Jove-Journal of Visualized Experiments最新文献

Imaging microcapillary networks of the skin in humans using nailfold capillaroscopy (NFC) has underscored the importance of microcirculation as a target organ system in critical systemic illnesses. Nailfold capillaroscopy is applied clinically to detect peripheral microvascular dysfunction and abnormalities in a range of systemic conditions, including rheumatic, cardiac, ocular (e.g., glaucoma), and endocrine disorders (e.g., hypertension and diabetes mellitus). NFC is useful not only in detecting peripheral systemic microvasculature disruption but also in assessing drug efficacy. However, translating clinical NFC findings to animal disease models can be challenging. Detecting microvascular dysfunction or abnormalities in animals is often invasive (e.g., endoscopic), carried out ex vivo (e.g., post-mortem imaging of tissues), or expensive, requiring specialized equipment such as those used in microcomputed tomography and photoacoustic imaging techniques. Developing quick, non-invasive, and inexpensive techniques to image peripheral microvasculature in animal models of disease is warranted to decrease research expenses and increase translatability to the clinic. Capillaroscopy has previously been used to visualize the nailfold microvasculature in animal models, including in guinea pigs and mice, thus demonstrating the capability of capillaroscopy as a non-invasive imaging tool in animal models. This study provides a protocol that applies capillaroscopy to a mouse nailbed, allowing researchers to easily and inexpensively assess the morphology of its microvasculature. Representative images of typical nailbed microvascular architecture in wild-type mice using two commonly used laboratory strains, SV129/S6 and C57/B6J, are provided. Further studies using this method are essential for applying nailbed capillaroscopy to a wide range of mouse disease models with peripheral microvascular abnormalities.

Soil-dwelling plant parasitic nematodes (PPNs) are important potato pests that cause lesions and/or change plant roots structure, leading to reduced crop fitness and productivity. Research on the cellular and subcellular mechanisms of PPNs infection and development can resort to field plants or seedlings under greenhouse conditions. Field studies are more representative of natural environments but are subjected to the unpredictability of environmental conditions that can heavily influence research outcomes. Greenhouse studies allow higher control over environmental variables and higher safety against contaminants or pathogens. However, in some hosts, genetic diversity becomes an important factor of variability and influences the host-parasite complex response. We have developed in vitro co-cultures of transgenic roots with PPNs as a reliable alternative that occupies less space, requires less time to obtain, and is free from contamination or from host genetic variability. Co-cultures are obtained by introducing aseptic PPNs to host in vitro transgenic roots. They can be maintained indefinitely, which makes them excellent support for keeping collections of reference PPNs. In the present work, a protocol is detailed for the controlled infection of in vivo potato roots with the root lesion nematode and for establishing in vitro co-cultures of potato transgenic roots with the root-knot nematode. The in vitro co-cultures provided a laboratory proxy for the natural potato infection condition and produced nematode life stages irrespective of season or climate conditions. Additionally, the methodology used for structural analysis is detailed using histochemistry and optical microscopy. The acid fuchsin dye is used to follow nematode attack sites on roots, while differential staining with Periodic acid-Schiff (PAS) and toluidine blue O highlights nematode structures in potato internal root tissue.

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. These methods enable detailed examinations of tissue morphologies and the localization of specific proteins within the tissue, which provide valuable insights into disease processes and mechanisms. Mice are the most widely used model for this purpose. However, because mouse eyeballs are small and mouse retinas are extremely delicate tissues, obtaining high-quality retinal sections and immunostaining images from mouse eyeballs is typically challenging. This study describes an improved protocol for cryosectioning mouse retinas and performing immunohistochemistry. An essential point of this protocol involves coating the eyeball with a layer of super glue, which prevents deformation of the eyeballs during the processes of cornea removal, lens extraction, and embedding. This step ensures the integrity of retinal morphologies is well preserved. This protocol highlights critical technical considerations and optimization strategies for consistently producing high-quality retinal sections and achieving excellent immunostaining results.

Pigs are increasingly used as a large animal model for pharmacologic CNS research due to the anatomical and physiological similarities between the porcine and human central nervous systems (CNS). However, accessing the cerebrospinal fluid (CSF) in larger pig breeds by conventional lumbar puncture techniques can be challenging due to an oblique orientation of the spinal spinous processes and a limited interlaminar space. Accordingly, an open surgical procedure for inserting a lumbar spinal catheter for continuous CSF sampling at the L4/L5 level in pigs is thoroughly described in this work. After positioning the pig and identifying the anatomical landmarks, a dorsal midline surgical incision is made to expose the spinous processes. By advancing the introducer needle, the spinal catheter is inserted inside the thecal sac of the spinal canal while leaving the bone structures of the spine intact. This method allows continuous infusion into or sampling from the porcine thecal sac with minimal bleeding or CSF leakage. The procedure is simple, time-efficient, and reproducible across different experimental setups, offering significant potential for various pre-clinical studies, including pharmacokinetic research, surgical training, and spinal cord injury models.

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including their interplay with significant human pathogens such as the human immunodeficiency virus (HIV). Compared to other immortalized cell lines of myeloid origin, THP-1 cells retain many intact inflammatory signaling pathways and display phenotypic characteristics that more closely resemble those of primary monocytes, including the ability to differentiate into macrophages when treated with phorbol-12-myristate 13-acetate (PMA). The use of CRISPR-Cas9 technology to engineer THP-1 cells through targeted gene knockout (KO) provides a powerful approach to better characterize immune-related mechanisms, including virus-host interactions. This article describes a protocol for efficient CRISPR-Cas9-based engineering using electroporation to deliver pre-assembled Cas9:sgRNA ribonucleoproteins into the cell nucleus. Using multiple sgRNAs targeting the same locus at slightly different positions results in the deletion of large DNA fragments, thereby increasing editing efficiency, as assessed by the T7 endonuclease I assay. CRISPR-Cas9-mediated editing at the genetic level was validated by Sanger sequencing followed by Inference of CRISPR Edits (ICE) analysis. Protein depletion was confirmed by immunoblotting coupled with a functional assay. Using this protocol, up to 100% indels in the targeted locus and a decrease of over 95% in protein expression were achieved. The high editing efficiency makes it convenient to isolate single-cell clones by limiting dilution.

Pathogenic variants in ion channel genes are associated with a high rate of sudden unexpected death in epilepsy (SUDEP). Mechanisms of SUDEP are poorly understood but may involve autonomic dysfunction and cardiac arrhythmias in addition to seizures. Some ion-channel genes are expressed in both the brain and the heart, potentially increasing the risk of SUDEP in patients with ion-channelopathies associated with epilepsy and cardiac arrhythmias. Transgenic rabbits expressing epilepsy variants provide a whole organism to study the complex physiology of SUDEP. Importantly, rabbits more closely replicate human cardiac physiology than do mouse models. However, rabbit models have additional health and anesthesia considerations when undergoing invasive monitoring procedures. We have developed a novel method to surgically implant a telemetry device for long-term simultaneous electroencephalogram (EEG) and electrocardiogram (ECG) monitoring in neonatal rabbit kits. Here, we demonstrate surgical methods to implant a telemetry device in P14 (weight range 175-250 g) kits with detailed attention to surgical approach, appropriate anesthesia and monitoring, and postoperative care, resulting in a low complication rate. This method allows for continuous monitoring of neural and cardiac electrophysiology during critical points in the development of cardiac arrhythmias, seizures, and potential SUDEP in rabbit models of genetic or acquired epilepsies.

This study outlines a detailed protocol for the fabrication of core-sheath 3D-bioprinted scaffolds designed to enhance chronic wound healing. The protocol involves isolating extracellular vesicles (EVs) from mesenchymal stem cells (MSCs), known for their regenerative and immunomodulatory properties. These EVs are then incorporated into a unique scaffold structure. The scaffold features a core composed of alginate loaded with EVs, surrounded by a sheath made of carboxymethyl cellulose and alginate lyase. This innovative design ensures controlled scaffold degradation while promoting efficient and controlled release of EVs at the wound site. The protocol covers key steps, including the preparation and characterization of the EVs, the formulation of bio-inks for 3D bioprinting, and the optimization of printing parameters to achieve the desired core-sheath architecture. By combining structural integrity and bioactivity, the scaffold aims to address the limitations of conventional wound dressings, offering a targeted approach to accelerate tissue regeneration and reduce inflammation in chronic wounds. This method provides a reproducible and scalable strategy for developing advanced biomaterials with potential clinical applications in chronic wound management. The protocol also highlights critical considerations for achieving consistent results, ensuring adaptability for future therapeutic applications.

Accurate preoperative planning in revision hip arthroplasty is crucial for achieving successful outcomes. To enhance the intuitive evaluation of acetabular bone defect severity and leverage previous successful experience in revision hip arthroplasty, this study proposes a novel approach based on expert surgical case database retrieval and is initially implemented in clinical application. In this study, five patients who required revision hip arthroplasty were preoperatively planned to employ the expert case database surgical planning system.The patient's imaging data was entered into the system and matched with cases in the expert case database. Based on the expert's surgical experience, a revision surgery plan was recommended. If no suitable case was found, the model and position of the prosthesis were planned based on patient-specific reconstruction results. A total of five patients were enrolled in this study, four males and one female, with a mean age of 50.6 years. The diagnosis was aseptic prosthesis loosening after hip arthroplasty. The mean operative time was 123.2 min, and the mean intraoperative hemorrhage was 672 mL. No intraoperative complications, such as vascular or nerve injury, were observed. In Case 2, for instance, the application of this innovative planning scheme enabled the surgeon to delineate the revision surgery plan for this patient in the preoperative period, thereby reducing the operative time and intraoperative hemorrhage. Furthermore, patients could be apprised of the outcomes of analogous cases in advance. Leveraging a big data analysis approach through our comprehensive case database enables automated identification of matching expert treatment plans throughout the entire process. This particularly benefits inexperienced orthopedic surgeons by providing accurate guidance on surgical strategies to assist them in selecting appropriate prosthetic sizes and mounting positions. Additionally, the matching results can offer patients visualizations depicting predicted postoperative outcomes.

This paper presents a robot-based experimental program aimed at developing an efficient and fast colorimetric gas sensor. The program employs an automated Design-Build-Test-learning (DBTL) approach, which optimizes the search process iteratively while optimizing multiple recipes for different concentration intervals of the gas. In each iteration, the algorithm generates a batch of recipe suggestions based on various acquisition functions, and with the increase in the number of iterations, the values of weighted objective function for each concentration interval significantly improve. The DBTL method begins with parameter initialization, setting up the hardware and software environment. Baseline tests establish performance standards. Subsequently, the DBTL method designs the following round of optimization based on the proportion of recipes in each round and tests performance iteratively. Performance evaluation compares baseline data to assess the effectiveness of the DBTL method. If the performance improvement does not meet expectations, the method will be performed iteratively; if the objectives are achieved, the experiment concludes. The entire process maximizes system performance through the DBTL iterative optimization process. Compared to the traditional manual developing process, the DBTL method adopted by this experimental process uses multi-objective optimization and various machine learning algorithms. After defining the upper and lower limits of component volume, the DBTL method dynamically optimizes iterative experiments to obtain the optimal ratio with the best performance. This method greatly improves efficiency, reduces costs, and performs more efficiently within the multi-formulation variable space when finding the optimal recipe.

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, anti-inflammatory, antiviral, antioxidant, and antineoplastic activities. In C-glycosides, the anomeric carbon of the sugar moiety is directly connected to an aglycone through carbon-carbon bonding. Compared with O-glycosides, C-glycosides are structurally stable and resistant to acids and enzymes. Consequently, they are typically unbreakable, resulting in poor absorbability and low bioavailability. Interestingly, some intestinal bacteria can cleave C-C glycosidic bonds, providing a specific and environmentally friendly biological approach to degrade C-glycosides. In this study, a set of standard operating procedures (SOPs) was developed for screening intestinal bacteria capable of cleaving C-C glycosidic bonds based on the biotransformation model of natural compounds. The SOPs include the preparation and enrichment of intestinal bacteria, activity-oriented screening, and activity validation in a low-carbon source medium. This methodology provides a foundational reference for researchers aiming to isolate and study these specialized functional bacteria.