Jove-Journal of Visualized Experiments最新文献

{"title":"Advanced Glycation End-Products Sensitize Human Sensory-Like Neuron Cells to Capsaicin-Induced Calcium Influx.","authors":"Gessica Sabrina de Assis Silva, Michelle Cristiane Bufalo, Marcelo Medina de Souza, Carlos DeOcesano-Pereira, Ana Marisa Chudzinski-Tavassi, Vanessa Olzon Zambelli","doi":"10.3791/68036","DOIUrl":"https://doi.org/10.3791/68036","url":null,"abstract":"<p><p>Increased collagen-derived advanced glycation end products (AGEs) are consistently linked to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. Human sensory-like neurons differentiated from the SH-SY5Y cell line gain pro-nociceptive functions when exposed to AGEs by releasing substance P and upregulating the transient receptor potential vanilloid 1 (TRPV1) expression. Here, we investigated whether this receptor was functionally active and whether the glycation process sensitizes sensory neurons to capsaicin excitation. Sensory-like neuron cells were obtained from the differentiation of SH-SY5Y cells with all-trans-retinoic acid and brain-derived neurotrophic factor. Incubation with glycated collagen extracellular matrix (ECM-GC) simulated a pro-nociceptive stimulus. Control cells were incubated with a non-glycated extracellular collagen matrix (ECM-NC). Fluo-8 Calcium Flux Assay Kit was used to assess calcium influx, which was stimulated by capsaicin. The results show that glycation increases calcium influx compared with cells treated with normal collagen, suggesting that sensory-like neurons express functional TRPV1 channels and that glycation increases capsaicin excitation. These data indicate AGEs hypersensitive sensory-like neuron cells, triggering pro-nociceptive signaling. Together, our results suggest that we established a functional model responsive to capsaicin that can be useful for screening candidates for managing painful conditions.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144102732","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":"Mesocosm-Scale Constructed Wetland Design for Wastewater Treatment.","authors":"Amy-Lynne Balaberda, Kaitlyn E Trepanier, Dani Degenhardt","doi":"10.3791/68195","DOIUrl":"https://doi.org/10.3791/68195","url":null,"abstract":"<p><p>Oil sands process-affected water (OSPW), a by-product of bitumen extraction through surface mining in Alberta, Canada, contains various constituents of concern, including naphthenic acid fraction compounds (NAFCs). These organic compounds are particularly worrisome due to their toxicity and persistence in the environment. Constructed wetland treatment systems (CWTS) use plants and their associated microbes to attenuate contaminants in wastewater. Field-scale CWTS have been presented as a potential large-scale treatment option for OSPW, specifically for degrading NAFCs. To optimize the use of CWTS for large-scale treatment of NAFCs in OSPW, it is essential to deepen our understanding of various design parameters and explore ways to enhance efficacy. Mesocosm-scale experiments serve as a valuable intermediary, bridging the gap between complex field trials and controlled laboratory settings. Mesocosms provide a controlled, replicable environment to study the effects of various parameters such as substrate, plant species, temperature, and retention time while incorporating ecological complexities in their design. Published and previous work has shown that this method is successful in evaluating the impacts of different parameters on the efficacy of CWTS to attenuate NAFCs in OSPW. This protocol outlines the design and setup of a surface flow wetland mesocosm, along with the experimental approach for treating NAFCs in OSPW. This method can be adapted to treat other wastewaters across diverse geographical locations.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144103015","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 Functional Endodermal Hepatic Organoids.","authors":"Soheil Akbari, Nevin Ersoy, Alper Bagriyanik, Nur Arslan, Tamer Tevfik Önder, Esra Erdal","doi":"10.3791/65027","DOIUrl":"https://doi.org/10.3791/65027","url":null,"abstract":"<p><p>Organoid technology has allowed us to generate a variety of human organ-like mini structures, such as for the liver, brain, and intestine, in vitro. The remarkable advances in organoid models have recently opened a new experimental era for various applications in disease modeling, developmental biology, and drug discovery. Adult stem cells or induced pluripotent stem cell (iPSC)-derived liver organoids govern the generation of hepatocytes to use for diverse applications. Here, we present a robust and reproducible protocol for generating hepatic organoids from pluripotent stem cells. This protocol is applicable to healthy and patient-derived cells. To achieve 3D endoderm-derived hepatic organoids (eHEPOs), iPSCs were directly first differentiated into endodermal cells, and then FACS-enriched EpCAM-positive (EpCAM+) cells were used to establish hepatic organoids using the expansion medium. We provide a fast and efficient method to generate hepatic organoids within 2 weeks. The generated organoids mimic the essential properties and functions of hepatocytes, such as albumin secretion, glycogen storage, and cytochrome P450 enzyme activity. Besides the liver-specific gene expression similarities, eHEPOs comprise polarized epithelial cells with bile canaliculi in between. In addition, eHEPOs can be expanded and serial passages long term (1 year) without losing their capacity to differentiate into mature hepatocytes. Thus, eHEPOs provide an alternative source to produce functional hepatocytes.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144102949","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":"Dermal Allograft and Biceps Superior Capsule Reconstruction for Massive Irreparable Rotator Cuff Tears.","authors":"Joe Chih-Hao Chiu, Poyu Chen, Chung-Yu Chen, Cheng-Pang Yang, You-Hung Cheng, Chen-Heng Hsu, Alvin Chao-Yu Chen","doi":"10.3791/68054","DOIUrl":"https://doi.org/10.3791/68054","url":null,"abstract":"<p><p>Since the use of autologous fascia lata for superior capsule reconstruction of massive irreparable rotator cuff tears (MIRCTs), the technique has evolved into various modifications, including dermal allografts, long head of the biceps tendon (LHBT), and combinations of both, which will be discussed in this article. After making sure the remnant cuff cannot be restored to the anatomical footprint of the supraspinatus, a double-loaded or triple-loaded, suture-based anchor is inserted 5-8 mm posterior to the bicipital groove to secure the long head of the biceps (LHBT) initially. A bone trough is made 5 mm posterior to the bicipital groove. One or two lasso loops are created through the LHBT before the complete release of the transverse humeral ligament without tenotomy of the LHBT distal to the fixation point, resulting in a posteriorly rerouted LHBT.Preservation of the proximal attachment of the biceps on the glenoid side is maintained, ensuring a native fixation. Subsequently, a 3 x 3 cm dermal allograft of 2 mm thickness is utilized to cover the rerouted LHBT, enhancing its strength and providing a tensile effect. Four anchors are then employed for fixation: two double-loaded anchors at the glenoid side and two lateral row anchors at the greater tuberosity. Following the introduction of the dermal allograft into the joint, the sutures from the glenoid anchors are secured, and the optimal tension of the allograft is gauged during the insertion of the lateral row anchors at 45° shoulder abduction. The dermal allograft can cover the LHBT to increase the spacer effect. Medial row anchors are not necessary. The remaining portions of the supraspinatus and infraspinatus can be repaired using sutures passed through the lateral row anchors or repaired with the dermal allograft together to enhance stability.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144102897","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":"Generating Primary Cultures for Keratinocyte Live Cell Imaging.","authors":"Brook Abegaze, Nwamaka Ijeh, Brooke Vittimberga, Ruby Ghadially","doi":"10.3791/67854","DOIUrl":"https://doi.org/10.3791/67854","url":null,"abstract":"<p><p>Tracking stem cells and committed progenitor behavior at the single-cell level in human skin has been challenging both in vivo and in vitro. Live-cell imaging has allowed for significant advances in the ability to identify differences between keratinocyte stem cells and committed progenitor behavior. Live-cell imaging is an evolving and somewhat challenging method to study keratinocyte behavior in vitro. This protocol has been developed to culture keratinocytes at low seeding density, enabling relatively long-term time-lapse photography and monitoring of individual cell behavior. Passage 0 keratinocytes are grown at clonal density, and time-lapse photography allows documentation of individual cell divisions and the time of their occurrence. For maximum biological relevance, freshly isolated human keratinocytes are placed in vitro. This approach focuses on proliferation. However, this protocol can be adapted for use in other live cell imaging applications measuring individual cell behavior, such as measuring cell migration, wound healing, and motility.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144102946","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":"Kidney Procurement in a Preclinical Large Animal Model.","authors":"Eleni M Drivas, Amanda H Loftin, Siavash Khaki, Alexandrea L Bailey, Michael Wallis, Jessica Izzi, Byoung Chol Oh, Gerald Brandacher","doi":"10.3791/67835","DOIUrl":"https://doi.org/10.3791/67835","url":null,"abstract":"<p><p>Machine perfusion has evolved as a viable strategy for ex vivo organ assessment, monitoring, treatment, optimization, as well as to prolong preservation times. Large animal models have been paramount for the development and optimization of these technologies. However, in order to ensure graft quality and data reproducibility, standardized and clinically translatable surgical techniques for organ and tissue procurement should be followed. Thus, here, we describe an optimized protocol for kidney procurement in a preclinical swine model. Kidney recovery is performed using mixed breed (Yorkshire cross/mix) pigs. Briefly, following sterile disinfection and draping of the surgical field, a complete midline incision is performed to gain optimal access to both kidneys. The ureter, renal vein, and artery are dissected until their origin from the inferior vena cava and the aorta, respectively. After complete renal dissection, the ureter is tied and cut distally. The donor animal is then fully heparinized with 100 IU per kg/body weight. Next, the renal artery is clamped close to the aorta, and the renal vein is clamped close to the vena cava using a Satinsky vascular clamp. The kidney graft is then resected, and the renal artery is immediately cannulated back table. The kidney will then be flushed with an ice-cold preservation solution and stored on ice until either machine perfusion or transplantation. Finally, the renal artery stump is ligated with a 2-0 silk ligature, and the vena cava is closed with a 6-0 polypropylene suture. This technique recovers kidneys and simulates either a living (single kidney) or deceased (dual kidney) donor setting. The single kidney recovery offers the advantage to perform a subsequent autotransplantation. In the deceased donor model, blood can be collected prior to euthanasia by inserting blood bag needles directly into the aorta, thereby exsanguinating the animal and providing blood for ex vivo machine perfusion.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144102999","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 Common Bile Duct Exploration Followed by Primary Suture Using a Modified Bile Duct Incision.","authors":"Guixing Chen, Meijiao Li, Zhanhui Chen, Shilong Tang, Songxu Qi, Rongjun Chen, Zheng Liang, Yurong Luo, Yongqiang Chen, Xingdong Song","doi":"10.3791/67549","DOIUrl":"https://doi.org/10.3791/67549","url":null,"abstract":"<p><p>Cholecystolithiasis is a common clinical disease, and 10-15% of patients with cholecystolithiasis have common bile duct (CBD) stones. Laparoscopic CBD exploration (LCBDE) followed by primary closure has proven to be safe and cost-effective for treating CBD stones and is typically performed via transcystic and transductal approaches. However, traditional LCBDE with choledochotomy may lead to biliary stricture and leakage, and performing choledochoscopy in transcystic LCBDE may be challenging because of the narrow cystic duct. To reduce the incidence rate of biliary stricture and leakage and increase the success rate of choledochoscopy, we propose a modified technique for bile duct incision. We performed LCBDE via a micro longitudinal incision extended along the cystobiliary junction toward the CBD instead of the traditional anterior wall of the CBD or purely transverse transcystic incision. Our micro incision size on CBD only ranged from 5 to 10 mm according to the size of the CBD stones. Using this micro incision technique, which is less invasive, resulted in easier exploration of the CBD and preservation of the ductal wall integrity. This approach encourages surgeons to use primary closure and may be considered a viable alternative choice for patients with ductal stones in the future.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144103010","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":"Postmortem Diagnosis of Rabies in Animals by the Updated, Multiplexed LN34 Real-Time Reverse Transcription-Polymerase Chain Reaction Assay.","authors":"Crystal M Gigante, Vaughn Wicker, Rene Edgar Condori, Kimberly Wilkins, Yu Li","doi":"10.3791/66761","DOIUrl":"https://doi.org/10.3791/66761","url":null,"abstract":"<p><p>Rabies is a fatal zoonotic disease caused by Lyssavirus rabies (RABV) and related negative strand RNA viruses from the Lyssavirus genus (family Rhabdoviridae). The LN34 assay targets the highly conserved leader region and nucleoprotein gene of the lyssavirus genome and utilizes degenerate primers and a TaqMan probe containing locked nucleotides to detect RNA across the diverse Lyssavirus genus. A negative finding for rabies should be made only if a full cross-section of the brain stem and three lobes of the cerebellum are examined; however, identification of lyssavirus RNA in any tissue is diagnostic of rabies infection. Tissue is collected and homogenized in TRIzol reagent, which also inactivates the virus. RNA extraction is performed using a spin column-based commercial extraction kit. Master mixes are prepared in a clean space and aliquoted into a 96 well plate before adding sample RNA. In clinical settings, each sample is tested by real-time RT-PCR for the presence of lyssavirus RNA in triplicate and singly for host β -actin mRNA. Positive and negative controls are included at extraction and real-time RT-PCR steps of the protocol. Data analysis involves manual adjustment of the thresholds to standardize Ct values across instrument runs. Positive results are determined by the presence of typical amplification in the pan-lyssavirus assay (Ct ≤ 35). Negative results are determined by the absence of typical amplification in the pan-lyssavirus assay and detection of host β-actin mRNA (Ct ≤ 33). Observation of values outside of these ranges or failure of the assay controls can invalidate the run or result in inconclusive results for a specimen. The protocol should be closely followed to ensure high assay sensitivity and specificity. Procedural modifications can affect assay performance and lead to false positive, false negative, or uninterpretable results.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144103024","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 Anatomical Resection of The Right Anterior Lobe Based on The Laennec Capsule Technique.","authors":"HongBo Liang, ChuYing Huang, ShuWen Lin","doi":"10.3791/67705","DOIUrl":"10.3791/67705","url":null,"abstract":"<p><p>This paper presents a laparoscopic anatomical resection technique for hepatocellular carcinoma (HCC) utilizing the Laennec capsule concept. The goal of this protocol is to improve surgical precision and safety during complex liver resections, particularly in tumors closely associated with vital vascular structures, by ensuring clear identification and dissection of critical anatomical landmarks. Surgical resection remains the primary curative treatment for hepatocellular carcinoma (HCC), but laparoscopic anatomical right anterior sectionectomy becomes particularly challenging when tumors are closely associated with major vascular structures, as this increases the risk of significant bleeding. Comprehensive preoperative treatment, including targeted therapy, immunotherapy, and hepatic arterial infusion chemotherapy (HAIC), can reduce tumor size and improve surgical outcomes, making previously borderline resectable tumors operable. In this case, a 75-year-old patient with a tumor in segment 8 (S8) of the liver, closely associated with major vascular structures, underwent two cycles of preoperative treatment. This reduced the tumor size from 6 cm by 5 cm to 4.5 cm by 3.1 cm. Laparoscopic anatomical right anterior sectionectomy was performed using the Laennec capsule, an anatomical structure that aids in vascular dissection. The procedure lasted 240 minutes with minimal blood loss (200 mL). The tumor was successfully resected with negative surgical margins, and the patient was discharged on the seventh postoperative day without complications. Postoperatively, the patient was monitored for signs of liver dysfunction and underwent routine imaging to assess for recurrence. Follow-up included liver function tests and regular CT scans, showing no recurrence after 6 months. This case demonstrates that the combination of the Laennec capsule technique and comprehensive preoperative treatment allows for precise, minimally invasive resections of HCC tumors closely associated with vascular structures, providing a safe and effective solution to challenging liver tumors with minimal intraoperative complications and promising postoperative outcomes.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144103003","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":"Isolated Pancreatic Islet Treatment and Apoptosis Measurement.","authors":"Jubril Akolade, Isaac Crossley, Nikki L Farnsworth","doi":"10.3791/67996","DOIUrl":"https://doi.org/10.3791/67996","url":null,"abstract":"<p><p>This study investigates the effect of pro-inflammatory cytokines on pancreatic islets, particularly insulin-producing β-cells, using a combination of fluorescence staining techniques and confocal microscopy to assess cell viability, apoptosis, and β-cell-specific death. Isolated mouse islets were treated with varying concentrations of a cytokine cocktail, including TNF-α, IL-1β, and IFN-γ, to mimic immune-mediated apoptosis during the development of type 1 diabetes. The viability of islet cells was evaluated with FDA/PI dual staining, where FDA conversion to fluorescein indicated viable cells, and PI marked membrane-compromised cells. YOPRO-1 and nuclear staining provided additional data on apoptosis, with Annexin-V confirming early apoptotic cells. Quantitative analysis revealed significant increases in apoptosis and cell death rates in cytokine-treated islets. To specifically assess effects on β-cells, Zn²⁺ selective indicator staining was used to label insulin-producing cells through the zinc association in insulin granules, revealing substantial β-cell loss following treatment of islets with pro-inflammatory cytokines for 24 h. These multi-staining protocols effectively capture and quantify the extent of cytokine-induced damage in islets and can be used to evaluate therapeutics designed to prevent β-cell apoptosis in early type 1 diabetes.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 219","pages":""},"PeriodicalIF":1.2,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144102996","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}