Jove-Journal of Visualized Experiments最新文献

{"title":"Technique for Intranasal Administration of α-Synuclein Aggregates.","authors":"Masanori Sawamura, Ryosuke Takahashi","doi":"10.3791/66943","DOIUrl":"https://doi.org/10.3791/66943","url":null,"abstract":"<p><p>Parkinson's disease (PD) is a neurodegenerative disorder characterized by the presence of Lewy bodies, which are aggregates of α-synuclein (α-Syn). Recently, the disease was proposed to develop and progress through the prion-like propagation of α-Syn aggregates from the olfactory bulb (OB) or dorsal nucleus of the vagus nerve. Although the origin of α-Syn aggregates in the OB remains unclear, their propagation from the olfactory mucosa has been recently suggested. We previously showed that intranasal administration of α-Syn aggregates in a mouse model induced α-Syn pathology in the OB of mice. In this study, we present a method of intranasal administration of α-Syn aggregates that induced α-Syn pathology in the OB of mice. Intranasal administration of α-Syn aggregates is a very simple and straightforward method, and we believe it will be a useful tool in the research for elucidating the origin of α-Syn pathology in the OB and the pathway of α-Syn propagation through the olfactory system.</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":"142711582","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":"IntelliSleepScorer, a Software Package with a Graphic User Interface for Mice Automated Sleep Stage Scoring.","authors":"Ziyue Zhu, Lei A Wang, Ryan Kern, Jen Q Pan","doi":"10.3791/66950","DOIUrl":"https://doi.org/10.3791/66950","url":null,"abstract":"<p><p>Sleep stage scoring in rodents is the process of identifying the three stages: nonrapid eye movement sleep (NREM), rapid eye movement sleep (REM), and wake. Sleep stage scoring is crucial for studying sleep stage-specific measures and effects. Sleep patterns in rodents differ from those in humans, characterized by shorter episodes of NREM and REM interspaced by waking, and traditional manual sleep stage scoring by human experts is time-consuming. To address this issue, previous studies have used machine learning-based approaches to develop algorithms to automatically categorize sleep stages, but high-performing models with great generalizability are often not publicly available/cost-free nor user-friendly for non-trained sleep researchers. Therefore, we developed a machine learning-based LightGBM algorithm trained with a large dataset. To make the model available to sleep researchers without coding experience, a software tool named IntelliSleepScorer (v1.2- newest version) was developed based on the model, which features an easy-to-use graphic user interface. In this manuscript, we present step-by-step instructions for using the software to demonstrate a convenient and effective automatic sleep stage scoring tool in mice for sleep researchers.</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":"142711371","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":"Harnessing the Power of MicroRNA Cargoes in Small Extracellular Vesicles Released from Fresh-Frozen Human Brain Sections.","authors":"Joseph Morgan, Toby Aarons, Gemma Lace, Arijit Mukhopadhyay","doi":"10.3791/67379","DOIUrl":"https://doi.org/10.3791/67379","url":null,"abstract":"<p><p>Small extracellular vesicles (sEVs) are crucial mediators of cell-cell communication, transporting diverse cargoes like proteins, lipids, and nucleic acids (microRNA, mRNA, DNA). The microRNA sEV cargo has potential utility as a powerful non-invasive disease biomarker due to sEV's ability to traverse biological barriers (e.g., blood-brain barrier) and become accessible through various body fluids. Despite numerous studies on sEV biomarkers in body fluids, identifying tissue or cell-specific sEV subpopulations remains challenging, particularly from the brain. Our study addresses this challenge by adapting existing methods to isolate sEVs from minimal amounts of frozen human brain sections using size exclusion chromatography (SEC). After ethical approval, approximately 250 µg of fresh-frozen human brain tissue (obtained from Manchester Brain Bank [UK]) was sliced from the 3 donor tissues and incubated in collagenase type 3/Hibernate-E solution, with intermediate agitation, followed by serial centrifugation and filtration steps. Then, sEVs were isolated using the SEC method and characterized by following MISEV guidelines. Before isolating RNA from within these sEVs, the solution was treated with Proteinase-K and RNase-A to remove any non-sEV extracellular RNA. The RNA quantity and quality were checked and processed further for qPCR and small RNA sequencing experiments. The presence of sEVs was confirmed through fluorescence nanoparticle tracking analysis (fNTA) and western blot for surface markers (CD9, CD63, CD81). Size distribution (50-200 nm) was confirmed by NTA and electron microscopy. The total RNA concentration within lysed sEVs ranged from 3-9 ng/µL and was used for successful quantification by qPCR for selected candidate microRNAs. Small RNA sequencing on MiSeq provided high-quality data (Q >32) with 1.4-5 million reads per sample. This method enables efficient isolation and characterization of sEVs from minimal brain tissue volumes, facilitating non-invasive biomarker research and holds promise for equitable disease biomarker studies, offering insights into neurodegenerative diseases and potentially other disorders.</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":"142711369","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":"Minimally Invasive Treatment for Thoracolumbar Burst Fracture Using Sagittal Alignment Screws and A Trauma Reduction Device.","authors":"Joji Ogawa, Yusuke Dodo, Yoshihisa Komuro, Chikara Hayakawa, Ichiro Okano","doi":"10.3791/66957","DOIUrl":"https://doi.org/10.3791/66957","url":null,"abstract":"<p><p>Thoracolumbar (TL) burst fracture is one of the most common indications for minimally invasive percutaneous pedicle screw fixation. Although the indication for surgical treatment of neurologically intact TL fractures remains under debate, studies have demonstrated that posttraumatic malalignment may lead to a deterioration in the patient's quality of life. For burst fractures with malalignment or fragments in the spinal canal, a reduction technique using ligamentotaxis is commonly used to improve long-term outcomes. The sagittal adjusting screw (SAS) system is a monoaxial screw system with a fixed head and concave sliding saddle that allows lordotic sliding of the rod in the sagittal plane after screw insertion. SAS also has a percutaneous option and has been used for TL spine fractures. Notably, the SAS only allows motion on the sagittal plane, allowing both secure fixation and angular reduction. The SAS has certain advantages over the conventional Schanz screw system or normal mono-/multiaxial pedicle screws for TL spine fracture treatment. In addition, specialized trauma reduction devices are available for the SAS system. In this video protocol, we discuss the indication for the SAS system in TL burst fracture and describe a technique of TL burst fracture reduction and fixation using the SAS system. Additionally, we describe our recent case series with radiological evaluation, including regional kyphotic angle and percent loss of anterior vertebral body height, to evaluate the newly introduced trauma reduction device.</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":"142711513","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":"An In Vitro Assay to Study Platelet Migration Using RGD-Functionalized Avidin-Biotin Tethers.","authors":"Shuxia Fan, Ben Raude, Florian Gaertner","doi":"10.3791/66757","DOIUrl":"https://doi.org/10.3791/66757","url":null,"abstract":"<p><p>Despite being anucleated cell fragments, platelets are now widely recognized for their multifaceted abilities. Not only do they form blood clots to prevent bleeding after injury, but they also fight infections and maintain vascular integrity during inflammatory diseases. While hemostatic plugs require the collective activation and aggregation of platelets, their role in protecting inflamed blood vessels is performed at the single-cell level. In this context, recent data have shown that platelets can migrate autonomously, a process dependent on the mechanosensing of their adhesive environment. Here, a detailed protocol for imaging single platelet migration is presented, utilizing a three-layer coating system consisting of a poly-L-lysine graft poly(ethylene glycol) (PLL-PEG)-biotin backbone (1), a fluorescent avidin linker (2), and biotin-cyclic Arg-Gly-Asp (cRGD) (3) as the platelet integrin-binding motif. This reductionist approach allows precise control of substrate adhesion properties and serves as a simple, standardized in vitro assay to study the mechanisms underlying platelet migration. The results indicate that migrating platelets binding to cRGD exert forces capable of disrupting the avidin-biotin bond. Furthermore, the density of biotin-cRGD significantly influences both platelet spreading and migration.</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":"142711427","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":"Non-invasive Skeletal Muscle Quantification in Small Animals Using Micro-computed Tomography.","authors":"Alexander Pereira-Rosa, Thamires S Oliveira, Matheus S Ferreira, Rodrigo J Vianna-Barbosa, Tula C Wilmart-Gonçalves, Tânia M Ortiga, Flavia F Bloise","doi":"10.3791/67393","DOIUrl":"https://doi.org/10.3791/67393","url":null,"abstract":"<p><p>Skeletal muscle size, mass, and composition are critical properties for studying metabolic and muscle-related diseases, as they directly impact the understanding of disease progression and treatment outcomes. Quantifying a live animal's lean, adipose, and skeletal mass is important in metabolic, physiology, pharmacologic, and geroscience studies. However, obtaining accurate body composition measurements, especially of lean mass, remains challenging due to the inherent limitations of conventional assessment techniques. Micro-computed tomography (micro-CT) is a non-invasive radiological technique that enables high-resolution visualization of internal structures in small animal models. A standardized micro-CT method can significantly enhance translational research with more reliable and impactful results, particularly during aging studies or at different time points within the same animal. Despite its potential, the lack of standardization in image acquisition and analysis methods significantly hinders the comparability of results across different studies. Herein, we present a comprehensive and detailed low-cost protocol for lean mass analysis using micro-CT to address these challenges and promote consistency in research involving small animal models.</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":"142711537","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":"Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis.","authors":"Rodrigo Escalona-Rivano, Delia I Chiarello, Lorena Carvajal, Ivo Carrasco-Wong, Jaime A Gutiérrez","doi":"10.3791/66454","DOIUrl":"https://doi.org/10.3791/66454","url":null,"abstract":"<p><p>During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.</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":"142711585","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":"Establishing a Mouse Model of Thin Endometrium.","authors":"Mingye Chen, Junjie Tang, Binfa Liang, Lianghui Diao, Jie Liu, Qing Sun, Xian Chen","doi":"10.3791/67084","DOIUrl":"10.3791/67084","url":null,"abstract":"<p><p>Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory treatment options are still urgently needed. Several animal models of TE have been developed, but the mouse model involving abdominal surgery and injection of 95% ethanol presents a formidable challenge due to the high mortality rate and risk of intrauterine adhesions if not performed correctly. Here, we describe a detailed protocol that produces reliable and reproducible TE with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus with varying infusion times. The results showed that all of the mice successfully developed TE with infusion times ranging from 1-3 min, characterized by a typical reduction in endometrial thickness and the number of glands, as well as excessive endometrial fibrosis. These findings suggest that this mouse model is suitable for studying thin endometrium and can serve as a platform for developing future TE treatments.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649406","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":"Confocal Microscopy Analysis of Protein Sorting to Plasmodesmata in Nicotiana benthamiana.","authors":"Yumin Kan, Eli Simons, Vitaly Citovsky","doi":"10.3791/67378","DOIUrl":"10.3791/67378","url":null,"abstract":"<p><p>Plasmodesmata are membranous nanopores that connect the cytoplasm of adjacent plant cells and enable the cell-to-cell trafficking of nutrients, macromolecules, as well as invading viruses. Plasmodesmata play fundamental roles in the regulation of intercellular communication, contributing to plant development, environmental responses, and interactions with viral pathogens. Discovering plasmodesmal localization of plant or viral proteins could provide useful functional information about the protein and is important for understanding the mechanisms of plant-virus interactions. To facilitate these studies, we describe a protocol for confocal microscopy-based analysis of different plasmodesmal targeting proteins to select the best plasmodesmal marker for studying the virus-plasmodesmata interactions or plasmodesmal transport. Specifically, the analyses of these events are illustrated using the cell-to-cell movement protein (MP) of the Turnip vein-clearing virus (TVCV), the Arabidopsis Plasmodesmata-Localized Protein 5 (PDLP5) and Plasmodesmata Callose-Binding Protein 1 (PDCB1). The protein plasmodesmal localization data are analyzed in parallel with the global visualization of plasmodesmata using aniline blue staining of the sampled tissues. These approaches can be easily adapted to analyze the plasmodesmal localization of any cellular or pathogen proteins in planta.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11581684/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649402","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Combined In Vivo Electroporation and Short-Term Reinnervation of the Cranial Levator Auris Longus Skeletal Muscle.","authors":"Jessica Mella, Francisca Bermedo-Garcia, Angelymar Medina-Moreno, Jorge Ojeda, Juan Pablo Henríquez","doi":"10.3791/66706","DOIUrl":"https://doi.org/10.3791/66706","url":null,"abstract":"<p><p>The neuromuscular junction (NMJ) is the peripheral synapse controlling the contraction of skeletal muscle fibers to allow the coordinated movement of many organisms. At the NMJ, a presynaptic motor axon terminal contacts a muscle postsynaptic domain and is covered by terminal Schwann cells. The integrity and function of the NMJ is compromised under several conditions, including aging, neuromuscular diseases, and traumatic injuries. To analyze the potential contribution of muscle-derived proteins to NMJ maintenance and regeneration, an in vivo gene transfer strategy has been combined with the denervation of the cranial levator auris longus (LAL) muscle after mechanical nerve injury. Previous findings showed that the forced expression of control fluorescent proteins does not alter NMJ organization or neurotransmission. This procedure aims to describe a detailed method of in vivo electroporation of the LAL muscle followed by transection or crushing of the specific branch of the facial nerve innervating cranial muscles, leading to NMJ denervation and reinnervation, respectively. The combination of these experimental strategies in the convenient LAL muscle constitutes an efficient method to study the potential contribution of muscle protein overexpression or silencing in the context of short-term NMJ reinnervation.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649401","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}