Jove-Journal of Visualized Experiments最新文献

To date, uterus transplantation is the only option for women with absolute uterine infertility, such as those with Rokitansky syndrome, to experience pregnancy and give birth. Despite the growing interest in uterus transplantation in recent years, several issues still require further research, including ischemia-reperfusion injury and its impact on graft quality and rejection. Recent literature has highlighted a thrombotic complication rate of up to 20% following uterus transplantation. This type of complication may result from hypoxia-induced endothelial cell damage, often leading to uterine graft rejection. Hypoxia is induced during static cold storage, which remains the gold standard for graft preservation in solid organ transplantation. Recently, dynamic preservation using machine perfusion has been shown to improve the long-term storage of conventional and marginal organs by reducing ischemic and hypoxic injury. In this protocol, we aim to describe every surgical step involved in porcine uterus procurement and dynamic preservation, based on both uterine pedicles, to enable the connection and initiation of the machine perfusion protocol.

The gold standard to assess the aerobic capacity in physically active subjects and athletes is the maximal oxygen consumption test (VO2-max), which involves analysis of exhaled-gases and cardiorespiratory variables obtained via the breath-by-breath method in an ergospirometer during an incremental exercise. However, this method cannot elucidate metabolic changes at the muscular level. Near-infrared spectroscopy (NIRS) has emerged as a valuable technology to evaluate local oxygen levels (Tissular Saturation Index, TSI) by quantifying the concentrations of oxygenated (O2-Hb) and deoxygenated (H-Hb) hemoglobin in the microvasculature of tissues. NIRS applications extend to respiratory and locomotor muscles, assessing metabolic changes associated with the cost of breathing (COB) and peripheral workload, respectively. Additionally, cerebral regions, such as the prefrontal cortex, have been explored with NIRS technology to assess physiological changes related to cognitive demand associated with planning or ideation of motor tasks linked to sports performance. Thus, by analyzing exercise-induced changes (D) in O2-Hb, H-Hb, and TSI, it is possible to identify central and peripheral exercise limitations, particularly when endurance training is the main component of physical fitness (e.g., running, cycling, triathlon, etc.). Addressing these factors is paramount for coaches and exercise physiologists to optimize athletic performance, incorporating training strategies focused on the primary exercise-limiting factors. This study outlines a protocol for utilizing wearables devices equipped with NIRS technology to analyze exercise changes in TSI, O2-Hb, and H-Hb, alongside cardiorespiratory variables typically registered in athletes during VO2-max tests. This approach offers a comprehensive method for identifying the primary systems involved in stopping exercise progression and sports performance improvement.

Fungi infect humans when environmental spores are inhaled into the lungs. The lung is a heterogeneous organ. Conducting airways, including bronchi and bronchioles, branch until terminating in the alveolar airspace where gas exchange occurs. Infections originating in the bronchioles or alveoli elicit distinct host responses and disease manifestations. Therefore, understanding precisely where spores naturally localize in the lungs, particularly soon after infection, expands opportunities for investigation of host-pathogen interactions. Herein, we detail an in-situ analysis of lungs from mice infected with Coccidioides posadasii cts2/ard1/cts3Δ arthroconidia. Conventional methods for histological preservation involve liquid inflation of the airways with a fixative solution, which displaces the natural location of aspirated fungal particles, pushing spores from proximal bronchioles to terminal airspaces. Conversely, this method of air-inflation with blood vasculature perfusion-fixation preserves the physiologic position of fungal spores within the bronchioles. Moreover, we describe a simple approach to cryopreserving, embedding, and imaging lung specimens. We also share high-throughput computational techniques via the open-source QuPath program to analyze the spatial distribution of fungal spores within the lung. The method presented here is simple and quick, requires minimal equipment to perform, and can be easily adapted for use with many respiratory fungal infection models.

Hemocytes are the circulating immune-competent cells in bivalve mollusks and play a key role in several important functions of cell-mediated innate immunity. During the early stages of the immune response, hemocytes actively migrate to the site of infection. This inherent motility is a fundamental characteristic of these cells. It represents a key cellular function that integrates multiple processes, such as cell adhesion, cell signaling, cytoskeletal dynamics, and changes in cell volume. Therefore, alterations in cell motility following exposure to drugs or pollutants can serve as a useful toxicological endpoint. Despite the fundamental role of cell motility in cellular physiology, it has been poorly investigated from a toxicological perspective. This work proposes a novel in vitro method for the rapid and sensitive assessment of the toxicity and ecotoxicity of pollutants, based on evaluating the hemocyte motility of Mytilus galloprovincialis. We developed a cell motility assay on hemocytes adhering to the bottom of a 96-well polystyrene microplate. Following exposure to increasing concentrations of drugs, cell trajectories, and velocities were quantified by cell tracking under time-lapse microscopy, allowing us to measure the effects on hemocyte motility. Due to the ease of hemocyte collection from the animals in a relatively non-invasive manner, the proposed method offers an alternative test for screening the effects and mechanisms of action of pollutants and drugs. It aligns with the 3Rs (Replacement, Reduction, and Refinement) criteria, addressing ethical concerns and contributing to the reduction of vertebrate in vivo animal testing.

The CUT&RUN technique facilitates detection of protein-DNA interactions across the genome. Typical applications of CUT&RUN include profiling changes in histone tail modifications or mapping transcription factor chromatin occupancy. Widespread adoption of CUT&RUN is driven, in part, by technical advantages over conventional ChIP-seq that include lower cell input requirements, lower sequencing depth requirements, and increased sensitivity with reduced background signal due to a lack of cross-linking agents that otherwise mask antibody epitopes. Widespread adoption of CUT&RUN has also been achieved through the generous sharing of reagents by the Henikoff lab and the development of commercial kits to accelerate adoption for beginners. As technical adoption of CUT&RUN increases, CUT&RUN sequencing analysis and validation become critical bottlenecks that must be surmounted to enable complete adoption by predominantly wet lab teams. CUT&RUN analysis typically begins with quality control checks on raw sequencing reads to assess sequencing depth, read quality, and potential biases. Reads are then aligned to a reference genome sequence assembly, and several bioinformatics tools are subsequently employed to annotate genomic regions of protein enrichment, confirm data interpretability, and draw biological conclusions. Although multiple in silico analysis pipelines have been developed to support CUT&RUN data analysis, their complex multi-module structure and usage of multiple programming languages render the platforms difficult for bioinformatics beginners who may lack familiarity with multiple programming languages but wish to understand the CUT&RUN analysis procedure and customize their analysis pipelines. Here, we provide a single-language step-by-step CUT&RUN analysis pipeline protocol designed for users with any level of bioinformatics experience. This protocol includes completing critical quality checks to validate that the sequencing data is suitable for biological interpretation. We expect that following the introductory protocol provided in this article combined with downstream peak annotation will allow users to draw biological insights from their own CUT&RUN datasets.

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human vascular networks. This three-dimensional (3D) model holds substantial potential for vascular pathology modeling and in vitro drug screening. Despite recent advances, a key technical challenge remains in reproducibly generating organoids with consistent quality, which is crucial for downstream assays and applications. Here, a modified protocol is presented that improves both the homogeneity and reproducibility of vascular organoid generation. The modified protocol incorporates the use of microwells and the CEPT cocktail (chroman 1, emricasan, polyamines, and the integrated stress response inhibitor, trans-ISRIB) to improve embryoid body formation and cell survival. Differentiated, mature vascular organoids generated using this protocol are characterized by whole-mount 3D immunofluorescence microscopy to analyze their morphology and complex vasculature. This protocol enables the production of high-quality vascular organoids in a scalable manner, potentially facilitating their use in disease modeling and drug screening applications.

Single cell Ca²⁺ imaging is essential for the study of Ca²⁺ channels activated by various stimulations like temperature, voltage, native compound and chemicals et al. It primarily relies on microscopy imaging technology and the related Ca²⁺ indicator Fura-2/AM (AM is the abbreviation for Acetoxymethyl ester). Inside the cells, Fura-2/AM is hydrolyzed by esterases into Fura-2, which can reversibly bind with free cytoplasmic Ca²⁺. The maximum excitation wavelength shifts from 380nm to 340nm (when saturated with Ca²⁺) upon binding. The emitted fluorescence intensity is quantitatively related to the concentration of bound Ca²⁺. By measuring the 340/380 ratio, the Ca²⁺ concentration in the cytoplasm can be determined, eliminating errors caused by variations in the loading efficiency of the fluorescent probe among different samples. This technology allows for real-time, quantitative, and simultaneous monitoring of Ca²⁺ changes in multiple cells. The results are stored in ".XLSX" format for subsequent analysis, which is fast and generates intuitive change curves, greatly improving the detection efficiency. From different experimental perspectives, this article lists the use of this technology to detect Ca²⁺ signals in cells with endogenous or overexpressed channel proteins. Meantime, different methods for activating cells were also showed and compared. The aim is to provide readers with a clearer understanding of the usage and applications of single cell Ca²⁺ imaging.

With the advancement of scientific research, the demand for gene-edited rabbit models is increasing. However, there are limited pregnancy and feeding management systems for gene-edited rabbits, leading to low survival rates among gene-edited rabbits prepared by many inexperienced researchers. Therefore, proper guidance is essential. This article summarizes the pregnancy and feeding practices for genetically modified rabbits developed in the author's laboratory and outlines a set of fundamental processes. These include pregnancy diagnosis, antenatal care, midwifery, assisted breastfeeding, weaning, and other procedures, along with the rescue and care of weak newborn rabbits. Compared to the traditional natural childbirth and nurturing methods used in rabbit farms, this approach involves more refined management, requiring additional time and effort but significantly increasing the survival rate of suckling rabbits. The methods described in this article are suitable for most laboratory breeding scenarios involving gene-edited or embryo-transferred rabbits and provide a straightforward and effective reference for other researchers.

The study of heat tolerance in Drosophila melanogaster has been of particular interest to researchers for decades, with a common approach to assessing heat tolerance being to monitor the time to knockdown (TKD) after exposure to an elevated temperature. Classically, flies are housed in individual vials and placed inside a heated water bath. TKD is then monitored manually by researchers. While very well-established, there remain problems of subjectivity and consistent application of a tangible definition of cessation of all movement, including muscular spasms, when implementing these manual assays. We have developed a high-throughput method for automating heat tolerance assays using the TriKinetics Drosophila Activity Monitors (DAM2). To accompany the DAM2 system, we have written a program and created an easy-use executable to automatically read the last time of movement from the activity data generated. This script then writes to a .csv file the time to heat paralysis (TKD) for each fly. Our data show that this automated DAM2 method is consistent and reliable. Meanwhile, activity profiles created from the activity count data are of interest. These activity profiles can be compiled and have the potential to expand heat tolerance assays to include the relatively unstudied behavioral components of heat tolerance. This protocol will describe in detail how to use the DAM2 system and the HoTDAM! software to estimate heat tolerance in D. melanogaster.

The use of laboratory mice with a natural microbiome, such as "Wildling mice", offers a promising research tool for both basic and applied science due to their close resemblance to the human superorganism. However, the breeding and maintenance of these mice, which harbor a diverse microbiome including bacteria, viruses, and parasites, pose significant challenges for animal husbandry facilities at research institutions. To address these challenges, a specialized facility concept was developed for housing "Wildling mice" at Charité - Universitätsmedizin Berlin. This approach involved designing a facility with specific structural features and operational protocols to effectively contain the natural microbiome, thereby protecting areas with higher hygiene standards. A methodology for blood sampling from both specified pathogen-free (SPF) and "Wildling mice" for immunophenotyping is demonstrated, highlighting the workflow and biocontainment measures implemented in the facility. Remarkable results reveal that "Wildling mice" exposed to a natural microbiome develop distinct immune cell populations, which are significantly reduced in mice bred and maintained under stringent hygiene conditions. The significance of this study lies in its potential to provide researchers with access to mice that possess a natural microbiome and a mature immune system similar to that of human adults. This approach could enhance the translatability of preclinical findings into clinical practice, thereby advancing the field of biomedical research.