Jove-Journal of Visualized Experiments最新文献

{"title":"Surgical Approach and Complications of Stand-alone Lateral Trans-Psoas Interbody Fusion.","authors":"Julius Gerstmeyer, Periklis Godolias, Thomas A Schildhauer, Clifford Pierre, Neel Patel, Donald David Davis, Bryan Anderson, Amir Abdul-Jabbar, Jens R Chapman, Rod J Oskouian","doi":"10.3791/66878","DOIUrl":"https://doi.org/10.3791/66878","url":null,"abstract":"<p><p>Interbody fusion of the lumbar spine is a standard procedure for symptomatic degenerative lumbar spine disease if conservative treatment fails. Surgical decompression and fusion of the segment can be achieved using several different techniques. Over the last decades, minimally invasive techniques, such as lateral interbody fusion (LLIF), have been developed to reduce tissue damage and complications and allow quicker patient recovery. With growing popularity, indications for LLIF have expanded to treat spinal deformities and foraminal/central stenosis. Mechanically, it allows for an unsurpassed fixation through the left-to-right apophyseal ring placement of the cage. LLIF utilizes a minimally disruptive retroperitoneal corridor and includes both trans-psoas and pre-psoas approaches. For the pre-psoas approach, the risk of damage from manipulation of the intramuscular lumbar plexus is reduced compared to a trans-psoas approach. However, increased risks of major vascular, ureteral, bowel injury and sympathetic plexus damage are reported. This article aims to provide a detailed and comprehensive guide on stand-alone lateral trans-psoas interbody fusion, including its indications, surgical procedure, potential complications, and outcomes based on a decade of experience from a single center.</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":"143606784","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":"Tumor Hypoxia Assessment: In Vivo 3D Oxygen Imaging Through Electron Paramagnetic Resonance.","authors":"Gabriela Dziurman, Aleksandra Bienia, Aleksandra Murzyn, Bartosz Płóciennik, Julia Kozik, Grzegorz Szewczyk, Małgorzata Szczygieł, Martyna Krzykawska-Serda, Boris Epel, Howard J Halpern, Martyna Elas","doi":"10.3791/67129","DOIUrl":"https://doi.org/10.3791/67129","url":null,"abstract":"<p><p>The precise and real-time measurement of oxygen partial pressure (pO2) brings valuable information in many pathologies, including cancer. Low tumor pO2 (i.e., hypoxia) is connected to tumor aggressiveness and poor response to therapy. The quantification of tumor pO2 allows the evaluation of treatment effectiveness. Electron Paramagnetic Resonance Imaging (EPRI), particularly Pulse EPRI, has emerged as an advanced three-dimensional (3D) method of assessing tissue oxygenation in vivo. This innovation was enabled by the technological developments in EPR (Electron Paramagnetic Resonance) and the application of the water-soluble oximetric spin probes from the triaryl family, offering fast and sensitive oxygenation data. The relaxation time of the spin probe (T1 and/or T2) provides accurate information about pO2 in selected voxels. Human glioblastoma LN229 tumors were grown in the interscapular fad pad of BALB/c nude mice. Ultrasound (US) imaging was used as a reference for tumor anatomical information. To image tissue pO2, the animals were placed in a fixed position in the animal bed with fiducials, enabling registration between the imaging modalities. After the OX071 contrast agent was administered, EPRI was performed, followed by US B-mode. Because of the low spin probe toxicity, the procedure can be repeated during tumor growth or treatment. Following imaging, the registration process was carried out using software written in MATLAB. Ultimately, the hypoxic fraction can be calculated for a specific tumor, and the histogram of pO2 tissue distribution can be compared over time. EPRI combined with ultrasound is an excellent tool for oxygen mapping of tumors in the preclinical setting.</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":"143606816","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":"Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes.","authors":"Patricia J Hare, Jonathan I Batchelder, Travis J LaGree, Nisha Mahey, Angela D Power, Yi I Wu, Wendy W K Mok","doi":"10.3791/67617","DOIUrl":"https://doi.org/10.3791/67617","url":null,"abstract":"<p><p>Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment that kills the other genetically identical cells. Bacterial persisters can resume replication once antibiotic treatment ends and are commonly thought to underlie clinical treatment failure. Recent work harnessing the power of time-lapse fluorescence microscopy, in which bacteria are labeled with fluorescent transcriptional reporters, translational reporters, and/or dyes for a variety of cellular features, has advanced our understanding of Escherichia coli persisters beyond what could be learned from population-level antibiotic survival assays. Such single-cell approaches, rather than bulk population assays, are essential for delineating the mechanisms of persister formation, damage response, and survival. However, methods for studying persisters in other important pathogenic species at this level of detail remain limited. This study provides an adaptable approach for time-lapse imaging of Pseudomonas aeruginosa (a gram-negative rod) and Staphylococcus aureus (a gram-positive coccus) during antibiotic treatment and recovery. We discuss molecular genetic approaches to introduce fluorescent reporters into these bacteria. Using these reporters, as well as dyes, we can track the phenotypic changes, morphological features, and fates of individual cells in response to antibiotic treatment. Additionally, we are able to observe the phenotypes of individual persisters as they resuscitate following treatment. In all, this work serves as a resource for those interested in tracking the survival and gene expression of individual antibiotic-treated cells, including persisters, both during and after treatment, in clinically important pathogens.</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":"143606793","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":"Generation of 3D Midbrain Organoids from Human-Induced Pluripotent Stem Cells.","authors":"Tsering Yangzom, Anbin Chen, Kristina Xiao Liang","doi":"10.3791/67228","DOIUrl":"https://doi.org/10.3791/67228","url":null,"abstract":"<p><p>The development of midbrain organoids (MOs) from human pluripotent stem cells (hPSCs) represents a significant advancement in understanding brain development, facilitating precise disease modeling, and advancing therapeutic research. This protocol outlines a method for generating midbrain-specific organoids using induced pluripotent stem cells (iPSCs), employing a strategic differentiation approach. Key techniques include dual-SMAD inhibition to suppress SMAD signaling, administration of fibroblast growth factor 8b (FGF-8b), and activation of the Sonic Hedgehog pathway using the agonist purmorphamine, guiding iPSCs towards a midbrain fate. The organoids produced by this method achieve diameters up to 2 mm and incorporate a diverse array of neuroepithelial cell types, reflecting the midbrain's inherent cellular diversity. Validation of these organoids as authentic midbrain structures involves the expression of midbrain-specific markers, confirming their identity. A notable outcome of this methodology is the effective differentiation of iPSCs into dopaminergic neurons, which are characteristic of the midbrain. The significance of this protocol lies in its ability to produce functionally mature, midbrain-specific organoids that closely replicate essential aspects of the midbrain, offering a valuable model for in-depth exploration of midbrain developmental processes and the pathophysiology of related disorders such as Parkinson's disease. Thus, this protocol serves as a crucial resource for researchers seeking to enhance our understanding of the human brain and develop new treatments for neurodegenerative diseases, making it an indispensable tool in the field of neurological research.</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":"143606747","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":"Planarian as an Animal Model for Experimental Acute Seizure.","authors":"Taylor Miller, Jenny Vu, Charles J Tran, Abheek Ritvik, Kenjy Cruz-Ham, Kaleem Haq, Bernadette Musto, Alberto E Musto","doi":"10.3791/67307","DOIUrl":"https://doi.org/10.3791/67307","url":null,"abstract":"<p><p>Epilepsy is among the most prevalent neurological disorders characterized by recurring spontaneous seizures. Seizures represent a clinical manifestation of uncontrolled, excessively synchronized neural cell activity. The extent of brain damage from seizures depends on their duration and intensity. Regrettably, there is no effective remedy for epilepsy. The aim of this investigation is to assess whether the planaria worm Dugesia dorotocephala could serve as a model to aid in identifying and developing treatments for epilepsy that can target acute seizures. Currently, various models, such as marine models, are used to evaluate antiseizure medications (ASM). However, they are very expensive, and there are ethical concerns. Alternatively, invertebrate models offer a cost-effective research opportunity in the drug discovery process for ASM. Planaria belong to the flatworm family and inhabit marine freshwater and terrestrial environments. Dugesia dorotocephala is the dominant species of aquatic planaria across North America. D. dorotocephala presents as a viable invertebrate model for epilepsy studies due to its cost-effectiveness, vertebrate-like neurons, and quantifiable behaviors, unlike other invertebrates or larger animals. They have been used in various pharmacology and environmental toxicology studies related to age, memory, and regeneration. In this study, planaria were exposed to different concentrations of pilocarpine, a common chemoconvulsant to study their behavior upon exposure. Following the observation, planaria were euthanized and preserved in either formaldehyde or Golgi solution for neurohistological assessment. Six distinct behavioral phenotypes were observed in planaria: dorsal oscillations, head oscillations, tail dorsal expansion, C-shape, head flick, and tail flick. Dorsal oscillation frequencies were significantly increased among experimental groups compared to the control and exhibited dose dependence. Additionally, pilocarpine disrupted the motility of the planaria. Pilocarpine-induced seizures in planaria can serve as a model to evaluate acute seizures and antiseizure medication, which is essential in developing therapeutic interventions for human patients suffering from epilepsy.</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":"143606764","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":"Isolation and Analysis of Aortic Arch and Root Lesions in an Atherosclerotic Mouse Model.","authors":"Yu Yan, Guofang Wang, Longhua Liu","doi":"10.3791/67875","DOIUrl":"https://doi.org/10.3791/67875","url":null,"abstract":"<p><p>Atherosclerosis, a leading cause of cardiovascular diseases, necessitates a detailed examination of lesion development and progression. This study introduces a comprehensive protocol for the isolation and histological analysis of aortic arch and root lesions in a widely used atherosclerotic mouse model, low-density lipoprotein receptor knock-out (Ldlr^-/-) mice. The aortic arch and root are key sites for atherosclerotic lesions, and their examination is critical for assessing the onset, progression, or regression of atherosclerosis, predicting cardiovascular event risks, and identifying potential therapeutic targets. This protocol outlines methods for quantifying atherosclerotic burden in the aortic arch and root, including tissue isolation, fixation, Oil Red O staining, aortic root sectioning, Hematoxylin and Eosin (HE) staining, Verhoeff-Van Gieson (VVG) staining, and image analysis. Oil Red O staining measures plaque area in the aortic arch, evaluating the severity of atherosclerosis, while HE staining of the aortic root reveals plaque components such as the lipid core and fibrous cap, facilitating the assessment of plaque stability and rupture risk. VVG staining can stain collagen fibers within tissues, providing further insights into plaque composition and related information. This thorough analysis offers valuable insights into the mechanisms of lesion development and may guide the creation of novel therapeutic strategies for preventing and treating atherosclerosis.</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":"143606749","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":"Experimental Investigation of the Hierarchical Control in DC Microgrids Using a Real-time Simulator.","authors":"Xiaoran Dai, Zhongcheng Lei, Wenshan Hu, Ningzhao Luo, Hong Zhou, Guo-Ping Liu","doi":"10.3791/67464","DOIUrl":"https://doi.org/10.3791/67464","url":null,"abstract":"<p><p>The rise of renewable energy sources has underscored the significance of microgrids, particularly DC variants, which are well-suited for integrating photovoltaic panels, battery storage systems, and other DC load solutions. This paper presents the development and experimentation of a DC microgrid with hierarchical control implemented in OPAL RT-Lab, a simulator. The microgrid includes distributed energy resources (DERs) interconnected via power converters, a DC bus, and DC loads. The primary control employs a droop control mechanism and double-loop Proportional-Integral (PI) control to regulate voltage and current, ensuring stable operation and proportional power sharing. The secondary control utilizes a consensus-based strategy to coordinate DERs to restore the bus voltage and ensure accurate power sharing, enhancing system reliability and efficiency. The experimental setup detailed in this paper includes circuit modeling, hardware implementation, and control strategies. The hardware platform's circuitry and controller parameters are specified, and the results can be observed through oscilloscope measurements. Two sets of experiments demonstrating the secondary control response with and without delay are conducted to validate the effectiveness of the control strategy. The outcomes confirm the successful implementation of hierarchical control in the microgrid. This study underscores the significance of a comprehensive experimental platform for advancing microgrid technology, providing valuable insights for future research and development.</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":"143606742","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":"Live Imaging of Synaptic Vesicle Recycling in the Neuromuscular Junction of Dissected Larval Zebrafish.","authors":"Yoshihiro Egashira, Fumihito Ono","doi":"10.3791/67633","DOIUrl":"https://doi.org/10.3791/67633","url":null,"abstract":"<p><p>Neuronal communication is mediated by synaptic transmission, which depends primarily on the release of neurotransmitters stored in synaptic vesicles (SVs) in response to an action potential (AP). Since SVs are recycled locally at the presynaptic terminal, coordination of SV exocytosis and endocytosis is important for sustained synaptic transmission. A pH-sensitive green fluorescent protein, called pHluorin, provides a powerful tool to monitor SV exo/endocytosis by targeting it to the SV lumen. However, tracking AP-driven SV recycling with the pHluorin-based probes is still largely limited to in vitro culture preparations because the introduction of genetically encoded probes and subsequent optical imaging is technically challenging in general for in vivo animal models or tissue preparations. Zebrafish is a model system offering valuable features, including ease of genetic manipulation, optical clarity, and rapid external development. We recently generated a transgenic zebrafish that highly expresses a pHluorin-labeled probe at motor neuron terminals and developed a protocol to monitor AP-driven SV exo/endocytosis at the neuromuscular junction (NMJ), a well-established synapse model that forms in vivo. In this article, we show how to prepare larval zebrafish NMJ preparation suitable for pHluorin imaging. We also show that the preparation allows time-lapse imaging under conventional upright epifluorescence microscope, providing a cost-effective platform for analyzing NMJ function.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143494447","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":"Enzymatic Isolation of Skeletal Muscle Interstitial Extracellular Vesicles.","authors":"Yaochao Zheng, Aiden Charles Streleckis, Hongyu Chen, Yao Yao","doi":"10.3791/67439","DOIUrl":"https://doi.org/10.3791/67439","url":null,"abstract":"<p><p>Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells to transport bioactive cargo, such as proteins, RNAs, and DNAs, for intercellular communication. Investigating EV-mediated crosstalk among cells in muscle homeostasis and diseases offers significant potential to enhance our understanding of muscle development, regeneration, and atrophy. However, current protocols for isolating skeletal muscle-derived EVs (SkM-EVs) face challenges in achieving high purity and yield, primarily due to difficulties in releasing EVs from muscle tissues without compromising cellular membranes. This article presents an efficient protocol for SkM-EV isolation, comprising mechanical detachment, enzymatic dissociation, filtration, and ultracentrifugation. These steps are optimized to enhance EV release from muscle tissues, yielding high-purity SkM-EVs. Subsequently, nano-flow cytometry, BCA assay, and Western blot assay are performed to characterize the quantity and quality of the isolated SkM-EVs. This protocol holds promise for establishing a reliable platform to obtain tissue-derived EVs for advancing basic research, disease diagnosis, and drug delivery.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143494452","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":"Visualizing Intracellular Sialylation with Click Chemistry and Expansion Microscopy.","authors":"Yannick Masson, Aude Sivery, Corentin Spriet, Anthony Treizebre, Christophe Biot, Cedric Lion","doi":"10.3791/67479","DOIUrl":"https://doi.org/10.3791/67479","url":null,"abstract":"<p><p>Metabolic labeling techniques allow the incorporation of bioorthogonal reporters into glycans, enabling the targeted bioconjugation of molecular dyes within cells through click and bioorthogonal chemistry. Metabolic oligosaccharide engineering (MOE) has attracted considerable interest due to the essential role of glycosylation in numerous biological processes that involve molecular recognition and its impact on pathologies ranging from cancer to genetic disorders to viral and bacterial infections. Although MOE is better known for the detection of cell surface glycoconjugates, it is also a very important methodology for the study of intracellular glycans in physiological and pathological contexts. Such studies greatly benefit from high spatial resolution. However, super-resolution microscopy is not readily available in most laboratories and poses challenges for daily implementation. Expansion microscopy is a recent alternative that enhances the resolution of microscopy by physically enlarging biological specimens labeled with fluorescent markers. By embedding the sample in a swellable gel and causing it to expand isotropically through chemical treatment, subcellular structures can be visualized with enhanced precision and resolution without the need for super-resolution techniques. In this work, we illustrate the capacity of expansion microscopy to visualize intracellular sialylated glycans through the combined use of MOE and click chemistry. Specifically, we propose a procedure for bioorthogonal labeling and expansion microscopy that employs a reporter targeting sialylation, which may be associated with immunofluorescence for co-localization studies. This protocol enables localization studies of sialoconjugate biosynthesis, intracellular trafficking, and recycling.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 216","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143494292","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}