Jove-Journal of Visualized Experiments最新文献

{"title":"Investigating Murine CD4 T Cell Differentiation Using CRISPR-Cas9 Ribonucleoprotein Complex-mediated Gene Ablation.","authors":"Jillian K Perry, Pamela L Schwartzberg, Dominic P Golec","doi":"10.3791/67380","DOIUrl":"https://doi.org/10.3791/67380","url":null,"abstract":"<p><p>The widespread accessibility of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 technology has made gene targeting in primary cells a routine method for evaluating gene function in T cells. Given the cost and limited availability of knockout (KO) mouse strains, testing preliminary hypotheses involving gene function in T cells can be prohibitive using gene-targeted animal models. However, using commercially available resources, including predesigned guide RNAs (gRNAs), researchers can conveniently generate gene-targeted naïve T cells that can be used for T cell activation and differentiation studies. Here we outline a protocol for using nucleofection-delivered CRISPR-Cas9 ribonucleoprotein complexes (RNPs) to efficiently generate gene KO murine naïve CD4 T cells that can be used to evaluate gene function in CD4 T cell differentiation, in vitro. Isolation of naïve CD4 T cells from mouse secondary lymphoid organs, followed by nucleofection with Cas9-gRNA complexes ensures gene KO is initiated before downstream T cell activation, offering a strategic advantage over retroviral-mediated gRNA delivery, which typically requires preactivation of T cells, preventing the evaluation of effects in naïve T cells. Furthermore, this nucleofection-based method bypasses potential developmental issues associated with gene KO animals. Following Cas9-gRNA delivery, we describe protocols for studying CD4 T cell differentiation into Th1, Th2, Th17, and Treg lineages using in vitro polarization. In addition, this protocol is adaptable to using gene-targeted CD4 or CD8 T cells for numerous downstream applications, including other T cell activation studies in vitro and adoptive transfer studies in vivo. The use of CRISPR-Cas9 methods has streamlined our ability to evaluate gene function in T cells and allows for the routine KO of many genes of interest, freeing researchers from limitations associated with studying gene KO animals.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 220","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144585356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Predicting and Validating the Regulation of Podocyte Injury and Treatment of Diabetic Kidney Disease by Yinhuo Tang.","authors":"Shukun Chen, Chunyan Liu, Yan He, Tingting Zhao, Shuang Yu, Zhaoman Wang, Chunhua He, Fan Li, Sichao Ma, Shoulin Zhang","doi":"10.3791/67939","DOIUrl":"https://doi.org/10.3791/67939","url":null,"abstract":"<p><p>Diabetic kidney disease (DKD) is a chronic kidney disease characterized by massive proteinuria and decreased glomerular filtration rate. The main pathological feature of DKD is renal microangiopathy, and ~40% of diabetic patients will develop DKD. The podocyte-associated proteins CD2AP and WT-1 play a crucial role in maintaining the normal function of podocytes and the integrity of the glomerular filtration barrier. When podocyte structure or function is disrupted-due to inflammation, oxidative stress, or other pathological insults-the slit diaphragm and actin cytoskeleton are damaged, leading to increased permeability of the glomerular filtration barrier and resulting in proteinuria. Yinhuo Tang (YHT) is a traditional Chinese medicine prescription that has the function of reducing proteinuria and delaying the deterioration of renal function; however, the mechanism of YHT in treating DKD is not clear. In this paper, based on network pharmacology and animal experiments, we examined the blood glucose, liver function, renal function, blood lipids, renal pathology, expression levels of podocyte marker proteins CD2AP and WT-1, as well as the PI3K/AKT/NF-κB signaling pathway in db/db mice after the administration of YHT for 8 weeks. Our results show that YHT reduces the expression levels of inflammatory factors by inhibiting the PI3K/AKT/NF-κB signaling pathway, thus reducing podocyte damage to play a role in delaying the progression of DKD.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 220","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144585387","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Novel Surgical Technique in a Sheep Model for Suburethral Graft Implantation.","authors":"Raouf Seyam, Christopher Chapple, Omar Alsulaiman, Falah Al-Mohanna, Sheila MacNeil, Waleed Altaweel","doi":"10.3791/67282","DOIUrl":"https://doi.org/10.3791/67282","url":null,"abstract":"<p><p>Suburethral polypropylene mesh tapes for women were initially introduced into clinical practice for the treatment of stress urinary incontinence without being tested in an appropriate animal model. The goal of this work has been to establish and evaluate a suitable animal model for the assessment of suburethral tape grafts. Allowing for the anatomical similarities between ewes and women and recognizing the inevitable differences in posture, but recognizing prior work, this report presents a step-by-step description of the surgery involved in an animal model that simulates the implantation of support materials for treating stress urinary incontinence in women. Seven ewes with a mean weight of 27.7 SD 5.1 kg underwent surgery. In the lithotomy position, a ventral vaginal wall incision was made. In five animals, using an introducer, a polyurethane implant was placed underneath the urethra and fixed with absorbable sutures. The incision was closed, and a betadine vaginal pack was placed after 24 h. Two animals received transvaginal polypropylene mesh using similar surgical steps but no fixation sutures. After surgery, all animals urinated freely and had regular bowel movements. No animals showed signs of vaginal erosion or graft infection during the three-month follow-up. This large animal model accurately reproduces the surgical technique used in women, thereby providing a valuable platform for evaluating new materials for stress urinary incontinence treatment.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 220","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144585318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unilateral Lung Volume Analysis Using Micro-CT for Enhanced Assessment of Pulmonary Fibrosis in Preclinical Models.","authors":"Xihui Huang, Shu Xia, Yiqian Yang, Chengcheng Lei, Penghui Yang, Shuang Li, Jianyi Niu, Jian Wu, Qun Luo, Zhenli Fu","doi":"10.3791/67837","DOIUrl":"10.3791/67837","url":null,"abstract":"<p><p>Pulmonary fibrosis (PF) is a progressive and irreversible interstitial lung disease characterized by poor prognosis and limited treatment options. The traditional mouse model of pulmonary fibrosis induced by intratracheal instillation of bleomycin can result in uneven drug distribution and high mortality rates. The refined unilateral intratracheal instillation method for bleomycin administration in mice enables the creation of a more uniform and controlled pulmonary fibrosis model, providing a better simulation of the disease in humans. Here we present a protocol to utilize micro-computed tomography (micro-CT) to quantify ventilated regions in a unilateral bleomycin-induced pulmonary fibrosis model via intratracheal instillation on the lesional side. This approach allows for the quantification of overall lung volume and ventilated lung regions, providing a sensitive and accurate measure of disease progression and treatment response. This study involves anesthesia, precise drug administration, micro-CT scanning, and post-processing using 3D Slicer software for image analysis. The unilateral lung volume analysis following bleomycin treatment demonstrated a significant reduction in lung volume compared to controls. Masson's Trichrome staining confirmed the presence and increased deposition of collagen in the BLM-treated mice. Herein, we evaluated pulmonary fibrosis in preclinical models by performing unilateral lung volumetric analysis using micro-CT, providing researchers with a precise and thorough assessment method for evaluating therapeutic interventions in PF studies.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 220","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144477454","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Endoscopic Ultrasound-Based Differentiation of Pancreatic Neuroendocrine Tumors and Pancreatic Duct Adenocarcinoma.","authors":"Danjie Shen, Can Yao, Ke Chen, Yunwei Sun, Xiaojuan Li, Junpei Zhang","doi":"10.3791/68199","DOIUrl":"https://doi.org/10.3791/68199","url":null,"abstract":"<p><p>Endoscopic ultrasound (EUS) is one of the most critical imaging modalities for evaluating pancreatic masses, providing high-resolution visualization of the pancreas. This study aimed to assess the feasibility of digital image analysis (DIA) in differentiating pancreatic neuroendocrine neoplasms (PNEN) from pancreatic ductal adenocarcinoma (PDAC) in solid pancreatic tumors observed via EUS. A retrospective analysis was conducted on all solid pancreatic tumors examined by EUS procedures from 2018 to 2023, including cases with confirmed pathological diagnoses of PDAC and PNEN. Adobe Photoshop was used to perform DIA. The gray ratio, defined as the gray-scale ratio of the lesion to a selected background, was calculated to ensure consistency. Circularity was determined by the ratio of area to perimeter. Additionally, gray-scale standard deviations were extracted for further analysis. Three quantitative parameters from EUS images demonstrated potential for distinguishing PNEN from PDAC: circularity, gray ratio, and gray-scale standard deviation. Specifically, a circularity greater than 0.68, a gray ratio exceeding 0.40, and a gray-scale standard deviation below 12.44 were indicative of potential PNEN.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 220","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144585337","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Gastrointestinal Motility Recording Using Balloon Insertion.","authors":"Tong Li, Xiaoxi Wang","doi":"10.3791/68404","DOIUrl":"https://doi.org/10.3791/68404","url":null,"abstract":"<p><p>Gastrointestinal motility is one of the most important physiological functions of the digestive system. The migrating motor complex (MMC) is a characteristic form of movement that occurs in the gastrointestinal tract during fasting. The MMC is present in many species, including humans. Although the physiological role of the MMC is not completely understood, its abnormalities are associated with various diseases. The measurement of gastrointestinal motility is important for the diagnosis of gastrointestinal diseases. Thus, a system was constructed using a rubber condom and polyethylene (PE) tube, with one side placed in the gastrointestinal tract of the mice and the other side connected to the pressure transducer. Gastrointestinal movements were visualized when the balloon was squeezed during gastrointestinal movements. This study work provides a detailed protocol of the system's design and operation and shows representative results demonstrating its effectiveness. By using this system device, gastrointestinal motor function can be recorded in vivo. This provides additional approaches for future studies of gastrointestinal disorders.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 220","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144585340","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-throughput Method for Observing Motility Phenotypes in Pseudomonas aeruginosa.","authors":"Océane Goncalves, Jean-Philippe Côté","doi":"10.3791/67761","DOIUrl":"https://doi.org/10.3791/67761","url":null,"abstract":"<p><p>Motility behaviors often play a significant role in the ability of a bacterium to exploit the resources available in its environment. This is particularly true for the versatile pathogen Pseudomonas aeruginosa, which can exhibit diverse types of motilities, including swarming and twitching, which are important pathogenic traits that contribute to surface colonization, biofilm formation, and evasion of host defenses. This manuscript presents a high-throughput motility protocol to study the motility behaviors of P. aeruginosa. The protocol allows simultaneous testing of multiple strains of P. aeruginosa from a genome-wide mutant library, for instance, to identify and analyze the genetic factors involved in its motility. The approach offers the possibility to study motility in a comprehensive manner and insights into the molecular mechanisms underlying P. aeruginosa's motility. The protocol described here can also be modified to accommodate different types of motility assays as well as other bacterial species, thus providing a powerful platform for advancing the understanding of bacterial behavior in various contexts.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 220","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144585341","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Prevention of Allodynia and Hyperalgesia by Cannabidiol in a Rat Model of Chemotherapy-Induced Peripheral Neuropathy.","authors":"Michael Ippolito, Charlie Maddox, Amal Inam, Mathieu E Wimmer, Sara Jane Ward","doi":"10.3791/68079","DOIUrl":"https://doi.org/10.3791/68079","url":null,"abstract":"<p><p>This study demonstrates the utility of a rat model of chemotherapy-induced peripheral neuropathy (CIPN) to assess the ability of the non-psychoactive cannabinoid cannabidiol (CBD) to modulate the development of this syndrome in vivo. The method utilizes the chemotherapeutic agent paclitaxel to generate an allodynic phenotype in the animals. This study describes how to handle and solubilize CBD, administer the chemotherapeutic agent, assess mechanical and cold sensitivity, and apply high-speed videography to measure nocifensive behavior in animals. Using the procedures outlined, the data support that CBD prevents the allodynic phenotype from developing in the treated animals. No difference was observed in the CBD-treated animals from day 0 (pre-paclitaxel baseline) to day 7 (post-sensitization) in mechanical or thermal sensitivity, while the vehicle-treated animals became significantly more sensitive. This response to treatment is durable up to the latest time point where data were collected (7 weeks). The addition of high-speed videography allows for a more granular and unbiased assessment of this behavioral phenotype (e.g., classification of analgesia and anti-allodynia). This demonstrates both the utility of this model for cannabinoid drug characterization and the potential role of CBD in mitigating neuropathic pain.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 220","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144585388","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Establishment and Comparison of Fluorescence-Based T6SS Activity Detection Methods in Acinetobacter baumannii.","authors":"Wenjing Yang, Jing Jie, Chunxiuli Li, Dong Chen, Shuang Zhao, Sanwei Gu, Jiancheng Xu, Lei Song","doi":"10.3791/67772","DOIUrl":"https://doi.org/10.3791/67772","url":null,"abstract":"<p><p>The Type VI Secretion System (T6SS) is a crucial mechanism mediating intercellular interactions in Gram-negative bacteria, particularly in pathogenic species such as Acinetobacter baumannii. Previous studies have shown that the large plasmid pAB3 in the A. baumannii ATCC 17978 strain encodes a TetR-like protein that inhibits the expression of core T6SS genes. In contrast, the WT^R- strain, which lacks pAB3, can stably express and secrete the T6SS effector protein Hcp and exhibits the ability to kill E. coli. The tssM gene, one of the core genes of T6SS, is essential for its activity; its deletion directly leads to the inactivation of T6SS. However, traditional T6SS activity detection methods, such as killing assays, suffer from low throughput and insufficient sensitivity. To address these limitations, we have developed quantitative detection methods based on fluorescent labeling. To improve T6SS activity detection, we developed three fluorescent labeling methods: (1) A quantitative detection method based on Luciferase labeling, which is characterized by high specificity, sensitivity, and reproducibility, making it suitable for high-throughput analysis; (2) A detection method based on green fluorescent protein (GFP) labeling, which, despite being susceptible to environmental interference, offers the advantage of high throughput; (3) Flow cytometry detection, which can quantitatively assess bacterial viability but is operationally complex and costly. After a comprehensive comparison, the Luciferase-based labeling method proved to be the most accurate, sensitive, and user-friendly. When applied to 20 clinical isolates of A. baumannii, this method was confirmed to rapidly and accurately evaluate T6SS activity.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 220","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144585338","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Establishment of Psoriasis Mouse Model by Imiquimod.","authors":"Mengyan Que, Yuhua Wei, Shijian Wu, Xiaoling Liang, Kongqing Zhang, Guanyi Wu","doi":"10.3791/68111","DOIUrl":"https://doi.org/10.3791/68111","url":null,"abstract":"<p><p>Psoriasis is a common, chronic, relapsing, and inflammatory skin condition characterized by thickened skin infiltration, scales, and erythema. The pathogenesis of the disease is highly complex, involving the interplay of various factors such as genetics, immunity, and environmental factors. To investigate the mechanisms underlying psoriasis, researchers have developed multiple animal models of the disease. However, the variability in methodologies has presented challenges for constructing these models consistently. In this study, we established and comprehensively evaluated a highly efficient and stable psoriasis mouse model using imiquimod (IMQ). 8-week-old SPF-grade female C57BL/6J mice were used to establish the psoriasis mouse model. After one week of acclimatization, mice with irritated or damaged skin in the depilated areas were excluded. Starting on day two, 5% IMQ cream (62.5 mg/d) was applied daily to the depilated neck and back regions for seven consecutive days. During the experiment, daily measurements of body weight, skin photography, skin scoring, and behavioral observations were conducted. Post-experiment, spleen index determination, skin histological analysis, serum cytokine detection, and skin mRNA level detection were performed. The results showed that mice in the IMQ group developed typical psoriasis-like symptoms, including erythema, scales, and skin thickening, accompanied by increased scratching behavior, weight loss, elevated spleen index, and increased skin thickness. And significant changes in the levels of TNF-α, IL-23, and IL-17A in serum, as well as the mRNA levels of IL-23, IL-17A, IL-17F, and IFN-γ in the skin. Serum cytokine levels (TNF-α, IL-23, IL-17A) and skin mRNA expression of pro-inflammatory markers (IL-23, IL-17A, IL-17F, IFN-γ) were notably upregulated. In contrast, the control group exhibited no such alterations. By optimizing the IMQ induction duration and standardizing the administration protocol, while dynamically monitoring skin changes, this study aims to provide a standardized framework for researchers to construct reliable psoriasis models.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 220","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-06-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144585339","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}