Jove-Journal of Visualized Experiments最新文献

Three-dimensional (3D) brain organoid cultures derived from induced pluripotent stem cells (iPSC) provide an important alternative in vitro tool for studying human brain development and pathogenesis of neurological diseases. However, the lack of incorporation of microglia in the human brain organoids is still a major hurdle for 3D models of neuroinflammation. Current approaches include either the incorporation of fully differentiated microglia into mature brain organoids or the induction of microglial differentiation from the early stage of iPSC-derived embryoid bodies (EBs). The first approach misses the stage when microglial differentiation interacts with the adjacent neural environment, and the later approach is technically challenging, resulting in inconsistency among the final organoids in terms of the quantity and quality of microglia. To model brain organoids with microglia to study the early interactions between microglial and neuronal development, highly pure hematopoietic progenitor cells (HPC) differentiated from human iPSCs were incorporated into iPSC-derived EBs to make brain organoids. Using immunostaining and single-cell RNA sequencing (sc-RNA-seq) analysis, we confirmed that HPCs were incorporated into the 3D organoids, which eventually developed into brain organoids with both microglia and neurons. Compared to brain organoids without HPCs, this approach produces significant microglial incorporation in the brain organoids. This novel 3D organoid model, which consists of both microglial and neural development properties, can be used to study the early interactions between innate immune and nervous system development and potentially as a model for neuroinflammation and neuroinfectious disorders.

Serial Two-photon tomography (STPT) is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic stage control and microtome slicing. We successfully implemented this technique for tracing the axonal projections in the marmoset brain. Here, the detailed experimental procedures that resulted in reliable volumetric imaging of the whole marmoset brain are described. A key process for successful imaging was the removal of meninges surrounding the brain, which interferes with slicing. A big advantage of this methodology is that the sliced sections can be used for additional staining. In the original set-up, the sliced sections are scrambled in the water bath. These sections can be correctly aligned in their original order according to the blood vessel patterns in the cortex. An example of effective histology is the visualization of myelin structure by simple light reflection, which can be combined with Nissl staining to define anatomical borders. These sections can also be used for immunological detection of non-fluorescent anterograde and retrograde tracers, which can be registered to the STPT data for layering of multiple data.

Perihilar cholangiocarcinoma (pCCA) is a highly malignant and aggressive tumor, with radical resection being the only curative treatment available. With continuous advancements in laparoscopic techniques and instruments, laparoscopic radical surgery for pCCA is now considered technically safe and feasible. However, due to the high complexity of the surgery and the lack of evidence-based clinical support, laparoscopic radical surgery for type IIIb pCCA is performed only in a few large hepatobiliary centers. Current guidelines recommend left hemihepatectomy combined with total caudate lobectomy and standardized lymphadenectomy for resectable type IIIb pCCA. Therefore, in this article, we provide a detailed description of the surgical steps and technical points of complete laparoscopic left hemihepatectomy combined with total caudate lobectomy, regional lymphadenectomy, and right hepatic duct-jejunal Roux-en-Y anastomosis in patients with type IIIb pCCA, using fluorescence navigation technology to enhance surgical precision and safety. By adhering to standardized surgical procedures and precise intraoperative techniques, we offer an effective means to improve patient outcomes.

The nuclear lamina is a network of filaments underlying the nuclear membrane, composed of lamins and lamin-associated proteins. It plays critical roles in nuclear architecture, nuclear pore positioning, gene expression regulation, chromatin organization, DNA replication, and DNA repair. Mutations in genes involved in the expression or post-translational processing of lamin proteins result in genetic disorders known as laminopathies. Specifically, mutations in the LMNA or ZMPSTE24 genes can lead to the accumulation of incompletely processed forms of lamin A that retain farnesyl and methyl groups, which are absent in fully processed lamin A. These incompletely processed lamin A proteins localize to the inner nuclear membrane instead of the nuclear lamina, where mature lamin A resides. Mislocalized lamin proteins profoundly disrupt nuclear function and structure, often resulting in nuclear blebbing. In severe cases, nuclear rupture can occur, causing a loss of compartmentalization and leakage of genomic DNA into the cytosol. Abnormal nuclear structure and compartmentalization loss can be identified through indirect immunofluorescence (IF) on fixed cells. This study outlines such a method, employing specific antibodies against a lamin protein and double-stranded DNA (dsDNA) to simultaneously visualize the nuclear envelope and DNA. This approach enables a rapid assessment of nuclear structural integrity and the potential leakage of nuclear DNA into the cytosol.

Strongyloides ratti is a parasitic nematode that naturally infects wild rats. However, most laboratory rat and mouse strains are fully susceptible to infection. Immunocompetent BALB/c and C57BL/6 mice terminate S. ratti infections within a month in the context of a canonical type 2 immune response and remain semi-resistant to a re-infection. The course of infection can be divided into three phases: (a) the tissue migration phase of the infective third-stage larvae during the first two days; (b) the early intestinal phase, including the molting to the adult parasites and embedding in the mucosa of the intestine in days 3 to 6 post-infection with reproduction starting by day 5 to 6 post-infection; (c) the later intestinal phase ending with the complete clearance of the parasites. Experimental infections of mice with S. ratti enable the precise study of host-parasite interactions throughout the whole life cycle at the different sites of infection, as well as immune evasion strategies employed by the parasite. The protocol presented here describes the maintenance of the parasite in Wistar rats, the infection of laboratory mice, and the detection and quantification of S. ratti parasites in the tissue migrating phase and during the intestinal phase.

Mitochondria, important cellular organelles found in most eukaryotic cells, are major sites of energy production through aerobic respiration. Beyond this well-known role as the 'cellular powerhouse,' mitochondria are also involved in many other essential cellular processes, including the regulation of cellular metabolism, proliferation, immune signaling, and hormonal signaling. Deterioration in mitochondrial function during aging or under mitochondrial stress is often characterized by distinct changes in mitochondrial morphology and volume. The nematode C. elegans is an ideal model for studying these changes due to its transparent body and short lifespan, which facilitate live microscopy throughout its lifetime. However, even within the C. elegans field, numerous transgenic constructs and methods for mitochondrial imaging are available, each with its own limitations. Here, single-copy, matrix-localized GFP constructs are presented as a robust and reliable method for imaging mitochondrial morphology in C. elegans. This study specifically focuses on experimentally controllable factors to minimize errors and reduce variability between replicates and across studies when performing mitochondrial imaging during the aging process. Additionally, mitoMAPR is recommended as a robust method to quantify changes in mitochondrial morphology across tissue types during aging.

Due to its anatomical and physiological similarities to the human eye, the porcine eye serves as a robust model for biomedical research and ocular toxicity assessment. An air/liquid corneal culture system using porcine eyes was developed, and ex vivo epithelial wound healing was utilized as a critical parameter for these studies. Fresh pig corneas were processed for organ culture, with or without epithelial wounding. The corneas were cultured in a humidified 5% CO2 incubator at 37 °C in MEM, with or without testing agents. Corneal permeability and wound healing rates were measured, and epithelial cells and/or whole corneas can be processed for immunohistochemistry, western blotting, and qPCR for molecular and cellular analyses. This study describes a detailed protocol and presents two studies using this ex vivo system. The data show that porcine corneal organ culture, combined with epithelial wound healing, is a suitable ex vivo model for chemical toxicity testing, studying diabetic keratopathy, and identifying potential therapies.

The extent of functional sequences within the human genome is a pivotal yet debated topic in biology. Although high-throughput reverse genetic screens have made strides in exploring this, they often limit their scope to known genomic elements and may introduce non-specific effects. This underscores the urgent need for novel functional genomics tools that enable a deeper, unbiased understanding of genome functionality. This protocol introduces the Insertion-based Screen for functional Elements and Transcripts (InSET), a method for identifying lentivirus integration sites within a lentivirus-based insertional mutagenesis cell library. InSET facilitates the capture of genome-wide lentiviral integration sites, with next-generation sequencing used to detect and quantify flanking sequences. InSET's design enables the analysis of integration site abundance variations in phenotypic screens on a large scale, establishing it as a robust tool for forward genetics and for identifying functional genomic elements. A key benefit of InSET is its capacity to reveal previously unidentified genomic elements, including novel functional exons of both protein-coding and non-coding RNAs, independent of prior annotation. Overall, InSET holds significant value in studying the intricate complexity of the human genome and transcriptome, where many genomic elements await functional characterization.

The therapeutic effectiveness of acupuncture relies on both safety and stability, making these factors essential in acupuncture manipulation research. However, manual manipulation introduces unavoidable inaccuracies, which can impact the reliability of research findings. To address this challenge, a unique lifting and thrusting manipulation control cannula was designed in this study, offering flexible adjustment of movement amplitude. The cannula was created using 3D printing technology, and its effectiveness in maintaining stability was verified by recording the acupuncture needle's movement range with optical sensor technology. The study's results show that the control cannula significantly enhances the stability of acupuncture manipulation, reducing human error. This innovation suggests that the cannula could serve as a valuable auxiliary tool for ensuring both the precision and safety of acupuncture-related experimental research. Its adoption could also contribute to the standardization of acupuncture practices, ensuring more consistent and accurate research outcomes, which is essential for future advancements in acupuncture research and clinical applications.

For noninvasive light-based physiological monitoring, optimal wavelengths of individual tissue components can be identified using absorption spectroscopy. However, because of the lack of sensitivity of hardware at longer wavelengths, absorption spectroscopy has typically been applied for wavelengths in the visible (VIS) and near-infrared (NIR) range from 400 to 1,000 nm. Hardware advancements in the short-wave infrared (SWIR) range have enabled investigators to explore wavelengths in the ~1,000 nm to 3,000 nm range in which fall characteristic absorption peaks for lipid, protein, and water. These molecules are difficult to visualize in the VIS-NIR and can provide label-free sources of biological contrast. Furthermore, lower SWIR absorption has been observed for melanin, the primary chromophore responsible for skin pigmentation. In vivo optical devices like clinically standard pulse oximeters have been found to have reduced accuracy in people with darkly pigmented skin, possibly because of the stronger melanin absorption in the VIS range. Thus, error associated with skin pigmentation could be reduced by using devices operating in the SWIR. Optical instrument design is facilitated by the understanding of the absorption properties of core tissue components from the VIS to the SWIR range. This article describes protocols and instrumentation for obtaining VIS-SWIR absorption spectra of common tissue absorbers: oxygenated hemoglobin, deoxygenated hemoglobin, melanin, water, and lipid.