Qitao Zhang, Daisy Y Shu, Richard A Bryan, John Y S Han, Gillian A Gulette, Kin Lo, Leo A Kim, Jason M L Miller
Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for photoreceptor survival. RPE mitochondrial dysfunction is a hallmark of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world, and proliferative vitreoretinopathy (PVR), a blinding complication of retinal detachments. RPE degenerative conditions have been well-modeled by RPE culture systems that are highly differentiated and polarized to mimic in vivo RPE. However, monitoring oxygen consumption rates (OCR), a proxy for mitochondrial function, has been difficult in such culture systems because the conditions that promote ideal RPE polarization and differentiation do not allow for easy OCR measurements. Here, we introduce a novel system, Resipher, to monitor OCR for weeks at a time in well-differentiated RPE cultures while maintaining the RPE on optimal growth substrates and physiologic culture media in a standard cell culture incubator. This system calculates OCR by measuring the oxygen concentration gradient present in the media above cells. We discuss the advantages of this system over other methods for detecting OCR and how to set up the system for measuring OCR in RPE cultures. We cover key tips and tricks for using the system, caution about interpreting the data, and guidelines for troubleshooting unexpected results. We also provide an online calculator for extrapolating the level of hypoxia, normoxia, or hyperoxia RPE cultures experience based on the oxygen gradient in the media above cells detected by the system. Finally, we review two applications of the system, measuring the metabolic state of RPE cells in a PVR model and understanding how the RPE metabolically adapts to hypoxia. We anticipate that the use of this system on highly polarized and differentiated RPE cultures will enhance our understanding of RPE mitochondrial metabolism both under physiologic and disease states.
{"title":"Long-term Monitoring of Oxygen Consumption Rates in Highly Differentiated and Polarized Retinal Pigment Epithelial Cultures.","authors":"Qitao Zhang, Daisy Y Shu, Richard A Bryan, John Y S Han, Gillian A Gulette, Kin Lo, Leo A Kim, Jason M L Miller","doi":"10.3791/67038","DOIUrl":"https://doi.org/10.3791/67038","url":null,"abstract":"<p><p>Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for photoreceptor survival. RPE mitochondrial dysfunction is a hallmark of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world, and proliferative vitreoretinopathy (PVR), a blinding complication of retinal detachments. RPE degenerative conditions have been well-modeled by RPE culture systems that are highly differentiated and polarized to mimic in vivo RPE. However, monitoring oxygen consumption rates (OCR), a proxy for mitochondrial function, has been difficult in such culture systems because the conditions that promote ideal RPE polarization and differentiation do not allow for easy OCR measurements. Here, we introduce a novel system, Resipher, to monitor OCR for weeks at a time in well-differentiated RPE cultures while maintaining the RPE on optimal growth substrates and physiologic culture media in a standard cell culture incubator. This system calculates OCR by measuring the oxygen concentration gradient present in the media above cells. We discuss the advantages of this system over other methods for detecting OCR and how to set up the system for measuring OCR in RPE cultures. We cover key tips and tricks for using the system, caution about interpreting the data, and guidelines for troubleshooting unexpected results. We also provide an online calculator for extrapolating the level of hypoxia, normoxia, or hyperoxia RPE cultures experience based on the oxygen gradient in the media above cells detected by the system. Finally, we review two applications of the system, measuring the metabolic state of RPE cells in a PVR model and understanding how the RPE metabolically adapts to hypoxia. We anticipate that the use of this system on highly polarized and differentiated RPE cultures will enhance our understanding of RPE mitochondrial metabolism both under physiologic and disease states.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142113785","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}
Frederic St-Denis-Bissonnette, Melanie Kirkby, Lisheng Wang, Jessie R Lavoie
Natural killer cell-derived extracellular vesicles (NK-EVs) are being investigated as cancer biotherapeutics. They possess unique properties as cytotoxic nanovesicles targeting cancer cells and as immunomodulatory communicators. A scalable biomanufacturing workflow enables the production of large quantities of high-purity NK-EVs to meet the pre-clinical and clinical demands. The workflow employs a closed-loop hollow-fiber bioreactor, enabling continuous production of NK-EVs from the NK92-MI cell line under serum-free, xeno-free, feeder-free, and antibiotic-free conditions in compliance with Good Manufacturing Practices standards. This protocol-driven study outlines the biomanufacturing workflow for isolating NK-EVs using size-exclusion chromatography, ultrafiltration, and filter-based sterilization. Essential NK-EV product characterization is performed via nanoparticle tracking analysis, and their functionality is assessed through a validated cell viability-based potency assay against cancer cells. This scalable biomanufacturing process holds significant potential to advance the clinical translation of NK-EV-based cancer biotherapeutics by adhering to best practices and ensuring reproducibility.
{"title":"Scalable Biomanufacturing Workflow to Produce and Isolate Natural Killer Cell-derived Extracellular Vesicle-based Cancer Biotherapeutics.","authors":"Frederic St-Denis-Bissonnette, Melanie Kirkby, Lisheng Wang, Jessie R Lavoie","doi":"10.3791/67227","DOIUrl":"10.3791/67227","url":null,"abstract":"<p><p>Natural killer cell-derived extracellular vesicles (NK-EVs) are being investigated as cancer biotherapeutics. They possess unique properties as cytotoxic nanovesicles targeting cancer cells and as immunomodulatory communicators. A scalable biomanufacturing workflow enables the production of large quantities of high-purity NK-EVs to meet the pre-clinical and clinical demands. The workflow employs a closed-loop hollow-fiber bioreactor, enabling continuous production of NK-EVs from the NK92-MI cell line under serum-free, xeno-free, feeder-free, and antibiotic-free conditions in compliance with Good Manufacturing Practices standards. This protocol-driven study outlines the biomanufacturing workflow for isolating NK-EVs using size-exclusion chromatography, ultrafiltration, and filter-based sterilization. Essential NK-EV product characterization is performed via nanoparticle tracking analysis, and their functionality is assessed through a validated cell viability-based potency assay against cancer cells. This scalable biomanufacturing process holds significant potential to advance the clinical translation of NK-EV-based cancer biotherapeutics by adhering to best practices and ensuring reproducibility.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142113787","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}
Julieta S Del Valle, Azra Husetic, Dina Diek, Laurens F Rutgers, Joyce D Asseler, Jeroen Metzemaekers, Norah M van Mello, Susana M Chuva de Sousa Lopes
The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound healing while restoring ovarian integrity. Additionally, the OSE may serve as the source of epithelial ovarian cancers. Although the OSE regenerative properties have been well studied in mice, understanding the precise mechanism of tissue repair in the human ovary remains hampered by limited access to human ovaries and suitable in vitro culture protocols. Tissue-specific organoids, miniaturized in vitro models replicating both structural and functional aspects of the original organ, offer new opportunities for studying organ physiology, disease modeling, and drug testing. Here, we describe a method to isolate primary human OSE (hOSE) from whole ovaries and establish hOSE organoids. We include a morphological and cellular characterization showing heterogeneity between donors. Additionally, we demonstrate the capacity of this culture method to evaluate hormonal effects on OSE-organoid growth over a 2-week period. This method may enable the discovery of factors contributing to OSE regeneration and facilitate patient-specific drug screenings for malignant OSE.
卵巢表面上皮(OSE)是卵巢的最外层,每次排卵时都会破裂,在卵巢伤口愈合和恢复卵巢完整性方面起着至关重要的作用。此外,OSE 可能是上皮性卵巢癌的来源。虽然对小鼠卵巢上皮细胞的再生特性进行了深入研究,但对人类卵巢组织修复的精确机制的了解仍然受到人类卵巢和合适的体外培养方案的限制。组织特异性器官体是一种微型体外模型,可复制原始器官的结构和功能,为研究器官生理学、疾病建模和药物测试提供了新的机会。在这里,我们介绍了一种从完整卵巢中分离原代人类卵巢组织(hOSE)并建立 hOSE 器官组织的方法。我们对形态学和细胞特性进行了分析,显示了不同供体之间的异质性。此外,我们还展示了这种培养方法在 2 周内评估激素对 OSE 有机体生长影响的能力。这种方法可以发现促进 OSE 再生的因素,并促进针对恶性 OSE 的病人特异性药物筛选。
{"title":"Human Ovarian Surface Epithelium Organoids as a Platform to Study Tissue Regeneration.","authors":"Julieta S Del Valle, Azra Husetic, Dina Diek, Laurens F Rutgers, Joyce D Asseler, Jeroen Metzemaekers, Norah M van Mello, Susana M Chuva de Sousa Lopes","doi":"10.3791/66797","DOIUrl":"10.3791/66797","url":null,"abstract":"<p><p>The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound healing while restoring ovarian integrity. Additionally, the OSE may serve as the source of epithelial ovarian cancers. Although the OSE regenerative properties have been well studied in mice, understanding the precise mechanism of tissue repair in the human ovary remains hampered by limited access to human ovaries and suitable in vitro culture protocols. Tissue-specific organoids, miniaturized in vitro models replicating both structural and functional aspects of the original organ, offer new opportunities for studying organ physiology, disease modeling, and drug testing. Here, we describe a method to isolate primary human OSE (hOSE) from whole ovaries and establish hOSE organoids. We include a morphological and cellular characterization showing heterogeneity between donors. Additionally, we demonstrate the capacity of this culture method to evaluate hormonal effects on OSE-organoid growth over a 2-week period. This method may enable the discovery of factors contributing to OSE regeneration and facilitate patient-specific drug screenings for malignant OSE.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142113782","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}
Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.
{"title":"Point-of-care CRISPR-based Diagnostics with Premixed and Freeze-dried Reagents.","authors":"Rodjarin Kongkaew, Chayasith Uttamapinant, Maturada Patchsung","doi":"10.3791/66703","DOIUrl":"10.3791/66703","url":null,"abstract":"<p><p>Molecular diagnostics by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based detection have high diagnostic accuracy and attributes that are suitable for use at point-of-care settings such as fast turnaround times for results, convenient simple readouts, and no requirement of complicated instruments. However, the reactions can be cumbersome to perform at the point of care due to their many components and manual handling steps. Herein, we provide a step-by-step, optimized protocol for the robust detection of disease pathogens and genetic markers with recombinase-based isothermal amplification and CRISPR-based reagents, which are premixed and then freeze-dried in easily stored and ready-to-use formats. Premixed, freeze-dried reagents can be rehydrated for immediate use and retain high amplification and detection efficiencies. We also provide a troubleshooting guide for commonly found problems upon preparing and using premixed, freeze-dried reagents for CRISPR-based diagnostics, to make the detection platform more accessible to the wider diagnostic/genetic testing communities.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142113786","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}
Jacy Fang, Isabella Salinas, Sarah San Vicente, Caroline Zielinski, Moshe Sade-Feldman
Human tumor samples hold a plethora of information about their microenvironment and immune repertoire. Effective dissociation of human tissue samples into viable cell suspensions is a required input for the single-cell RNA sequencing (scRNAseq) pipeline. Unlike bulk RNA sequencing approaches, scRNAseq enables us to infer the transcriptional heterogeneity in tumor specimens at the single-cell level. Incorporating this approach in recent years has led to many discoveries, such as identifying immune and tumor cellular states and programs associated with clinical responses to immunotherapies and other types of treatments. Moreover, single-cell technologies applied to dissociated tissues can be used to identify accessible chromatin regions T and B cell receptor repertoire, and the expression of proteins, using DNA barcoded antibodies (CITEseq). The viability and quality of the dissociated sample are critical variables when using these technologies, as these can dramatically affect the cross-contamination of single cells with ambient RNA, the quality of the data, and interpretation. Moreover, long dissociation protocols can lead to the elimination of sensitive cell populations and the upregulation of a stress response gene signature. To overcome these limitations, we devised a rapid universal dissociation protocol, which has been validated on multiple types of human and murine tumors. The process begins with mechanical and enzymatic dissociation, followed by filtration, red blood lysis, and live dead enrichment, suitable for samples with a low input of cells (e.g., needle core biopsies). This protocol ensures a clean and viable single-cell suspension paramount to the successful generation of Gel Bead-In Emulsions (GEMs), barcoding, and sequencing.
人体肿瘤样本蕴含着大量有关其微环境和免疫反应的信息。将人体组织样本有效分离成有活力的细胞悬浮液是单细胞 RNA 测序(scRNAseq)管道所需的输入。与大量 RNA 测序方法不同,scRNAseq 使我们能够在单细胞水平上推断肿瘤标本的转录异质性。近年来,这种方法的应用带来了许多发现,例如确定与免疫疗法和其他类型治疗的临床反应相关的免疫和肿瘤细胞状态和程序。此外,应用于解离组织的单细胞技术可利用 DNA 条形码抗体(CITEseq)鉴定可访问的染色质区域、T 细胞和 B 细胞受体谱系以及蛋白质的表达。使用这些技术时,解离样本的存活率和质量是关键变量,因为它们会极大地影响单细胞与环境 RNA 的交叉污染、数据质量和解释。此外,长时间的解离方案可能会导致敏感细胞群的消失和应激反应基因特征的上调。为了克服这些局限性,我们设计了一种快速通用解离方案,该方案已在多种类型的人类和鼠类肿瘤上得到验证。该过程首先是机械和酶解,然后是过滤、红细胞裂解和活死细胞富集,适用于输入细胞较少的样本(如针芯活检样本)。该方案可确保获得洁净、有活力的单细胞悬浮液,这对成功生成凝胶珠乳剂(GEM)、条形码和测序至关重要。
{"title":"Dissociation of Human and Mouse Tumor Tissue Samples for Single-cell RNA Sequencing.","authors":"Jacy Fang, Isabella Salinas, Sarah San Vicente, Caroline Zielinski, Moshe Sade-Feldman","doi":"10.3791/66766","DOIUrl":"https://doi.org/10.3791/66766","url":null,"abstract":"<p><p>Human tumor samples hold a plethora of information about their microenvironment and immune repertoire. Effective dissociation of human tissue samples into viable cell suspensions is a required input for the single-cell RNA sequencing (scRNAseq) pipeline. Unlike bulk RNA sequencing approaches, scRNAseq enables us to infer the transcriptional heterogeneity in tumor specimens at the single-cell level. Incorporating this approach in recent years has led to many discoveries, such as identifying immune and tumor cellular states and programs associated with clinical responses to immunotherapies and other types of treatments. Moreover, single-cell technologies applied to dissociated tissues can be used to identify accessible chromatin regions T and B cell receptor repertoire, and the expression of proteins, using DNA barcoded antibodies (CITEseq). The viability and quality of the dissociated sample are critical variables when using these technologies, as these can dramatically affect the cross-contamination of single cells with ambient RNA, the quality of the data, and interpretation. Moreover, long dissociation protocols can lead to the elimination of sensitive cell populations and the upregulation of a stress response gene signature. To overcome these limitations, we devised a rapid universal dissociation protocol, which has been validated on multiple types of human and murine tumors. The process begins with mechanical and enzymatic dissociation, followed by filtration, red blood lysis, and live dead enrichment, suitable for samples with a low input of cells (e.g., needle core biopsies). This protocol ensures a clean and viable single-cell suspension paramount to the successful generation of Gel Bead-In Emulsions (GEMs), barcoding, and sequencing.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142113779","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}
The acquisition of reading skills is an intricate process that demands the cultivation of various domain-general and domain-specific abilities. Consequently, it is unsurprising that many children grapple with maintaining proficiency at the grade level, particularly when confronted with challenges spanning multiple abilities across both domains, as observed in individuals with reading difficulties. Strikingly, despite reading difficulties being among the most prevalent neurodevelopmental disorders affecting school-aged children, the majority of available diagnostic tools lack a comprehensive framework for assessing the full spectrum of cognitive skills linked to dyslexia, with minimal computerized options. Notably, there are currently limited tools with these features available for Spanish-speaking children. The aim of this study was to delineate the protocol for diagnosing Spanish-speaking children with reading difficulties using the Sicole-R multimedia battery. This tool for elementary grades focuses on evaluating cognitive skills that are associated with dyslexia as prescribed by the scientific literature. Specifically, it concentrates on assessing a range of cognitive abilities that studies have demonstrated to be linked to dyslexia. This focus is based on the observation that individuals with dyslexia typically exhibit deficits in several of the cognitive areas evaluated by this digital tool. The robust internal consistency and multidimensional internal structure of the battery were demonstrated. This multimedia battery has proven to be a fitting tool for diagnosing children with reading difficulties in primary education, offering a comprehensive cognitive profile that is valuable not only for diagnostic purposes but also for tailoring individualized instructional plans.
{"title":"Advancing Dyslexia Assessment in Children through Computerized Testing.","authors":"Juan E Jiménez, Eduardo García, Jennifer Balade","doi":"10.3791/67031","DOIUrl":"https://doi.org/10.3791/67031","url":null,"abstract":"<p><p>The acquisition of reading skills is an intricate process that demands the cultivation of various domain-general and domain-specific abilities. Consequently, it is unsurprising that many children grapple with maintaining proficiency at the grade level, particularly when confronted with challenges spanning multiple abilities across both domains, as observed in individuals with reading difficulties. Strikingly, despite reading difficulties being among the most prevalent neurodevelopmental disorders affecting school-aged children, the majority of available diagnostic tools lack a comprehensive framework for assessing the full spectrum of cognitive skills linked to dyslexia, with minimal computerized options. Notably, there are currently limited tools with these features available for Spanish-speaking children. The aim of this study was to delineate the protocol for diagnosing Spanish-speaking children with reading difficulties using the Sicole-R multimedia battery. This tool for elementary grades focuses on evaluating cognitive skills that are associated with dyslexia as prescribed by the scientific literature. Specifically, it concentrates on assessing a range of cognitive abilities that studies have demonstrated to be linked to dyslexia. This focus is based on the observation that individuals with dyslexia typically exhibit deficits in several of the cognitive areas evaluated by this digital tool. The robust internal consistency and multidimensional internal structure of the battery were demonstrated. This multimedia battery has proven to be a fitting tool for diagnosing children with reading difficulties in primary education, offering a comprehensive cognitive profile that is valuable not only for diagnostic purposes but also for tailoring individualized instructional plans.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142113778","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}
Xia Li, Yun Liu, Kun Liu, Longhua Du, Tao Lv, Bing Zhu, Xinyan Gao
The dorsal root ganglia (DRG), housing primary sensory neurons, transmit somatosensory and visceral afferent inputs to the dorsal horn of the spinal cord. They play a pivotal role in both physiological and pathological states, including neuropathic and visceral pain. In vivo calcium imaging of DRG enables real-time observation of calcium transients in single units or neuron ensembles. Accumulating evidence indicates that DRG neuronal activities induced by somatic stimulation significantly affect autonomic and visceral functions. While lumbar DRG calcium imaging has been extensively studied, thoracic segment DRG calcium imaging has been less explored due to surgical exposure and stereotaxic fixation challenges. Here, we utilized in vivo calcium imaging at the thoracic1 dorsal root ganglion (T1-DRG) to investigate changes in neuronal activity resulting from somatic stimulations of the forelimb. This approach is crucial for understanding the somato-cardiac reflex triggered by peripheral nerve stimulations (PENS), such as acupuncture. Notably, synchronization of cardiac function was observed and measured by electrocardiogram (ECG), with T-DRG neuronal activities, potentially establishing a novel paradigm for somato-visceral reflex in the thoracic segments.
{"title":"In Vivo Thoracic Dorsal Root Ganglia (DRG) Calcium Imaging and ECG Recording for Studying Peripheral Nerve Stimulation.","authors":"Xia Li, Yun Liu, Kun Liu, Longhua Du, Tao Lv, Bing Zhu, Xinyan Gao","doi":"10.3791/67283","DOIUrl":"10.3791/67283","url":null,"abstract":"<p><p>The dorsal root ganglia (DRG), housing primary sensory neurons, transmit somatosensory and visceral afferent inputs to the dorsal horn of the spinal cord. They play a pivotal role in both physiological and pathological states, including neuropathic and visceral pain. In vivo calcium imaging of DRG enables real-time observation of calcium transients in single units or neuron ensembles. Accumulating evidence indicates that DRG neuronal activities induced by somatic stimulation significantly affect autonomic and visceral functions. While lumbar DRG calcium imaging has been extensively studied, thoracic segment DRG calcium imaging has been less explored due to surgical exposure and stereotaxic fixation challenges. Here, we utilized in vivo calcium imaging at the thoracic1 dorsal root ganglion (T1-DRG) to investigate changes in neuronal activity resulting from somatic stimulations of the forelimb. This approach is crucial for understanding the somato-cardiac reflex triggered by peripheral nerve stimulations (PENS), such as acupuncture. Notably, synchronization of cardiac function was observed and measured by electrocardiogram (ECG), with T-DRG neuronal activities, potentially establishing a novel paradigm for somato-visceral reflex in the thoracic segments.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142113783","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}
Bo Wang, Yu-Zhao Xu, Yun-Chao Zhou, Shan Ha, Xiao-Shi Qin, Jie Cai, Bin Cong, Jian-Hua Chen, Jian-Qiang Deng
This corrects the article 10.3791/66737.
这更正了文章 10.3791/66737。
{"title":"Erratum: DNA Extraction and Comparison Between Old and Fresh Necrophilic Fly Samples.","authors":"Bo Wang, Yu-Zhao Xu, Yun-Chao Zhou, Shan Ha, Xiao-Shi Qin, Jie Cai, Bin Cong, Jian-Hua Chen, Jian-Qiang Deng","doi":"10.3791/6604","DOIUrl":"10.3791/6604","url":null,"abstract":"<p><p>This corrects the article 10.3791/66737.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141972132","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}
Endometrial organoids offer valuable insights into the development and pathophysiology of endometrial diseases and serve as platforms for drug testing. While human and mouse endometrial organoids have been developed, research on rat endometrial organoids remains limited. Given that rats can better simulate certain endometrial pathologies, such as intrauterine adhesions, this study aimed to establish rat endometrial organoids. We present a detailed protocol for the isolation and culture of rat endometrial epithelial stem cells (reESCs) and the generation of rat endometrial organoids. Using a refined reESCs expansion medium, we successfully isolated and stably expanded reESCs, demonstrating their long-term culture potential. The reESC-generated organoids exhibited typical structural and functional characteristics of the endometrium, including hormone responsiveness. Our results showed that rat endometrial organoids could be cultured over a long term with stable proliferation, maintaining the glandular structure, cell polarity, and functional characteristics of the endometrial epithelium. This novel rat-derived endometrial organoid model provides a valuable platform for studying endometrial diseases and testing therapeutic interventions, with potential applications across various mammalian species.
{"title":"Generation and Characterization of Rat Uterus Organoids from Rat Endometrial Epithelial Stem Cells.","authors":"Man Yang, Qinghua Liu, Yong Chen, Jun Li, Wen He","doi":"10.3791/66928","DOIUrl":"https://doi.org/10.3791/66928","url":null,"abstract":"<p><p>Endometrial organoids offer valuable insights into the development and pathophysiology of endometrial diseases and serve as platforms for drug testing. While human and mouse endometrial organoids have been developed, research on rat endometrial organoids remains limited. Given that rats can better simulate certain endometrial pathologies, such as intrauterine adhesions, this study aimed to establish rat endometrial organoids. We present a detailed protocol for the isolation and culture of rat endometrial epithelial stem cells (reESCs) and the generation of rat endometrial organoids. Using a refined reESCs expansion medium, we successfully isolated and stably expanded reESCs, demonstrating their long-term culture potential. The reESC-generated organoids exhibited typical structural and functional characteristics of the endometrium, including hormone responsiveness. Our results showed that rat endometrial organoids could be cultured over a long term with stable proliferation, maintaining the glandular structure, cell polarity, and functional characteristics of the endometrial epithelium. This novel rat-derived endometrial organoid model provides a valuable platform for studying endometrial diseases and testing therapeutic interventions, with potential applications across various mammalian species.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142001092","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}
Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in this field's research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.
{"title":"In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice.","authors":"Shanshan Han, Ning Ding, Ce Li, Peng Yuan","doi":"10.3791/66916","DOIUrl":"https://doi.org/10.3791/66916","url":null,"abstract":"<p><p>Real-time approaches are typically needed in studies of learning and memory, and in vivo calcium imaging provides the possibility to investigate neuronal activity in awake animals during behavior tasks. Since the hippocampus is closely associated with episodic and spatial memory, it has become an essential brain region in this field's research. In recent research, engram cells and place cells were studied by recording the neural activities in the hippocampal CA1 region using the miniature microscope in mice while performing behavioral tasks including open-field and linear track. Although the dentate gyrus is another important region in the hippocampus, it has rarely been studied with in vivo imaging due to its greater depth and difficulty for imaging. In this protocol, we present in detail a calcium imaging process, including how to inject the virus, implant a GRIN (Gradient-index) lens, and attach a base plate for imaging the dentate gyrus of the hippocampus. We further describe how to preprocess the calcium imaging data using MATLAB. Additionally, studies of other deep brain regions that require imaging may benefit from this method.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142001094","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}