Jove-Journal of Visualized Experiments最新文献

{"title":"Assessment of Glutamine as a Fuel Source for Alveolar Macrophages Exposed to Chronic Ethanol Using an Extracellular Flux Bioanalyzer.","authors":"Kathryn M Crotty, Samantha M Yeligar","doi":"10.3791/67579","DOIUrl":"https://doi.org/10.3791/67579","url":null,"abstract":"<p><p>Alveolar macrophages (AMs) are the first line of cellular defense in the lower airway against pathogens. However, chronic and excessive alcohol use impairs the ability of AMs to phagocytize and clear pathogens from the alveolar space, in part through dysregulated fuel metabolism and bioenergetics. Our prior work has shown that chronic ethanol (EtOH) consumption impairs mitochondrial bioenergetics and increases lactate levels in AMs. Further, we recently demonstrated that EtOH increases glutamine dependency and glutamine-dependent maximal respiration while decreasing flexibility, shifting away from pyruvate-dependent respiration and towards glutamine-dependent respiration. Glutaminolysis is an important compensatory pathway for mitochondrial respiration when pyruvate is used for lactic acid production or when other fuel sources are insufficient. Using a mouse AM cell line, MH-S cells, exposed to either no EtOH or EtOH (0.08%) for 72 h, we determined the dependency of mitochondrial respiration and bioenergetics on glutamine as a fuel source using an extracellular flux bioanalyzer. Real-time measures were done in response to bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), an inhibitor of glutaminase 1, which prevents the enzymatic conversion of glutamine to glutamate, in media vehicle or in response to vehicle alone, followed by testing mitochondrial stress. The step-by-step protocol provided herein describes our methods and calculations for analyzing average levels of glutamine-dependent basal mitochondrial respiration, mitochondrial ATP-linked respiration, maximal mitochondrial respiration, and mitochondrial spare respiratory capacity across multiple biological and experimental replicates.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142773680","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 Splenectomy with Pericardial Devascularization for Hypersplenism and Esophageal Variceal Hemorrhage Due to Portal Hypertension.","authors":"Kangshou Liu, Zhen Li, Fei Li, Leyang Xiang, Qiang Li, Mingrong Cao, Youzhu Hu, Zhilong Liu, Junjie Liang","doi":"10.3791/67603","DOIUrl":"https://doi.org/10.3791/67603","url":null,"abstract":"<p><p>Hypersplenism and esophageal variceal hemorrhage caused by portal hypertension are common and serious complications of decompensated cirrhosis. In recent years, with the widespread application of various therapeutic methods such as drugs, endoscopy, splenic artery embolization, transjugular intrahepatic portal shunt, and liver transplantation, the role of surgery in the treatment of portal hypertension has gradually diminished, and the indications for surgical treatment have become more strictly defined. However, according to the clinical practice in China, surgical treatment of portal hypertension still holds an important role that other treatments cannot fully replace. In fact, surgical treatment of portal hypertension is widely performed in hospitals at all levels in China, saving numerous lives. Splenectomy combined with pericardial devascularization (SPD) is the most common surgical method for treating hypersplenism and esophageal variceal hemorrhage caused by portal hypertension. Long-term clinical practice has proven that SPD is a safe and effective treatment for hypersplenism and esophageal variceal rupture and hemorrhage due to portal hypertension. With the rapid development of laparoscopic techniques, the minimally invasive advantages of laparoscopic splenectomy combined with pericardial devascularization (LSPD) have become increasingly evident. However, the successful performance of LSPD mainly depends on the skill and proficiency of the surgeon. In this context, this article presents detailed techniques for LSPD.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142773837","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 Mouse Model of Hemorrhagic Transformation Induced by Acute Hyperglycemia Combined with Transient Focal Ischemia.","authors":"Yaojian Sun, Changlong Leng, Kang Ma, Wei Liu","doi":"10.3791/67371","DOIUrl":"https://doi.org/10.3791/67371","url":null,"abstract":"<p><p>Hemorrhagic transformation (HT) is a serious complication that can occur as a result of thrombolytic therapy following ischemic stroke (IS), and it poses significant limitations on the clinical application of recombinant tissue plasminogen activator (rt-PA). Unfortunately, there are currently no effective interventions available for HT in clinical practice. Therefore, there is an urgent need for stable and reliable experimental animal models to elucidate the pathogenesis of HT and develop effective intervention strategies. This study presented a protocol for establishing a mouse model of HT induced by acute hyperglycemia combined with transient focal ischemia (tMCAO). Male C57BL/6J mice were injected with 30% glucose to induce hyperglycemia and then subjected to 60 min of tMCAO with reperfusion. The infarct volume, integrity of the blood-brain barrier (BBB), and degree of intracranial hemorrhage were assessed at 24 h after MCAO. The results showed that glucose injection led to transient hyperglycemia (14.3-20.3 mmol/L), which significantly increased both the infarct volume and the incidence of HT. Hematoxylin-eosin (H&E) staining indicated significant hemorrhagic lesions within the infarction zone in hyperglycemic mice. Additionally, hyperglycemic mice exhibited aggravated BBB disruption, as shown by more severe leakage of Evans blue (EB) and FITC-Dextran. In conclusion, acute hyperglycemia reliably and consistently resulted in macroscopic HT in a mouse model of tMCAO. This reproducible model offers a valuable tool for investigating the pathological mechanisms of HT and developing corresponding therapeutic interventions.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142774030","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":"Application of Microwave Ablation in Laparoscopic Partial Splenectomy.","authors":"Zhiwei Chen, Zhilong Liu","doi":"10.3791/67563","DOIUrl":"https://doi.org/10.3791/67563","url":null,"abstract":"<p><p>Splenic hemangioma is the most common pathological classification of splenic tumors, and its surgical indication and treatment have been controversial. Before, open splenectomy was usually used to treat splenic hemangioma. Following the rapid development of laparoscopic techniques, people's requirements for minimally invasive treatment have gradually increased, and laparoscopic splenectomy has gradually become the main treatment method. However, through the deeper study of spleen function, it was found that partial splenectomy can cut down the incidence of postoperative thrombocythemia and decrease side effects on the physiological function of the body, so laparoscopic partial splenectomy came into being. However, due to the special anatomical structure, the incidence of hemorrhage during partial splenectomy is greater. Therefore, during the operation, we removed part of the splenic blood vessels, combined with microwave ablation, which perfectly solved the problem of intraoperative bleeding. Laparoscopic partial splenectomy combined with microwave ablation not only achieves the requirements of minimally invasive treatment but also reduces the risk of intraoperative bleeding, meriting clinical application and promotion.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142774033","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":"Neuronavigated Focalized Transcranial Direct Current Stimulation Administered During Functional Magnetic Resonance Imaging.","authors":"Filip Niemann, Alireza Shahbabaie, Sven Paßmann, Steffen Riemann, Robert Malinowski, Harun Kocataş, Leonardo M Caisachana Guevara, Mohamed Abdelmotaleb, Daria Antonenko, Felix Blankenburg, Rico Fischer, Gesa Hartwigsen, Shu-Chen Li, Michael A Nitsche, Axel Thielscher, Dagmar Timmann, Anna Fromm, Dayana Hayek, Ann-Kathrin Hubert, Andrew K Martin, Alexander Hunold, Agnes Flöel, Marcus Meinzer","doi":"10.3791/67155","DOIUrl":"https://doi.org/10.3791/67155","url":null,"abstract":"<p><p>Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that allows the modulation of the excitability and plasticity of the human brain. Focalized tDCS setups use specific electrode arrangements to constrain the current flow to circumscribed brain regions. However, the effectiveness of focalized tDCS can be compromised by electrode positioning errors on the scalp, resulting in significant reductions of the current dose reaching the target brain regions for tDCS. Electrode placement guided by neuronavigation based on the individual's head and brain anatomy derived from structural magnetic resonance imaging (MRI) data may be suited to improve positioning accuracy. This protocol describes the method of neuronavigated electrode placement for a focalized tDCS setup, which is suitable for concurrent administration during functional MRI (fMRI). We also quantify the accuracy of electrode placement and investigate electrode drift in a concurrent tDCS-fMRI experiment. Critical steps involve the optimization of electrode positions based on current modeling that considers the individual's head and brain anatomy, the implementation of neuronavigated electrode placement on the scalp, and the administration of optimized and focal tDCS during fMRI. The regional precision of electrode placement is quantified using the Euclidean norm (L²Norm) to determine deviations of the actual from the intended electrode positions during a concurrent tDCS-fMRI study. Any potential displacement of electrodes (drift) during the experiment is investigated by comparing actual electrode positions before and after the fMRI acquisition. In addition, we directly compare the placement accuracy of neuronavigated tDCS to that achieved by a scalp-based targeting approach (a 10-20 Electroencephalography (EEG) system). These analyses demonstrate superior placement accuracy for neuronavigation compared to scalp-based electrode placement and negligible electrode drift across a 20 min scanning period.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142773844","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":"Control and Disposal of Invasive Japanese Knotweed Reynoutria japonica Houtt. Using Microwave Treatment.","authors":"Krzysztof Słowiński, Beata Grygierzec, Sylwester Tabor, Szymon Bucior, Agnieszka Synowiec","doi":"10.3791/67660","DOIUrl":"https://doi.org/10.3791/67660","url":null,"abstract":"<p><p>The study aims to assess the effectiveness of microwave treatment (MWT) at a frequency of 2.45 GHz and a power of 800 W to control Japanese knotweed (Reynoutria japonica Houtt.) using a self-propelled device that was built in the in-house facility. The MWT was applied in the field population of knotweed in July 2022. First, plants were mechanically moved from the area of 1 m^2, and next, the cut shoots around 4 cm high were microwave-treated for 25 min, 20 min, and 15 min. The control treatments were: 1) only cut plants and 2) rhizomes dug out to 30 cm deep. The effectiveness of the microwave treatments was observed for the next 11 months by counting the number of newly grown shoots. The results showed that a 25 min MWT was 100% effective in Japanese knotweed loss of vitality, while a 15 min MWT microwave treatment stimulated plant growth by around 50%, compared to controls. Rhizomes were dug out in a separate in vitro experiment for laboratory testing. The rhizomes were categorized by thickness and subjected to a 60 s MWT using a commercial microwave, after which their temperature and vitality were assessed. The temperature of rhizomes following MWT depended on their thickness. Those rhizomes that warmed to temperatures above 42 °C were effectively destroyed. Summing up, the time plants are exposed to microwaves plays a major role in the effectiveness of this method. The longer the exposure to MWT, the better control. The thinner the rhizomes, the more effective the in vitro MWT rhizomes disposal.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142773682","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 Functional Assessment of Rat Masseter Muscle Following Surgical Creation of a Volumetric Muscle Loss (VML) Injury.","authors":"Sydney B Shriver, Ramzi J Khairallah, George J Christ","doi":"10.3791/66902","DOIUrl":"https://doi.org/10.3791/66902","url":null,"abstract":"<p><p>Volumetric Muscle Loss (VML) is prevalent in civilian and military populations and represents a debilitating skeletal muscle injury surpassing the body's natural regenerative capacity. These injuries disrupt not only muscle fibers but also nerves, blood vessels, and the extracellular matrix, overwhelming the regenerative capacity of skeletal muscle and leading to severe fibrosis and permanent deficiencies in muscle structure and function. Current clinical management has many limitations, and thus, research is ongoing to develop more effective therapeutic approaches. Notably, however, much of the preclinical emphasis on VML injuries has focused on limb and trunk muscles, with limited investigation into craniofacial muscles. Differences in developmental biology and regenerative capacity between craniofacial and limb/trunk muscles may provide crucial insights that drive more injury-specific VML treatment options. Moreover, evaluation of functional recovery is critical to establishing therapeutic efficacy. In this regard, in vivo testing of muscle contraction with percutaneous nerve stimulation is a minimally invasive method that allows for repeated functional assessment over the course of a study - in the same animal. In light of these considerations, this paper describes a method for the in vivo assessment of muscle function in the rat masseter muscle before and after a VML injury. This protocol is the first published instance to detail the creation and functional evaluation of a biologically relevant craniofacial VML injury in the rat.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142773822","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":"Improving Student Outcomes with an Adaptable Molecular Cloning Course-Based Undergraduate Research Experience.","authors":"Christopher B Cummings, Samuel S Catania, Eirene M Q Ednacot, Austin J Kinsella-Johnson, Claire E Meeds, Jack W Reynolds, Ava E Sanderson, Rachel A Johnson, Katharine R Watts","doi":"10.3791/67067","DOIUrl":"https://doi.org/10.3791/67067","url":null,"abstract":"<p><p>The continuous advancement of molecular biology techniques requires that molecular biology curricula are regularly refined to effectively prepare students to enter the workforce with modern competencies. In particular, the emergence of Gibson Assembly, a highly customizable and adaptive molecular cloning technique, has advanced the landscape of molecular cloning in numerous research environments. Thus, we created a Gibson Assembly cloning module for deployment in a molecular biology laboratory course at California Polytechnic State University, San Luis Obispo and evaluated student learning outcomes from the module. Over three iterations of the course, students participated in an experiment-based independent project that involved cloning three unique plasmid libraries to support research projects in natural products biosynthesis. Students were given pre- and post-questionnaires to evaluate their understanding of molecular cloning and their confidence in molecular biology terms and techniques. Students' responses showed a significant increase in both learning molecular cloning concepts and in self-reported confidence with molecular cloning terms and techniques. This module framework can be generalized to teach Gibson Assembly for various applications, providing instructors with a toolkit for teaching an adaptable and emergent cloning technology while advancing their research projects.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142773816","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":"Retraction: Assisted Selection of Biomarkers by Linear Discriminant Analysis Effect Size (LEfSe) in Microbiome Data.","authors":"","doi":"10.3791/6606","DOIUrl":"https://doi.org/10.3791/6606","url":null,"abstract":"<p><p>The article Assisted Selection of Biomarkers by Linear Discriminant Analysis Effect Size (LEfSe) in Microbiome Data (10.3791/61715) has been retracted by the journal upon the authors' request due to a conflict regarding the data and methodology.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142630473","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":"Co-culture Model Using Two Types of Adherent Cell Lines.","authors":"Zhuo Song, Lisha Zhao, Jingwen Hu, Xi Zheng, Yingyu Liu, Hao Wang, Xiaoyan Chen","doi":"10.3791/67314","DOIUrl":"https://doi.org/10.3791/67314","url":null,"abstract":"<p><p>Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications between various cell types within the decidua are crucial for regulating endometrial receptivity; however, the molecular mechanisms mediating this interaction are still unclear. In this regard, a model to study the implantation process is needed to establish a comprehensive in vitro model that can recapitulate the biology of endometrial epithelium-stroma interaction. This model is composed of regular cell-culture plates and a matching scaffold, which is generated by three-dimensional (3D) printing from low-cost materials. Here, we detail a set of protocols for model construction, cell preparation, cell seeding, cell culture, observation, and evaluation. Furthermore, we have included representative results with cells exhibiting good growth conditions under the microscope. This study aimed to develop in vitro models that would mimic the interaction between endometrial stromal cells and epithelial cells, as well as between trophoblast cells and endometrial cells.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142711434","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}