Jove-Journal of Visualized Experiments最新文献

{"title":"Development of an Uncomplicated Mild Traumatic Brain Injury Model Modified by Weight-Drop Method and Evidenced by Magnetic Resonance Imaging.","authors":"Pin-Hui Kuo, Tzu-Hsuan Tang, Shu-Hui Huang, Bao-Yu Hsieh, Chia-Feng Lu, Yu-Chieh Jill Kao","doi":"10.3791/67011","DOIUrl":"https://doi.org/10.3791/67011","url":null,"abstract":"<p><p>Mild traumatic brain injury (mTBI), known as concussion, accounts for more than 85% of brain injuries globally. Specifically, uncomplicated mTBI showing negative findings in routine clinical imaging in the acute phase hinders early and appropriate care in these patients. It has been acknowledged that different impact parameters may affect and even accelerate the progress of subsequent neuropsychological symptoms following mTBI. However, the association of impact parameters during concussion to the outcome has not been extensively examined. In the current study, an animal model with closed-head injury (CHI) modified from the weight-drop injury paradigm was described and demonstrated in detail. Adult male Sprague-Dawley rats (n = 20) were randomly assigned to CHI groups with different impact parameters (n = 4 per group). Longitudinal MR imaging studies, including T2-weighted imaging and diffusion tensor imaging, and sequential behavioral assessments, such as modified neurological severity score (mNSS) and the beam walk test, were conducted over a 50-day study period. Immunohistochemical staining for astrogliosis was performed on day 50 post-injury. Worse behavioral performance was observed in animals following repetitive CHI compared to the single injury and sham group. By using longitudinal magnetic resonance imaging (MRI), no significant brain contusion was observed at 24 h post-injury. Nevertheless, cortical atrophy and alteration of cortical fractional anisotropy (FA) were demonstrated on day 50 post-injury, suggesting the successful replication of clinical uncomplicated mTBI. Most importantly, changes in neurobehavioral outcomes and image features observed after mTBI were dependent on impact number, inter-injury intervals, and the selected impact site in the animals. This in vivo mTBI model combined with preclinical MRI provides a means to explore brain injury on a whole-brain scale. It also allows the investigation of imaging biomarkers sensitive to mTBI across varying impact parameters and severity levels.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144020394","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":"Screening of Lentil Fields for Presence of Fusarium Wilt and Root Rot in Türkiye under Terrestrial Climate.","authors":"Sevim Atmaca, Yusuf Yanar, Ali Endes, Bekir Aktaş","doi":"10.3791/68156","DOIUrl":"https://doi.org/10.3791/68156","url":null,"abstract":"<p><p>Lentil is an important self-pollinated legume crop plant. Its production is limited by various biotic factors, especially fungal agents causing the wilt and root rot complex. The study aimed to understand the regional epidemiology and etiology of phytopathogenic fungal agents to develop control strategies against soilborne Fusarium spp. This study investigated 83 lentil sowing localities in Yozgat province for wilt, root and crown rot diseases caused by common Fusarium species during 2022 and 2023. Symptomatic lentil plants were collected for fungal isolation and identification. The Fusarium isolates were grouped according to colony morphology and cultured on PDA medium. Moreover, genomic DNAs obtained from Fusarium isolates were analyzed using PCR and compared with other Fusarium isolates registered in the NCBI GenBank. Genetic relationships among Fusarium isolates were determined using the Maximum Parsimony (MP) method in the Mega 11 program. The results, mean incidence and disease severity rate of wilt and root rot diseases in Yozgat province were determined to be 16.9% and 38.6%, respectively. Fusarium isolates were found in 95.4% of the samples. There was 99.5% to 100% nucleotide sequence homogeneity among F. oxysporum, F. culmorum, F. graminearum, F. acuminatum and F. solani isolates, and the most isolated species was F. oxysporum. The MP dendrogram of Fusarium isolates was divided into two main branches, the first branch included all F. solani isolates. The second main branch included other Fusarium species isolated in the present study and in NCBI GenBank. The study suggests periodic local surveys to determine the frequency of Fusarium wilt for suppression in lentils. Timely suppression of Fusarium-based damages is strongly suggested to control the disease and conserve the lentil production system.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144054686","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":"Cryosectioning and Immunostaining Mouse Inner Ear Tissue: From Embryonic to Adult Stages.","authors":"Mehmet Alp Demirhan, Thomas M Coate","doi":"10.3791/67647","DOIUrl":"https://doi.org/10.3791/67647","url":null,"abstract":"<p><p>This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is to provide a straightforward and standardized method for inner ear tissue processing for researchers, possibly new to the field, interested in working with the mouse cochlea. Included here are protocols for dissection, fixation, embedding, cryosectioning, and immunostaining. Section immunostaining is one of the best methods for examining individual cells within the context of the entire cochlea. Key steps include dissecting the inner ear away from the skull, fixing the tissue using 4% paraformaldehyde, embedding with Optimal Cutting Temperature compound, cryosectioning, and immunostaining with antibodies targeting specific proteins expressed by the cochlea. Included in our methods are special considerations made for the cochlea given its shape and structure. Reasonably good focus and dissection skills are needed, as tissue damage can affect the quality of immunostaining and consistency among samples. However, with the procedures we outline, most users can be trained in a few weeks to make beautiful preparations. Overall, this protocol offers a valuable way to promote research to understand auditory development, function, and disease in mouse models.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144052992","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":"Oncogene Expression Analysis with Alterations in pH in a Pancreatic Ductal Cell Line.","authors":"Renuka Goudshelwar, Sheethal Galande, Karuna Rupula, Sundeep Lakhtakia, D Nageshwar Reddy, Mitnala Sasikala","doi":"10.3791/67843","DOIUrl":"https://doi.org/10.3791/67843","url":null,"abstract":"<p><p>The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor prognosis and is characterized by a rigid stromal microenvironment, which represents a tangible challenge in its treatment. Chronic pancreatitis patients have a 10-fold greater risk of developing PDAC; in these patients, the ductal pH decreases from pH 8.0 to pH 6.0 due to bicarbonate insufficiency and the inflammatory milieu. Our goal was to understand the role of the acidic environment observed in chronic pancreatitis on oncogene expression in a pancreatic ductal cell line. Therefore, 80% confluent human pancreatic ductal epithelial cells were incubated at pH 6.0 to pH 7.2 for 6 h. Total RNA from the cells was processed to enrich the total mRNA in the samples. Gene expression was evaluated via next-generation sequencing (NGS) of biological replicates. RNA-seq analysis was carried out with the aid of an online tool, and the differentially expressed genes (FCs < ± 2.0) were identified; there were 90, 148, and 109 upregulated genes and 20, 14, and 23 downregulated genes at pH 6.0, 6.5, and 7.0, respectively. Four oncogenes were upregulated at pH 6.0, seven were upregulated at pH 6.5, and seven were upregulated at pH 7.0. The common genes that were upregulated at pH 6.0, pH 6.5, and pH 7.0 were lymphocyte cell-specific protein-tyrosine kinase (LCK) [pH 6.0, FC: 2.93; pH 6.5, FC: 2.93; pH 7.0, FC: 3.32], FGR proto-oncogene, Src family tyrosine kinase (FGR) [pH 6.0, FC: 4.17; pH 6.5, FC: 5.25; pH 7.0, FC: 5.09], and ArfGAP With SH3 domain, ankyrin repeat, and PH domain 3 (ASAP3) [pH 6.0, FC: 2.37; pH 6.5, FC: 3.84; pH 7.0, FC: 2.51]. The acidic environment triggers the activation of proto-oncogenes, which may trigger tumor initiation in chronic pancreatitis.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144046758","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 Simple and Efficient Method for Testing Immunomodulatory Agents for Generation of Tolerogenic Dendritic Cells from Human CD14+ Monocytes.","authors":"Sihan Jia, Peter Deak","doi":"10.3791/68159","DOIUrl":"https://doi.org/10.3791/68159","url":null,"abstract":"<p><p>Tolerogenic dendritic cells (tolDCs) are a subset of dendritic cells (DCs) that are known to influence naïve T cells toward a regulatory T cell (Treg) phenotype. TolDCs are currently under investigation as therapies for autoimmunity and transplantation, both as a cell therapy and method to induce tolDCs from endogenous DCs. To date, however, the number of known agents to induce tolDCs from naïve DCs is relatively small and their potency to generate Tregs in vivo has been inconsistent, particularly therapies that induce tolDCs from endogenous DCs. This provides an opportunity to explore novel compounds to generate tolerance. Here we describe a method to test novel immunomodulatory compounds on monocyte-derived DCs (moDCs) in vitro and validate their functionality to generate autologous Tregs. First, we obtain PBMCs and isolate CD14⁺ monocytes and CD3⁺ T cells using commercially available magnetic separation kits. Next, we differentiate monocytes into moDCs, treat them with an established immunomodulator, such as rapamycin, dexamethasone, IL-10, or vitamin D3, for 24 h and test their change in tolerogenic markers as a validation of the protocol. Finally, we co-culture the induced tolDCs with autologous T cells in the presence of anti-CD3/CD28 stimulation and observe changes in Treg populations and T cell proliferation. We envision this protocol being used to evaluate the efficacy of novel immunomodulatory agents to reprogram already differentiated DCs towards tolDC.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144023231","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 Gaze-Contingent Display Framework for Perceptual Learning Research with Simulated Central Vision Loss.","authors":"Marcello Maniglia, Samyukta Jayakumar, Pinar Demirayak, Elliot Maxwell, Diya Anand, Jamison Cortez, Jason E Vice, Kristina M Visscher, Aaron R Seitz","doi":"10.3791/67596","DOIUrl":"https://doi.org/10.3791/67596","url":null,"abstract":"<p><p>Macular degeneration (MD) is one of the leading causes of vision impairment in the Western world. Patients with MD tend to develop spontaneous eye movement strategies to compensate for their vision loss, including adopting a preferred retinal locus, or PRL, a spared peripheral region that they use more frequently to replace the damaged fovea. However, not all patients are successful in developing a PRL, and even when they do, it might take them months. Currently, no gold standard rehabilitative therapy exists, and MD research is further hindered by issues of recruitment, compliance, and comorbidity. To help address these issues, a growing body of research has used eye tracking-guided, gaze-contingent displays in a simulated central vision loss paradigm in individuals with intact vision. While simulated vision loss is qualitatively different than pathological central vision loss, our framework provides for a highly controlled model through which to study compensatory eye movements and test possible rehabilitation interventions in low vision. By developing a comprehensive framework, rather than relying on isolated and disconnected tasks, we create a cohesive environment where we can test larger-scale hypotheses, allowing us to examine interactions between tasks, assess training effects across multiple measures, and establish a consistent methodology for future research. Furthermore, participants in simulated central vision loss studies show similarities in their oculomotor compensatory behaviors compared to patients with MD. Here, we present a framework for conducting gaze-contingent studies related to simulated central vision loss. We emphasize the utilization of the framework to test behavioral and oculomotor performance of healthy individuals on a wide range of perceptual tasks encompassing different levels of visual processing. We also discuss how this framework can be adapted for training MD patients.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144058105","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":"Microfabrication of Implantable Optics Integrated in a Microstructured Imaging Window for Advanced In Vivo Imaging.","authors":"Alessandra Nardini, Behjat Sadat Kariman, Mario Marini, Conci Conci, Marco Grassi, Margaux Bouzin, Maddalena Collini, Roberto Osellame, Giulio Cerullo, Manuela Teresa Raimondi, Giuseppe Chirico, Rebeca Martínez Vázquez","doi":"10.3791/67975","DOIUrl":"https://doi.org/10.3791/67975","url":null,"abstract":"<p><p>In the context of biomaterials and drug testing in animal models, this study presents a streamlined protocol for fabricating a novel implantable integrated imaging window. The micro-device comprises a sophisticated system of microlenses coupled with micro-scaffolds specifically designed for in vivo quantification of the immune response using advanced non-linear excitation microscopy. The protocol is based on two-photon polymerization (2PP) of the biocompatible photoresist SZ2080, which enables the fabrication of micro-scaffolds and micro-lenses in a continuous sequence to enhance manufacturing efficiency and precision. To further improve speed, accuracy, and structural integrity, a hybrid optics fabrication approach was implemented, involving the 2PP of the microlens outer shell followed by UV bulk crosslinking of the inner core. This innovative technique optimizes the optical properties of the microlenses while streamlining the production process. The resulting micro-device demonstrates high reproducibility and mechanical stability, making it an effective method for prototyping microscale optical systems for a range of biomedical applications.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144035139","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":"Transconjunctival Approach for Injection into the Rat Optic Nerve.","authors":"Elaine Binkley, H Culver Boldt, Lorena Haefeli, Lola P Lozano, Emily Meyering, Luke Wiley, Budd Tucker","doi":"10.3791/68048","DOIUrl":"https://doi.org/10.3791/68048","url":null,"abstract":"<p><p>The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. Accessing the rat optic nerve for experimental manipulations and injections is challenging due to the small size of the orbit and the vascularity of the surrounding orbital tissues. Previous techniques have involved a cutaneous incision, which carries a higher risk of infection and requires wound closure. This study aims to describe a unique, less invasive, and potentially more efficient approach to accessing the rat optic nerve. A transconjunctival incision, along with a rat spinal cord hook, is used to isolate the optic nerve for experimental injections. In these experiments, an adenoviral vector is injected into the optic nerve with the goal of using this technique for direct gene therapy delivery in the future. This technique can also be applied by investigators requiring access to the rat optic nerve for other experimental studies, including drug delivery and optic nerve injury research.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144058025","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":"Patch-Clamp Techniques for Single Endolysosomal Vesicle Analysis.","authors":"Hsuan-Ti Wang, Cheng-Chang Chen","doi":"10.3791/67519","DOIUrl":"https://doi.org/10.3791/67519","url":null,"abstract":"<p><p>Endolysosomal ion channels are critical for endolysosomal ion and pH homeostasis, membrane potential regulation, and vesicle trafficking. However, electrophysiologically accessing these channels within small intracellular vesicles has been a challenge. The development of endolysosomal patch-clamp techniques has been instrumental in overcoming this barrier, allowing for the direct measurement of ion channel activity in endolysosomal membranes. Compared to existing planar patch-clamp techniques, endolysosomal patch-clamp can simultaneously record multiple cells and easily combine with other measurement methods. Manual operation offers the advantage of visualizing targeted vesicles. It also addresses the limitation of the indispensable presence of Ca²⁺ on one side of the endolysosomal membrane, increasing the flexibility of experimental design. Utilizing endolysosomal patch-clamp techniques enables the direct measurement and analysis of ion channel activity within endolysosomes. Given the close link between aberrant endolysosomal ion channel function and diseases such as neurodegenerative diseases and metabolic disorders, investigating and modulating these channels may unveil new drug targets. By restoring intracellular ion balance, we may alleviate or cure related diseases. Therefore, this technique is pivotal for discovering new drug targets and developing relevant medications.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144020400","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":"Trans-Arterial Indocyanine Green Staining for Precise Anatomical Hepatectomy of Liver Segments VII and VIII.","authors":"Kaijun Huang, Renguo Guan, Zhen Zhao, Pu Shen, Ye Lin","doi":"10.3791/67448","DOIUrl":"https://doi.org/10.3791/67448","url":null,"abstract":"<p><p>Anatomical hepatectomy for resectable hepatocellular carcinoma (HCC) is performed with precision using the Glissonean system, ultrasound imaging, and indocyanine green (ICG) fluorescence staining to define liver segments, enhancing surgical radicality and preserving liver volume. However, laparoscopic clamping and ultrasound-guided ICG staining for deep-seated tumors in liver segments VII and VIII pose challenges due to the depth of the Glissonean ducts and other technical limitations. This study aimed to overcome these obstacles by exploring a protocol using trans-arterial ICG staining, a technique specifically tailored for complex anatomical hepatectomy of liver segments VII and VIII. In this method, an interventionalist accessed the right femoral artery and advanced to the celiac trunk for arteriography, followed by strategic placement of a microcatheter into the tumor's blood supply vessel to facilitate surgical resection. During the operation, ICG was injected through the microcatheter to fluorescently label the tumor's liver segment, enabling precise anatomical resection under fluorescence guidance. This trans-arterial ICG staining approach allows for the accurate identification of tumor subsegments, facilitates complete resection, and optimizes liver function preservation, ultimately improving oncological outcomes without increasing surgical complication rates.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 218","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143991426","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}