Neutral Comet Assay in Cultured Cells
在培养细胞中使用中性彗星试验测量 DNA 损伤
Maintenance of DNA integrity is of pivotal importance for cells to circumvent detrimental processes that can ultimately lead to the development of various diseases. In the face of a plethora of endogenous and exogenous DNA damaging agents, cells have evolved a variety of DNA repair mechanisms that are responsible for safeguarding genetic integrity. Given the relevance of DNA damage and its repair for disease pathogenesis, measuring them is of considerable interest, and the comet assay is a widely used method for this. Cells treated with DNA damaging agents are embedded into a thin layer of agarose on top of a microscope slide. Subsequent lysis removes all protein and lipid components to leave ‘nucleoids’ consisting of naked DNA remaining in the agarose. These nucleoids are then subjected to electrophoresis, whereby the negatively charged DNA migrates towards the anode depending on its degree of fragmentation, creating shapes resembling comets, which can be visualized and analysed by fluorescence microscopy. The comet assay can be adapted to assess a wide variety of genotoxins and repair kinetics, and both DNA single-strand and double-strand breaks. In this protocol, we describe in detail how to perform the neutral comet assay to assess double-strand breaks and their repair using cultured human cell lines. We describe the workflow for assessing the amount of DNA damage generated by ionizing radiation or present endogenously in the cells, and how to assess the repair kinetics after such an insult. The procedure described herein is easy to follow and cost-effective.
细胞生物学
Live Imaging of Apoptotic Extrusion and Quantification of Apical Extrusion in Epithelial Cells
上皮细胞凋亡挤压的活体成像和根尖挤压的定量分析
Apoptotic cell death eliminates unhealthy cells and maintains homeostatic cell numbers within tissues. Epithelia, which serve as fundamental tissue barriers for the body, depend on a physical expulsion of dying cells (apoptotic cell extrusion) to remain sealed and intact. Apoptotic cell extrusion has been widely studied over recent years, with researchers using various approaches to induce apoptotic cell death. Unfortunately, the majority of chemical-based approaches for cell death induction rely on sporadically occurring apoptosis and extrusion, making imagining lengthy, often unsuccessful, and difficult to capture in high-quality images because of the frequent frame sampling needed to visualise the key molecular processes that drive extrusion. Here, we present a protocol that describes steps needed for laser-mediated induction of apoptosis in a cell of choice, followed by imaging of apoptotic extrusion in confluent monolayers of epithelial cells. Moreover, we provide the description of a new approach involving the mixing of labelled and unlabelled cells. In particular, this protocol characterises how cells surrounding apoptotic cells behave, with high spatial and temporal resolution. This can be achieved without the optical interference that apoptotic cells cause as they are physically expelled from the monolayer and move out of focus for imaging. Finally, the protocol is accompanied by detailed procedures describing cell preparation for apoptotic extrusion experiments, as well as post-acquisition analysis required to evaluate rates of successful extrusion.
Pericyte Mapping in Cerebral Slices with the Far-red Fluorophore TO-PRO-3
用远红荧光体TO-PRO-3绘制大脑切片中的周细胞地图
This protocol describes a method for high-resolution confocal imaging of pericytes with the far-red fluorophore TO-PROTM-3 Iodide 642/661 in cerebral slices of murine. Identification of pericytes with TO-PRO-3 is a short time-consuming, high cost-effective and robust technique to label pericytes with no need for immunostaining or generation of reporter mice. Since the TO-PRO-3 stain resists immunofluorescence, and lacks spectral overlap, the probe is well suited for multiple labelling. Our procedures also combine TO-PRO-3-staining of pericytes with fluorescent markers for astrocytes and vessels in brain slices. These approaches should enable the assessment of pericyte biology in gliovascular unit.
Gamete Fusion Assay in Mice
小鼠配子融合分析
Gamete fusion, which is the final event of fertilization, is a crucial physiological event in the creation of a new fetus. In mammals, sperm IZUMO1 and oocyte IZUMO1R (JUNO) recognition play a role in triggering this process. Gamete fusion occurs through a complex but steady and unfailing intermolecular reaction because fertilization must ensure species specificity, in which fusion takes place between gametes of the same species only. Although many factors involved in this process have recently been identified, their specific contributions remain largely unknown. The current article describes detailed methods for assessment of gamete fusion in mice, visualized by fluorescent dye transfer, from unfertilized oocyte to spermatozoa. These methods are applicable not only for fixed cells but also live imaging of gametes.
发育生物学
Isolation of Primary Leydig Cells from Murine Testis
从小鼠睾丸中分离出原发性睾丸间质细胞
In males, Leydig cells are the primary source of testosterone, which is necessary for testis development, masculinization, and spermatogenesis. Leydig cells are a valuable cellular model for basic research; thus, it is important to develop an improved method for isolation and purification of Leydig cells from testes. The available methods for Leydig cell isolation have some drawbacks, including the need for sophisticated instruments, high cost, tediousness, and time consumption. Here, we describe an improved protocol for isolation of primary Leydig cells from testicular tissue by digestion with collagenase IV.
Wholemount in situ Hybridization for Spatial-temporal Visualization of Gene Expression in Early Post-implantation Mouse Embryos
全座原位杂交技术对植入后早期小鼠胚胎中基因表达的时空可视化
Regionalized distribution of genes plays crucial roles in the formation of the spatial pattern in tissues and embryos during development. In situ hybridization has been one of the most widely used methods to screen, identify, and validate the spatial distribution of genes in tissues and embryos, due to its relative simplicity and low cost. However, acquisition of high-quality hybridization signals remains a challenge while maintaining good tissue morphology, especially for small tissues such as early post-implantation mouse embryos. In this protocol, we present a detailed RNA in situ hybridization protocol suitable for wholemount early post-implantation mouse embryos and other small tissue samples. This protocol uses digoxigenin (DIG) labeled riboprobes to hybridize with target transcripts, alkaline phosphatase-conjugated anti-DIG antibodies to recognize DIG-labeled nucleotides, and nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl-phosphate (BCIP) chromogenic substrates for color development. Specific steps and notes on riboprobe preparation, embryo collection, probe hybridization, and color development are all included in the following protocol.Graphic abstract: Overview of Wholemount in situ Hybridization in Early Mouse Embryos.
免疫学
CD45 Immunohistochemistry in Mouse Kidney
小鼠肾脏中的 CD45 免疫组织化学
CD45 is a pan-leukocyte marker, and CD45 stain is widely used to determine the extent of inflammatory cell infiltration and its association with tissue injury. In this manuscript, we share a reliable immunohistochemistry (IHC) protocol for CD45 staining in sections of paraffin-embedded mouse kidney. A rat anti-CD45 antibody was used as primary antibody, and a mouse adsorbed biotin-conjugated goat anti-rat IgG was selected as secondary antibody. A horseradish peroxidase (HRP)-linked avidin/biotin detection system was used to amplify the signal, which was detected with 3,3′-Diaminobenzidine (DAB). With this protocol, we show that the CD45 antibody recognizes cells of hematolymphoid lineage in bone marrow, as well as monocyte/macrophages in liver and lung tissue. The utility of this protocol in pathology research was indicated by dramatically increased CD45-positive (CD45+) cells in the kidneys of a mouse model of diabetes. Double staining for CD45 and injury marker KIM-1 showed accumulated CD45+ cells around injured tubular cells. CD45 and F4/80 macrophage staining on adjacent tissue sections revealed overlap of CD45+ cells with other inflammatory cells.
微生物学
Analysis of Protein Stability by Synthesis Shutoff
通过合成关闭分析蛋白质稳定性
In this protocol, we describe the analysis of protein stability over time, using synthesis shutoff. As an example, we express HA-tagged yeast mitofusin Fzo1 in Saccharomyces cerevisiae and inhibit translation via cycloheximide (CHX). Proteasomal inhibition with MG132 is performed, as an optional step, before the addition of CHX. Proteins are extracted via trichloroacetic acid (TCA) precipitation and subsequently separated via SDS-PAGE. Immunoblotting and antibody-decoration are performed to detect Fzo1 using HA-specific antibodies. We have adapted the method of blocking protein translation with cycloheximide to analyze the stability of high molecular weight proteins, including post-translational modifications and their impact on protein turnover.
神经科学
Visual-stimuli Four-arm Maze test to Assess Cognition and Vision in Mice
视觉刺激四臂迷宫测试评估小鼠的认知和视觉
Visual impairments, notably loss of contrast sensitivity and color vision, were documented in Alzheimer’s disease (AD) patients yet are critically understudied. This protocol describes a novel visual-stimuli four-arm maze (ViS4M; also called visual x-maze), which is a versatile x-shaped maze equipped with spectrum- and intensity-controlled light-emitting diode (LED) sources and dynamic grayscale objects. The ViS4M is designed to allow the assessment of color and contrast vision along with locomotor and cognitive functions in mice. In the color testing mode, the spectral distributions of the LED lights create four homogenous spaces that differ in chromaticity and luminance, corresponding to the mouse visual system. In the contrast sensitivity test, the four grayscale objects are placed in the middle of each arm, contrasting against the black walls and the white floors of the maze. Upon entering the maze, healthy wild-type (WT) mice tend to spontaneously alternate between arms, even under equiluminant conditions of illumination, suggesting that cognitively and visually intact mice use both color and brightness as cues to navigate the maze. Evaluation of the double-transgenic APPSWE/PS1ΔE9 mouse model of AD (AD+ mice) reveals substantial deficits to alternate in both color and contrast modes at an early age, when hippocampal-based memory and learning is still intact. Profiling of timespan, entries, and transition patterns between the different arms uncovers variable aging and AD-associated impairments in color discrimination and contrast sensitivity. The analysis of arm sequences of alternation reveals different pathways of exploration in young WT, old WT, and AD+ mice, which can be used as color and contrast imprints of functionally intact versus impaired mice. Overall, we describe the utility of a novel visual x-maze test to identify behavioral changes in mice related to cognition, as well as color and contrast vision, with high precision and reproducibility.Graphic abstract: Exploratory behavior of AD+ mice versus age- and sex-matched WT mice is tracked (top left: trajectory from a 5-min video file) in a novel visual-stimuli four-arm maze (ViS4M; also named visual x-maze) equipped with spectrum- and intensity-controlled LED sources or grayscale objects. Consecutive arm entries reveal that APPSWE/PS1ΔE9 (AD+) mice alternate less between arms, as opposed to WT mice. Sequence analysis, according to the three alternation pathways (depicted by white, yellow, and brown arrows) under different conditions of illumination, uncovers specific deficits linked to color vision in AD+ mice, evidenced by a color imprint chart.
Knockoff: Druggable Cleavage of Membrane Proteins
Knockoff：膜蛋白的药物裂解
Comparative cell biology relies on methods that disrupt protein function. Traditional approaches target the gene that encodes the protein of interest via conventional knockout (KO) methodology, conditional Cre-lox system, or recently, flexible protocols based on CRISPR/Cas9. However, these technologies lack precise temporal control (hours), whereby the slow half-lives of proteins may confound measurements, possibly resulting in misleading phenotypes. Targeting the protein itself bypasses issues pertaining to protein half-life, resulting in more acute disruption. An ideal system would enable controllable protein disruption, dependent on the presence or absence of a small molecule, with high temporal control achieved through washout/addition of the small molecule. Here, we outline the use of knockoff, a general method to disrupt membrane proteins based on the NS3/4A protease of the hepatitis C virus. This technique has been used in post-mitotic cells to study the function of long-lived integral membrane proteins and is suitable for the study of other membrane-bound proteins.Graphic abstract: Removal of the protease inhibitor induces cleavage from the membrane.General model of knockoff method. Inh, Inhibitor; POI, Protein of Interest; NS3/4A, Hepatitis C viral protease.
植物科学
Preparation and Transfection of Populus tomentosa Mesophyll Protoplasts
杨树中间叶原生质体的制备和转染
Mesophyll protoplasts freshly isolated from leaves are a useful research system in plants. However, cell walls in woody plants contain more pectin, making mesophyll protoplasts isolation difficult in Populus. This has limited their application in biochemical, molecular, cellular, genetic, genomic, transcriptomic, and proteomic assays. In this protocol, a simple and efficient method to prepare and transfect mesophyll protoplasts of Populus tomentosa is presented in detail. Leaves of P. tomentosa plants grown in tissue culture media were pre-treated in D-mannitol solution and then digested with an enzyme solution. After washing with W5 and MMg buffers, the protoplasts were incubated in PEG/Ca2+ solution with plasmid for transfection. The mesophyll protoplasts isolated were used to express the histone variant H2B fused with green fluorescent protein (GFP) for confocal microscopy imaging. This “P. tomentosa mesophyll protoplasts preparation and transfection” system provides a useful tool for studying woody plants using a variety of applications, including gene expression, subcellular localization, protein-protein interaction, chromatin immunoprecipitation, western blot, single-cell sequencing, and genome editing.
Imaging of Lipid Uptake in Arabidopsis Seedlings Utilizing Fluorescent Lipids and Confocal Microscopy
利用荧光脂质和共聚焦显微镜对拟南芥幼苗的脂质摄取进行成像
Eukaryotic cells use a diverse set of transporters to control the movement of lipids across their plasma membrane, which drastically affects membrane properties. Various tools and techniques to analyze the activity of these transporters have been developed. Among them, assays based on fluorescent phospholipid probes are particularly suitable, allowing for imaging and quantification of lipid internalization in living cells. Classically, these assays have been applied to yeast and animal cells. Here, we describe the adaptation of this powerful approach to characterize lipid internalization in plant roots and aerial tissues using confocal imaging.Graphic abstract: Fluorescent lipid uptake in Arabidopsis seedlings. Scale bars: seedling, 25 mm; leaf, 10 μm; root, 25 μm.
干细胞
Isolation of CD31+ Bone Marrow Endothelial Cells (BMECs) from Mice
从小鼠中分离 CD31+ 骨髓内皮细胞 (BMECs)
In the bone marrow microenvironment, endothelial cells (ECs) play a pivotal role in regulating the production of both growth and inhibiting factors. They are held together by adherence molecules that interact with hematopoietic progenitor cells. The study of ECs in the hematopoietic stem cell niche is limited due to the lack of efficient protocols for isolation. In this protocol, we developed a two-step approach to extract bone marrow endothelial cells (BMECs) to unlock the challenges researchers face in understanding the function of the endothelial vascular niche in in-vitro studies.