Jove-Journal of Visualized Experiments最新文献

{"title":"Rapid in vivo Drug Response Prediction Using Leukemia Cell Grafts in Zebrafish Embryos.","authors":"Sophia Sartorius, Luca Bramè, Jutta Proba, Anton Gauert, Nadine Olk, Juan Lazaro, Angelika Eggert, Cornelia Eckert, Anja H Hagemann","doi":"10.3791/67451","DOIUrl":"https://doi.org/10.3791/67451","url":null,"abstract":"<p><p>Zebrafish xenotransplantation is a pivotal technique for investigating human cancer pathogenesis and predicting individual drug responses. This document introduces a streamlined protocol (ZefiX) for expanding primary B-cell precursor acute lymphoblastic leukemia (BCP-ALL) patient samples or immortalized cell lines in transiently immunosuppressed zebrafish embryos, utilizing flow cytometry for high-resolution single-cell analysis of treatment responses. Compared to solid tumor engraftments, leukemia cells profit significantly from a morpholino antisense oligonucleotide-based suppression of macrophage and neutrophil differentiating factors during the assay. Flow cytometry analysis of dissociated graft cells enables precise evaluation of cell count, proliferation rate, and vitality after treatment on a per-cell basis. This approach has been validated using targeted therapeutics such as venetoclax and dasatinib, with treatment outcomes compared to clinical records of related patient samples and traditional 2D culture controls. Notably, the protocol is completed within 7 days, aligning with clinical decision-making timelines. The methodology is adaptable for testing selected drugs in various cancer types, including solid tumors, thereby supporting personalized therapeutic strategies. However, limitations on the number of drugs that can be assessed, likely due to pharmacokinetic constraints in zebrafish embryos, should be considered.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259191","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":"Mechanism of Shu-Mu Brain-Kidney Acupoint in Treating Oligoasthenospermia in Rats via the Hypothalamic-Pituitary-Testicular Axis.","authors":"Kaiying Li, Nan Yang, Wenjing Sun, Zhenning Zhao, Hongyu Li, Xiaoge Wei, Lili Zhao, Yan Wang, Jing Mu, Huisheng Ma","doi":"10.3791/67912","DOIUrl":"https://doi.org/10.3791/67912","url":null,"abstract":"<p><p>Oligoasthenospermia is a common cause of male infertility. The hypothalamic-pituitary-testicular (HPT) axis regulates gonadal differentiation and maturation through reproductive hormone synthesis and release, playing a vital role in male fertility. Disrupting HPT axis stability impairs sperm production, reducing semen quality. Investigating electroacupuncture's effect on HPT axis regulation may provide insights into treating oligoasthenospermia. Fifty 8-week-old male SD rats were randomly divided into blank, model, Shu-Mu Brain-Kidney, non-acupoint, and L-carnitine groups (n = 10 per group). Except for the blank group, rats received adenine intragastrically for 28 days to establish the model. Post-modeling, the Shu-Mu Brain-Kidney group underwent electroacupuncture at designated acupoints, while the non-acupoint group received sham treatment for 30 min daily. The L-carnitine group received L-carnitine (10 mL/kg) intragastrically once daily. Treatments continued for 28 days. General conditions, organ coefficients, and semen quality were assessed. HE staining analyzed tissue morphology, and ELISA detected serum hormone changes. Compared to the model group, the Shu-Mu Brain-Kidney, and L-carnitine groups exhibited significant improvements in spirit, diet, and bowel movements, with increased body weight, while the non-acupoint group showed no significant change. Renal organ coefficients decreased significantly in the Shu-Mu Brain-Kidney and L-carnitine groups but remained unchanged in the non-acupoint group. Testicular organ coefficients showed no significant differences among treatment groups. Sperm count, density, survival, and motility rates improved significantly in the Shu-Mu Brain-Kidney and L-carnitine groups, but not in the non-acupoint group. H&E staining showed ameliorated kidney and testicular tissue damage in the Shu-Mu Brain-Kidney and L-carnitine groups. ELISA revealed increased T, GnRH, and INHB and decreased LH, FSH, E2, and PRL levels in these groups (p < 0.001), with no significant changes in the non-acupoint group. These findings indicate that Shu-Mu Brain-Kidney acupoint therapy improves sperm quality by regulating the HPT axis, offering a potential treatment for oligoasthenospermia.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259180","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":"Rapid Detection of Fecal Antigen of Helicobacter pylori Infection Based on Double Antibody Sandwich Detection Technology.","authors":"Xiuxian Chen, Caiping Gong, Xiaoxiao Wang, Ziyan Li, Xiaozhen Jiang, Jinxin Lai, Yanfei Luo","doi":"10.3791/68219","DOIUrl":"https://doi.org/10.3791/68219","url":null,"abstract":"<p><p>Helicobacter pylori can be parasitic in gastric mucosa, which can cause a series of gastrointestinal diseases after infection and is closely related to gastritis, gastric ulcer, and gastric cancer. The high prevalence of H. pylori infection in regions with poor medical infrastructure and inadequate sanitation areas remains a significant public health concern. Consequently, the development of rapid, simple, and cost-effective screening methods for H. pylori detection in resource-limited settings is of paramount importance. In this study, we conducted a community-based screening in Shitan Village, Guangdong Province, a remote and underdeveloped area characterized by a permanent population of approximately 300 residents with generally low educational attainment. We employed a double antibody sandwich assay for the detection of H. pylori fecal antigen, a method chosen for its potential applicability in low-resource settings. A total of 261 participants from the village were enrolled, and their fecal samples were analyzed using this technique. For comparative validation, the same samples were subjected to quantitative polymerase chain reaction (qPCR) analysis. Compared with qPCR results, the sensitivity and specificity of the detection of H. pylori antigen in feces were 60.24% and 80.08%. The results demonstrated a strong agreement between the fecal antigen detection method and qPCR (Kappa = 0.630). This study systematically elucidated the principles, procedures, methodologies, and clinical applications of fecal antigen detection for H. pylori infection, aiming to explore latex-based double antibody sandwich technology and establish novel strategies and practical guidelines for its auxiliary diagnosis.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259190","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":"Rapid Whole-Mount High-Resolution Imaging of Small Animal Vasculature for Quantitative Studies.","authors":"Daibo Zhang, Stephanie E Lindsey","doi":"10.3791/68206","DOIUrl":"https://doi.org/10.3791/68206","url":null,"abstract":"<p><p>In small animal models of cardiovascular development and diseases, subject-specific computational simulations of blood flow enable quantitative assessments of hemodynamic metrics that are difficult to measure experimentally. Computational fluid dynamic simulations shed light on the critical roles of mechanics in cardiovascular function and disease progression. Acquiring high-quality volumetric images of the vessels of interest is central to the accuracy and reproducibility of morphological measurement and flow quantitation results. This study proposes a rapid, cost-effective, and accessible method for whole-mount high-resolution imaging of small animal vasculature using light-sheet fluorescence microscopy. The modified iDISCO+ (immunolabeling-enabled three-dimensional imaging of solvent-cleared organs) light-sheet sample preparation protocol involves (1) labeling vasculature with a fluorescent agent, (2) preserving the sample, and (3) rendering the sample transparent. Unlike classical iDISCO+, which uses immunohistochemical staining, the authors label vascular endothelium with FITC-tagged poly-L-lysine, an affordable non-specific fluorescent dye that is highly resistant to photo-bleaching, in a process termed \"endo-painting.\" The rapid labeling reduces sample preparation time from approximately four weeks to less than 3 days. Furthermore, the use of minimally hazardous solvent ethyl cinnamate (ECi) as the clearing agent and imaging solution makes the samples safer to handle and compliant with a wider range of imaging facilities. The proposed protocol is applied to obtain highly resolved light-sheet fluorescence microscopy image stacks of the cardiovascular system in chick embryos ranging from day 3 (HH18) to day 8 (HH34). This study further demonstrates the suitability of this method for vascular quantitation through 3D reconstruction and computational hemodynamic modeling of a day 5 (HH 26) chick embryo.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259192","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":"Using Laser Scanning Microscopy to Determine Electromigration in Molybdenum Disilicide.","authors":"Julia Baldauf, Dennis Mitrenga, Andreas T Winzer","doi":"10.3791/68158","DOIUrl":"https://doi.org/10.3791/68158","url":null,"abstract":"<p><p>With increasing the current density and decreasing the size of chips electromigration becomes more and more important. Electromigration is the movement of atoms in an electrically conductive material caused by current flowing. For aluminum and copper the electromigration parameters and their dependencies have been investigated by a multitude of people and methods. For other materials, this is not the case. Electromigration experiments often use very long times to stress the lines under test in the median to failure experiments. These experiments only give surface-level information about electromigration. More sophisticated methods look at the microscopic or nanoscale effects and influences. Usually, expensive equipment such as scanning electron microscopes (SEM), synchrotrons, or X-ray microtomography are used for these investigations. A workflow enabling the investigation of electromigration on the microscopic scale by using a laser scanning microscope has been developed. With this laser scanning technique, it is possible to achieve results with slightly less accuracy than SEM but with much less effort in preparing the samples. With the electromigrated volume being known, the same procedures as with the electromigrated volumes determined via SEM can be used to calculate electromigration parameters. By varying different experimental variables, a multitude of information about electromigration can be achieved. In this work determining the length for the onset of electromigration is shown.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259197","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":"Localizing Function-specific Targets for Transcranial Magnetic Stimulation in the Absence of Navigation Equipment.","authors":"Bingbing Zhou, Jiajia Qi, Kangjia Chen, Hong Li, Jiahui Liu, Qian Zhou, Zujuan Ye, Jue Wang","doi":"10.3791/67888","DOIUrl":"https://doi.org/10.3791/67888","url":null,"abstract":"<p><p>Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive technique that modulates neural activity in the brain. Studies have shown that rTMS can regulate neural plasticity, promote neural network reorganization, and has been widely applied to neuropsychiatric disorders such as stroke. Although some studies suggest that rTMS can aid in stroke rehabilitation, its efficacy remains uncertain, possibly because of limitations in the traditional localization of the hand motor hotspot. The hand motor hotspot is determined by motor evoked potentials (MEPs), which reflect the conductivity of the corticospinal or pyramidal tract, representing non-voluntary movement. In contrast, functional magnetic resonance imaging (fMRI) activation points from a motor task define function-specific targets, which involve both perception and motor execution, representing voluntary movement. Based on this, we propose the concept of function-specific targets -- targets identified through brain imaging techniques aimed at specific functions. Function-specific targets exhibit stronger and more extensive functional connectivity with brain regions related to motor cognition, potentially offering more effective regulatory effects than the hotspots. We explored and validated the modulatory effects of function-specific targets in previous study. However, institutions without navigation equipment are unable to utilize these function-specific targets. Therefore, we have developed a non-navigated localization method for function-specific targets, specifically designed to define and localize rTMS targets in the post-stroke ipsilateral hemisphere, addressing the challenges faced by institutions lacking navigation equipment when applying function-specific targeted rTMS.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259179","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":"Ocular Therapeutic Delivery and Advanced Tissue Retrieval in Adult Rats.","authors":"Annie K Ryan, Tyler Heisler-Taylor, Stephanie M Small, Zeina A Elshaer, Ethan J Jackson, Vidhya Kannan, Stacey Meeker, Colleen M Cebulla, Matthew A Reilly","doi":"10.3791/68049","DOIUrl":"https://doi.org/10.3791/68049","url":null,"abstract":"<p><p>Therapeutic delivery to the posterior segment of the eye, including the retina and optic nerve, is complicated by the presence of blood-brain and blood-retinal barriers. Small animal models, such as rats, are utilized for studying various ocular pathologies. While therapeutic delivery to the posterior eye is challenging, achieving it is essential for treating ocular disorders, many of which require validation in small animal models for translational relevance. Therefore, two posterior therapeutic delivery techniques are presented: intravitreal injection (IVI) and retrobulbar injection (RBI) for use in adult rats. Additionally, a method for the en bloc removal of the eyes and optic nerves is introduced for various histological and molecular analysis techniques. The dissection protocol enables full observation of the neuro-visual system while minimizing post-mortem injury to retinal and optic nerve tissues. Successful delivery of the therapeutic cyclosporine to the retina and optic nerve was achieved, with detectable concentrations observed twenty-four hours after injection using both IVI and RBI. Furthermore, en bloc retina and nerve samples were successfully extracted for full eye histological tissue analysis, facilitating comprehensive observation of the retina and the wider neuro-visual system.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259184","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 Validation of a Rat Model of Pulmonary Arterial Hypertension Associated with Pulmonary Fibrosis.","authors":"Qian Jiang, Dongmei Wen, Fang Peng, Wei Hong, Xin Fu, Yuanhui Xiong, Chongyuan Ren, Xuanyi Li, Dansha Zhou, Jian Wang, Yuqin Chen","doi":"10.3791/67747","DOIUrl":"https://doi.org/10.3791/67747","url":null,"abstract":"<p><p>Patients with pulmonary fibrosis are at a higher risk of developing pulmonary hypertension, a complication with poor prognosis. At present, the mechanism of this link is still poorly understood. A major obstacle to progress in this area is the lack of a reliable animal model to replicate PF-PH. This study aimed to establish a stable PF-PH rat model. Rats were fasted overnight prior to intervention. Under sodium pentobarbital anesthesia (45 mg/kg), the trachea was intubated with a PE50 tube inserted to a depth of 3 cm (the distance from the glottis to the tube). Bleomycin (BLM) was administered intratracheally as a single dose (5 mg/kg, dissolved in 0.2 mL of 0.9% NaCl). Following the injection, the rats were immediately rotated to ensure even distribution of the BLM. At 35 days after the BLM injection, the rats exhibited progressive impairment of lung function and increased right ventricular systolic pressure and right ventricular hypertrophy, revealing the pathological characteristics of pulmonary hypertension. We provide a general and reliable method to establish a rat model of PF-PH.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259171","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":"Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes.","authors":"Yu Yuan, Ziyu Jiang, Yuxin Zeng, Jiawen Tang, Jiang Luo, Conghua Xie, Yan Gong","doi":"10.3791/67982","DOIUrl":"https://doi.org/10.3791/67982","url":null,"abstract":"<p><p>The CRISPR-Cas9 system has been harnessed and repurposed into a powerful genome editing tool. By leveraging this technology, researchers can precisely cut, paste, and even rewrite DNA sequences within living cells. Nevertheless, the application of CRISPR screen technology goes far beyond mere experimentation. It serves as a pivotal tool in the fight against genetic diseases, systematically dissecting complex genetic landscapes, empowering researchers to unravel the molecular mechanisms underlying biological phenomena, and enabling scientists to identify and target the root causes of illnesses such as cancer, cystic fibrosis, and sickle cell anemia. Among all, cancer poses a formidable challenge for medicine, spurring eradication efforts. Radiotherapy, as a traditional treatment, yields results but has limitations. It eradicates cancer cells but also damages healthy tissues, causing adverse effects that reduce quality of life. Additionally, not all cancer cells respond to radiotherapy, and some may develop resistance, worsening the condition. To address this, a comprehensive whole-genome CRISPR screen technology is introduced, as it enables the efficient identification of radiosensitive and radioresistant genes, thereby advancing the field of cancer research and treatment. A genome-wide CRISPR screen was conducted in lung adenocarcinoma cells exposed to irradiation following the described protocol, through which both radioresistance- and radiosensitivity-associated genes were identified.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259173","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":"Laparoscopic Anatomical Liver Segment VII Resection with Liver Parenchymal Transection Following a Priority Approach.","authors":"Qing Ye, Shengfeng Zhou, Jie Cheng, Junming He, Jianxin Peng, Jiaqiang Mo, Zhangyuanzhu Liu, Jianyi Wang, Haorong Xie, Xuefang Chen, Jingfang Diao","doi":"10.3791/66808","DOIUrl":"https://doi.org/10.3791/66808","url":null,"abstract":"<p><p>Laparoscopic liver resection procedures have rapidly developed, including surgical techniques ranging from early local resection of the liver margin and superficial lesions to semi-hepatectomy and even complex liver segment resection. Segment VII of the liver is located at the base of the subphrenic space, making it difficult to access and resulting in a lengthy and challenging resection procedure. This liver segment resection involves high volumes of blood loss throughout the procedure. To reduce bleeding caused by the anatomy of the hepatic porta, our center uses a surgical technique that is guided by the right hepatic vein. This technique is unaffected by anatomical variations, such as the dorsal branch of the hepatic pedicle crossing the right hepatic vein to innervate hepatic segment VII. Here, we provide a method and approach for the anatomical resection of segment VII of the liver with anatomical variations of the portal vein, which can be recommended for clinical practice.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144259176","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}