Jove-Journal of Visualized Experiments最新文献

We introduce an advanced immunocompetent lung-on-chip model designed to replicate the human alveolar structure and function. This innovative model employs a microfluidic-perfused biochip that supports an air-liquid interface mimicking the environment in the human alveoli. Tissue engineering is used to integrate key cellular components, including endothelial cells, macrophages, and epithelial cells, to create a representative tissue model of the alveolus. The model facilitates in-depth examinations of the mucosal immune responses to various pathogens, including viruses, bacteria, and fungi, thereby advancing our understanding of lung immunity. The primary goal of this protocol is to provide details for establishing this alveolus-on-chip model as a robust in vitro platform for infection studies, enabling researchers to closely observe and analyze the complex interactions between pathogens and the host's immune system within the pulmonary environment. This is achieved through the application of microfluidic-based techniques to simulate key physiological conditions of the human alveoli, including blood flow and biomechanical stimulation of endothelial cells, alongside maintaining an air-liquid interface crucial for the realistic exposure of epithelial cells to air. The model system is compatible with a range of standardized assays, such as immunofluorescence staining, cytokine profiling, and colony-forming unit (CFU)/plaque analysis, allowing for comprehensive insights into immune dynamics during infection. The Alveolus-on-chip is composed of essential cell types, including human distal lung epithelial cells (H441) and human umbilical vein endothelial cells (HUVECs) separated by porous polyethylene terephthalate (PET) membranes, with primary monocyte-derived macrophages strategically positioned between the epithelial and endothelial layers. The tissue model enhances the ability to dissect and analyze the nuanced factors involved in pulmonary immune responses in vitro. As a valuable tool, it should contribute to the advancement of lung research, providing a more accurate and dynamic in vitro model for studying the pathogenesis of respiratory infections and testing potential therapeutic interventions.

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry. In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1⁺ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or immunomodulatory effector proteins into host cells. The T3SS is a target for antimicrobial discovery campaigns since it is accessible extracellularly and largely absent from non-pathogenic bacteria. Recent studies demonstrated that the T3SS of Yersinia and Salmonella are regulated by factors responsive to iron and oxygen, which are important niche-specific signals encountered during mammalian infection. Described here is a method for iron starvation of Yersinia pseudotuberculosis, with subsequent optional supplementation of inorganic iron. To assess the impact of oxygen availability, this iron starvation process is demonstrated under both aerobic and anaerobic conditions. Finally, incubating the cultures at the mammalian host temperature of 37 °C induces T3SS expression and allows quantification of Yersinia T3SS activity by visualizing effector proteins released into the supernatant. The steps detailed here offer an advantage over the use of iron chelators in the absence of iron starvation, which is insufficient for inducing robust iron starvation, presumably due to efficient Yersinia iron uptake and scavenging systems. Likewise, acid-washing laboratory glassware is detailed to ensure the removal of residual iron, which is essential for inducing robust iron starvation. Additionally, using a chelating agent is described to remove residual iron from media, and culturing the bacteria for several generations in the absence of iron to deplete bacterial iron stores. By incorporating standard protocols of trichloroacetic acid-induced protein precipitation, SDS-PAGE, and silver staining, this procedure demonstrates accessible ways to measure T3SS activity. While this procedure is optimized for Y. pseudotuberculosis, it offers a framework for studies in pathogens with similar robust iron uptake systems. In the age of antibiotic resistance, these methods can be expanded to assess the efficacy of antimicrobial compounds targeting the T3SS under host-relevant conditions.

To control and decrease the public health impact of human protozoan diseases such as Chagas disease, leishmaniasis, and human African trypanosomiasis, expediting the development of new drugs and vaccines is necessary. However, this process is filled with difficulties such as highly complex parasite biology and disease pathogenesis and, as typical for neglected tropical diseases, comparatively limited funding for research and development. Thus, in vitro and in vivo study models that can sufficiently reproduce infection and disease key features while providing rational use of resources are essential for progressing research for these conditions. One example is the in vivo bioluminescence imaging (BLI) mouse model for Chagas disease, which provides highly sensitive detection of long wavelength light generated by Trypanosoma cruzi parasites expressing luciferase. Despite this technique becoming the standard approach for drug efficacy in vivo studies, research groups might still struggle to implement it due to a lack of proper practical training on equipment handling and application of quality control procedures, even when suitable BLI equipment is readily available. Considering this scenario, this protocol aims to guide from planning experiments to data acquisition and analysis, with details that facilitate the implementation of protocols in research groups with little or no experience with BLI, either for Chagas disease or for other infectious disease mouse models.

Idiopathic facial paralysis is the most common type of facial nerve injury, accounting for approximately 70% of peripheral facial paralysis cases. This disease can not only lead to a change in facial expression but also greatly impact the psychology of patients. In severe cases, it can affect the normal work and life of patients. Therefore, the research on facial nerve injury repair has important clinical significance. In order to study the mechanism of this disease, it is necessary to carry out relevant animal experiments, among which the most important task is to establish an animal model with the same pathogenesis as human disease. The compression of the facial nerve within the petrous bone, especially the nerve trunk at the junction of the distal end of the internal auditory canal and the labyrinthine segment, is the pathogenesis of idiopathic facial paralysis. In order to simulate this common disease, a compression injury model of the main extracranial segment of the facial nerve was established in this study. The neurological damage was evaluated by behavioral, neuroelectrophysiological, and histological examination. Finally, 50 g constant force and 90 s clamp injury were selected as the injury parameters to construct a stable idiopathic facial paralysis model.

Ulcerative colitis (UC) is a chronic immune-mediated disease that affects the entire colon and rectum with a relapsing and remitting course, causing lifelong morbidity. When medical treatment is ineffective, especially in cases of massive gastrointestinal bleeding, perforation, toxic megacolon, or carcinogenesis, surgery becomes the last line of defense to cure UC. Total colorectal resection and ileal pouch-anal anastomosis (IPAA) offer the best chance for long-term treatment. Pouchitis is the most common and troublesome postoperative complication. In this investigation, microsurgery is employed to create an ileal pouch model in experimental rats via IPAA surgery. Subsequently, a sustained rat model of pouchitis is established by inducing inflammation of the ileal pouch with dextran sulfate sodium (DSS). The successful establishment of rat pouchitis is validated through analysis of postoperative general status, weight, food and water intake, fecal data, as well as pouch tissue pathology, immunohistochemistry, and inflammatory factor analysis. This experimental animal model of pouchitis provides a foundation for studying the pathogenesis and treatment of the condition.

Prior hyperscanning studies that record the brain activities of caregivers and children concurrently have primarily been conducted within the confines of the laboratory, thus limiting the generalizability of results to real-life settings. Here, a comprehensive protocol for capturing synchronized electroencephalography (EEG), electrocardiography (ECG), and behavioral recordings from infant-caregiver dyads during various interactive tasks at home is proposed. This protocol demonstrates how to synchronize the different data streams and report EEG data retention rates and quality checks. Additionally, critical issues and possible solutions with respect to the experimental setup, tasks, and data collection in home settings are discussed. The protocol is not limited to infant-caregiver dyads but can be applied to various dyadic constellations. Overall, we demonstrate the flexibility of EEG hyperscanning setups, which allow experiments to be conducted outside of the laboratory to capture participants' brain activities in more ecologically valid environmental settings. Yet, movement and other types of artifacts still constrain the experimental tasks that can be performed in the home setting.

Craniofacial volumetric muscle loss (VML) injuries can occur as a result of severe trauma, surgical excision, inflammation, and congenital or other acquired conditions. Treatment of craniofacial VML involves surgical, functional muscle transfer. However, these procedures are unable to restore normal function, sensation, or expression, and more commonly, these conditions go untreated. Very little research has been conducted on skeletal muscle regeneration in animal models of craniofacial VML. This manuscript describes a rat model for the study of craniofacial VML injury and a protocol for the histological evaluation of biomaterials in the treatment of these injuries. Liquid hydrogel and freeze-dried scaffolds are applied at the time of surgical VML creation, and masseters are excised at terminal time points up to 12 weeks with high retention rates and negligible complications. Hematoxylin and eosin (HE), Masson's Trichrome, and immunohistochemical analysis are used to evaluate parameters of skeletal muscle regeneration as well as biocompatibility and immunomodulation. While we demonstrate the study of a hyaluronic-acid-based hydrogel, this model provides a means for evaluating subsequent iterations of materials in VML injuries.

Cerebral conditions often require precise diagnosis and monitoring, necessitating advanced imaging techniques. Current modalities may not adequately detect early signs of reversible tissue damage, underlining the need for innovative diagnostic tools that can quantify changes in cerebral blood flow (CBF) with high specificity and sensitivity. This study integrates three-dimensional arterial spin labeling (3D-ASL) with structural MRI to develop comprehensive CBF atlases that cover all main functional regions of the brain. This innovative magnetic resonance imaging- arterial spin labeling (MRI-ASL) methodology provides a rapid and noninvasive means of quantifying region-specific CBF, offering a detailed view of CBF levels across different functional regions.The comparison between chronic cerebral ischemia (CCI) patients and healthy subjects revealed significantly diminished CBF across the cerebral functional regions in the constructed CBF atlases for the former. This approach not only allows for the efficient identification of CCI by analyzing concurrent decreases in CBF across critical areas relative to healthy distributions but also enables the tracking of treatment responses and rehabilitation progress through longitudinal CBF atlases.The CBF atlas developed using the MRI-ASL technique represents a novel advancement in the field of cerebral diagnostics and patient care. By comparing regional CBF levels against normative standards, this method enhances diagnostic capabilities, enabling clinicians to provide personalized care to patients with cerebral conditions.

Synthetic droplets and condensates are becoming increasingly common constituents of advanced biomimetic systems and synthetic cells, where they can be used to establish compartmentalization and sustain life-like responses. Synthetic DNA nanostructures have demonstrated significant potential as condensate-forming building blocks owing to their programmable shape, chemical functionalization, and self-assembly behavior. We have recently demonstrated that amphiphilic DNA "nanostars", obtained by labeling DNA junctions with hydrophobic moieties, constitute a particularly robust and versatile solution. The resulting amphiphilic DNA condensates can be programmed to display complex, multi-compartment internal architectures, structurally respond to various external stimuli, synthesize macromolecules, capture and release payloads, undergo morphological transformations, and interact with live cells. Here, we demonstrate protocols for preparing amphiphilic DNA condensates starting from constituent DNA oligonucleotides. We will address (i) single-component systems forming uniform condensates, (ii) two-component systems forming core-shell condensates, and (iii) systems in which the condensates are modified to support in vitro transcription of RNA nanostructures.