Jove-Journal of Visualized Experiments最新文献

{"title":"Stab-Wound Mouse Model for Studying Hemorrhage and Inflammation in Traumatic Brain Injury.","authors":"Kei Hashimoto, Mari Nakashima, Yasunori Miyamoto, Hiroko Ikeshima-Kataoka","doi":"10.3791/67797","DOIUrl":"https://doi.org/10.3791/67797","url":null,"abstract":"<p><p>Traumatic brain injury (TBI) results from physical damage, often caused by accidents or sports-related incidents. The causes of TBI are diverse, including concussions, brain contusions, hematomas, and skull fractures. To replicate these different causes, various TBI mouse models have been developed using distinct protocols. Physical brain injury leads to both primary and secondary brain injuries, which exacerbate neuronal loss. Primary injury occurs immediately after the damage, often due to hemorrhage, and subsequently triggers secondary injuries, including inflammation around the lesion. Developing a TBI model suitable for assessing hemorrhage extension and inflammatory severity is therefore crucial. This protocol introduces a method for mimicking penetrating brain injury, referred to as the stab-wound TBI mouse model, to study mechanisms of hemorrhage, inflammation, and neuronal loss associated with TBI pathology. This model is created by puncturing the skull and brain with needles and is simple to execute without the need for specialized experimental equipment. Additionally, the minor injury inflicted on the mouse cerebral cortex using a needle does not affect the animal's behavior post-surgery. This feature allows researchers to study the localized effects of brain injury without concerns about broader behavioral consequences. Sample data from stab-wounded mouse cerebral cortices demonstrate the model's effectiveness in assessing blood leakage into the parenchyma, glial activation, and inflammatory cytokine production. Furthermore, this protocol facilitates the evaluation of blood coagulants and anti-inflammatory compounds, aiding in the development of therapeutic agents for TBI.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143598094","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":"Assessing the Accuracy of Fitness Smartwatch Data for Cardiovascular and Physical Activity Monitoring: A Validation Study in Digital Health.","authors":"Afrina Adlyna Mohamad Matrol, Melanie Koh, Wei Ping Eddy Tan, Hang Cheng Ong, Letchumy Praba Ramanaidu, Shier Nee Saw","doi":"10.3791/67674","DOIUrl":"https://doi.org/10.3791/67674","url":null,"abstract":"<p><p>This study aims to validate the accuracy of low-cost fitness smartwatches by comparing their data with gold-standard measurements for cardiovascular and physical activity parameters. The study enrolled 50 subjects, 26 undergoing validation testing for heart rate, blood oxygen saturation (SpO2), and sleep data against polysomnography (PSG). Additionally, 24 subjects participated in the 3-Minute Walk Test (3MWT) and Stairs Climbing (SC), with step counts validated against manual video calculations. Results showed no significant difference between the device's measurements and gold standard values for shallow sleep, deep sleep, REM time, mean heart rate, minimum heart rate, and SpO2. However, the device significantly underestimated manually counted steps (p = 0.009 (3MWT); p = 0.012 (SC)), total sleep duration (p = 0.004), and wake time (p = 8.94 × 10^-8) while overestimating maximum heart rate (p = 0.011). These findings highlight the importance of accurate validation and interpretation of wearable device data in clinical contexts. Given these limitations, excluding the device's readings in future analyses is recommended to maintain data reliability and research integrity. This study underscores the need for ongoing validation and improvement of wearable technology to ensure its reliability and effectiveness in healthcare.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143598003","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":"In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast.","authors":"Felipe Muñoz-Guzmán, Pablo Quintrel, José Benavides-Parra, Catalina Muñoz-Tapia, Luis F Larrondo, Francisco A Cubillos","doi":"10.3791/68161","DOIUrl":"https://doi.org/10.3791/68161","url":null,"abstract":"<p><p>Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase¹, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143598006","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 Modified Mouse Model of Intracranial Aneurysm Based on Hemodynamic Change and Hypertension.","authors":"Yongquan Han, Hao Qin, Junshuan Cui, Xi Zeng, Bing Zhao","doi":"10.3791/68087","DOIUrl":"https://doi.org/10.3791/68087","url":null,"abstract":"<p><p>Intracranial aneurysm (IA) poses a significant health risk due to morbidity and mortality associated with aneurysm rupture. However, the molecular mechanisms underlying IA development remain unclear, and a suitable mouse model is required. A mouse model of IA was established by ligating the pterygopalatine artery (PPA) to induce additive hemodynamic changes, combined with hypertension induction. In C57BL/6 male mice, vessels, including the right PPA, external carotid artery (ECA), occipital artery (OcA), and the left contralateral common carotid artery (CCA), were ligated to induce hemodynamic changes. One week later, the bilateral posterior branches of the renal artery (pRA) were ligated, and an 8% salt diet was introduced to induce hypertension. Magnetic resonance angiography (MRA), stereomicroscopy, and immunohistochemical (IHC) staining were performed to evaluate the morphological and pathological changes in IA three months after induction. In the experimental group, four mice died after the initial induction. IA at different locations was detected in five of the eleven remaining mice. Both microscopic and MRA examinations confirmed IA formation. Pathological and IHC analyses revealed disruption of the internal elastic lamina, disconnection of collagen fibers, and infiltration of CD86-positive M1 macrophages, findings consistent with those observed in human IA. This mouse model of IA replicates the pathological changes observed in human samples and may serve as a valuable tool for investigating the molecular mechanisms of IA formation and progression.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143597865","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":"Measurement of Mitochondrial Membrane Potential In Vivo using a Genetically Encoded Voltage Indicator.","authors":"Run-Zhou Yang, Dian-Dian Wang, Sen-Miao Li, Pei-Pei Liu, Jian-Sheng Kang","doi":"10.3791/67911","DOIUrl":"https://doi.org/10.3791/67911","url":null,"abstract":"<p><p>Mitochondrial membrane potential (MMP, ΔΨm) is critical for mitochondrial functions, including ATP synthesis, ion transport, reactive oxygen species (ROS) generation, and the import of proteins encoded by the nucleus. Existing methods for measuring ΔΨm typically use lipophilic cation dyes, such as Rhodamine 800 and tetramethylrhodamine methyl ester (TMRM), but these are limited by low specificity and are not well-suited for in vivo applications. To address these limitations, we have developed a novel protocol utilizing genetically encoded voltage indicators (GEVIs). Genetically encoded voltage indicators (GEVIs), which generate fluorescent signals in response to membrane potential changes, have demonstrated significant potential for monitoring plasma membrane and neuronal potentials. However, their application to mitochondrial membranes remains unexplored. Here, we developed protein-based mitochondrial-targeted GEVIs capable of detecting ΔΨm fluctuations in cells and the motor cortex of living animals. The mitochondrial potential indicator (MPI)offers a non-invasive approach to study ΔΨm dynamics in real-time, providing a method to investigate mitochondrial function under both normal and pathological conditions.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143598091","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":"The Creation of a Rat Model for Osteosarcopenia via Ovariectomy.","authors":"Xiaoping Ma, Chong Lu, Yu Yang, Hao Zheng, Haijian Cui, Yu Wang, Siwei Wang, Xiaosheng Yao, Xiaonan Qi","doi":"10.3791/67539","DOIUrl":"https://doi.org/10.3791/67539","url":null,"abstract":"<p><p>Osteosarcopenia (OS), a complex degenerative disorder, is characterized by the concurrent decline in skeletal muscle mass and bone mineral density (BMD), posing an enormous health hazard for the elderly population. Despite its clinical relevance, the pathophysiological mechanisms underlying OS are not fully understood, underscoring the necessity for a deeper comprehension of its etiology to facilitate effective treatment strategies. The development of a reliable animal model is pivotal in this endeavor. This study presents a refined protocol for the induction of postmenopausal osteosarcopenia in rats through bilateral ovariectomy, a method known to accelerate the onset of age-related muscle and bone loss. In this study, rats aged 12 weeks were stratified by body weight and randomly assigned to either a sham operation group or an ovariectomized (OVX) group. Tissue samples from the quadriceps and triceps muscles of the left hind limb, as well as the left femur, were systematically collected at 4, 8, and 12 weeks post-surgery. This methodical approach ensures a comprehensive evaluation of the effects of ovariectomy on muscle and bone health. Histological evaluation of muscle fiber atrophy and femoral morphology was conducted using hematoxylin and eosin (HE) staining, while bone mineral density was quantified using dual-energy X-ray absorptiometry (DXA). The temporal progression of OS was meticulously monitored at the aforementioned intervals, providing insights into the dynamic interplay between muscle and bone degeneration. This model not only accurately reflects the clinical manifestations of OS but also serves as a robust platform for investigating novel therapeutic approaches and their underlying mechanisms.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143598095","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":"Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells.","authors":"Jaya Lakshmi Thangaraj, Dan S Kaufman","doi":"10.3791/67438","DOIUrl":"https://doi.org/10.3791/67438","url":null,"abstract":"<p><p>Natural killer (NK) cells are innate immune cells that play a crucial role in the body's defense against tumors and viral infections. The generation of human induced pluripotent stem cell (iPSC)-derived chimeric antigen receptor (CAR) expressing NK cells has emerged as a promising avenue for \"off the shelf\" cancer immunotherapy. Here, we utilized an NK cell-optimized CAR construct that includes the transmembrane domain of NKG2D, the 2B4 co-stimulatory domain, and the CD3ζ signaling domain, which has been demonstrated to stimulate robust antigen-specific NK cell-mediated antitumor activity. The use of iPSCs for CAR NK cell generation offers several advantages, including homogenous CAR expression, scalability, reproducibility, and the potential for clinical application. This detailed step-by-step protocol from cell engineering to differentiation enables the generation of NK cell-optimized iPSC-derived CAR-expressing NK cells, providing a standardized and targeted cancer immunotherapy with improved antitumor activity and highlighting their potential as a promising treatment option for various malignancies.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143606712","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":"Cranial Neural Crest Cells Three-Dimensional In Vitro Differentiation Protocol for Multiplexed Assay.","authors":"Saverio Fortunato, Jean-Christophe Deschemin, Antoine Zalc","doi":"10.3791/67695","DOIUrl":"https://doi.org/10.3791/67695","url":null,"abstract":"<p><p>With their remarkable capacity to generate both ectodermal and mesenchymal derivatives, cranial neural crest cells (CNCC) have attracted a lot of interest in studying the mechanisms regulating cell fate decisions and plasticity. Originating in the dorsal neuroepithelium, this cell population is transient and relatively rare in the developing embryo - making functional tests, genomic screens, and biochemistry assays challenging to perform in vivo. To overcome these limitations, several methods have been developed to model CNCC development in vitro. Neurosphere (NS) based culturing methods provide a complex microenvironment that recapitulates the developing anterior neuroepithelium in 3D. These systems allow the growth of many NS in the same plate to generate a large amount of CNCC, but the produced NS present a high variability in shape, size, and number of CNCC formed - making quantitative assays difficult to perform. This protocol outlines a reproducible method for generating NS from mouse embryonic stem cells (mESC) in a 96-well format. NS generated in 96-well plates produce cranial neural crest cells (CNCC), which can be further cultured. By controlling the number of starting cells, this approach reduces variability in the size and shape between NS and increases reproducibility across experiments. Finally, this culture system is adaptable to several applications and offers a higher degree of flexibility, making it highly customizable and suitable for multiplexing experimental conditions.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143606713","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":"Prion Safety Laboratory Swipe Test.","authors":"Sara M Simmons, Qi Yuan, Jason C Bartz","doi":"10.3791/67889","DOIUrl":"https://doi.org/10.3791/67889","url":null,"abstract":"<p><p>Transmission of iatrogenic prion disease has occurred from contaminated neurosurgical tools, transplant materials, and occupational exposure to prion-contaminated laboratory tools. Prions cause disease by the templated misfolding of the normal cellular form of the prion protein, PrP^C, into the misfolded and pathogenic form PrP^Sc and are invariably fatal. Reducing iatrogenic and occupational prion transmission is challenging. First, prions can bind to and persist on surfaces for long periods of time. Second, prions are highly resistant to inactivation. Given this, surfaces can retain infectivity for long periods of time following ineffective decontamination. Not only can this pose a potential occupational risk for prion laboratory workers, but it could potentially cross-contaminate laboratory experiments utilizing sensitive prion amplification techniques. The protocol described here for a prion safety laboratory swipe test includes steps for the identification and documentation of high-traffic laboratory areas, recommended swabbing controls to ensure the validity of results, steps to identify proper responses to positive surface swabbing sites, representative results from prion swipe testing, as well as potential artifactual results. Overall, the prion safety laboratory swipe test can be implemented as part of a broader prion safety program to assess decontamination of surfaces, monitor common spaces for prion contamination, and implement the documentation of prion decontamination status.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143606768","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 Modified Method for Intrathecal Catheterization in Rats.","authors":"Lijun Yin, Quan Zhou, Wenwu Liu, Lize Xiong, Hui Chen","doi":"10.3791/66487","DOIUrl":"https://doi.org/10.3791/66487","url":null,"abstract":"<p><p>Intrathecal catheterization has been widely applied in animal experiments, especially those on neuropathic pain. However, the traditional methods still have several limitations. Although some investigators have attempted to improve the traditional methods, the available methods still need to be modified. Herein, we introduce a modified method for intrathecal catheterization in rats. This method uses a 20 cm long stainless-steel wire (0.2 mm in diameter), a 15 cm long plastic PE10 tube, a self-made sealing cap, and a 0.3 cm × 0.5 cm anti-allergic band. Our modified method for intrathecal catheterization has several advantages. First, introducing a stainless-steel wire to PE10 tube increases the elasticity of the tube, improves the success rate of intrathecal catheterization, reduces the amount of space required for the operation, and minimizes the damage to the tissues around the lumbar spine. Second, the length of PE10 tube is determined before the surgery, and catheter indwelling time can be longer than one week. Third, the PE10 tube is fixed by a figure-8 suture, 4 times, which prevents tube movement and retraction when the animal moves. Fourth, a self-made sealing cap is used to seal the PE10 tube, which not only prevents cerebrospinal fluid leakage but also reduces the need for repeated cutting of PE10 tube. Finally, the extracorporeal end of PE10 tube is tied with a band, which prevents tube retraction when the animal moves. This method can increase the catheterization success rate in rats, as approximately 80% of PE10 tubes remained in place even 28 days after surgery. Thus, this modified method may represent a simple, convenient, and reliable approach for repetitive intrathecal drug administration.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143606708","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}