Julian Rapp, Jan Ness, Paula Liang, Martin J Hug, Hansjürgen Agostini, Günther Schlunck, Clemens Lange, Felicitas Bucher
Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets. This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.
{"title":"Investigating Angiogenesis on a Functional and Molecular Level by Leveraging the Scratch Wound Migration Assay and the Spheroid Sprouting Assay.","authors":"Julian Rapp, Jan Ness, Paula Liang, Martin J Hug, Hansjürgen Agostini, Günther Schlunck, Clemens Lange, Felicitas Bucher","doi":"10.3791/66954","DOIUrl":"https://doi.org/10.3791/66954","url":null,"abstract":"<p><p>Angiogenesis plays a crucial role in both physiological and pathological processes within the body including tumor growth or neovascular eye disease. A detailed understanding of the underlying molecular mechanisms and reliable screening models are essential for targeting diseases effectively and developing new therapeutic options. Several in vitro assays have been developed to model angiogenesis, capitalizing on the opportunities a controlled environment provides to elucidate angiogenic drivers at a molecular level and screen for therapeutic targets. This study presents workflows for investigating angiogenesis in vitro using human umbilical vein endothelial cells (HUVECs). We detail a scratch wound migration assay utilizing a live cell imaging system measuring endothelial cell migration in a 2D setting and the spheroid sprouting assay assessing endothelial cell sprouting in a 3D setting provided by a collagen matrix. Additionally, we outline strategies for sample preparation to enable further molecular analyses such as transcriptomics, particularly in the 3D setting, including RNA extraction as well as immunocytochemistry. Altogether, this framework offers scientists a reliable and versatile toolset to pursue their scientific inquiries in in vitro angiogenesis assays.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141332303","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}
Hin Hung Sik, Stavros Skouras, Junling Gao, Hang Kin Leung, Siu Man Ng, Kin Cheung Lee, Bonnie Wai Yan Wu
This protocol presents a multi-modal neuroimaging approach to explore the potential brain activity associated with repetitive religious chanting, a widespread form of mind training in both Eastern and Western cultures. High-density electroencephalogram (EEG), with its superior temporal resolution, allows for capturing the dynamic changes in brain activity during religious chanting. Through source localization methods, these can be attributed to various alternative potential brain region sources. Twenty practitioners of religious chanting were measured with EEG. However, the spatial resolution of EEG is less precise, in comparison to functional magnetic resonance imaging (fMRI). Thus, one highly experienced practitioner underwent an fMRI scanning session to guide the source localization more precisely. The fMRI data helped guide the selection of EEG source localization, making the calculation of K-means of the EEG source localization in the group of 20 intermediate practitioners more precise and reliable. This method enhanced EEG's ability to identify the brain regions specifically engaged during religious chanting, particularly the cardinal role of the posterior cingulate cortex (PCC). The PCC is a brain area related to focus and self-referential processing. These multimodal neuroimaging and neurophysiological results reveal that repetitive religious chanting can induce lower centrality and higher delta-wave power compared to non-religious chanting and resting state conditions. The combination of fMRI and EEG source analysis provides a more detailed understanding of the brain's response to repetitive religious chanting. The protocol contributes significantly to the research on the neural mechanisms involved in religious and meditative practices, which is becoming more prominent nowadays. The results of this study could have significant implications for developing future neurofeedback techniques and psychological interventions.
{"title":"Religious Chanting and Self-Related Brain Regions: A Multi-Modal Neuroimaging Study.","authors":"Hin Hung Sik, Stavros Skouras, Junling Gao, Hang Kin Leung, Siu Man Ng, Kin Cheung Lee, Bonnie Wai Yan Wu","doi":"10.3791/66221","DOIUrl":"https://doi.org/10.3791/66221","url":null,"abstract":"<p><p>This protocol presents a multi-modal neuroimaging approach to explore the potential brain activity associated with repetitive religious chanting, a widespread form of mind training in both Eastern and Western cultures. High-density electroencephalogram (EEG), with its superior temporal resolution, allows for capturing the dynamic changes in brain activity during religious chanting. Through source localization methods, these can be attributed to various alternative potential brain region sources. Twenty practitioners of religious chanting were measured with EEG. However, the spatial resolution of EEG is less precise, in comparison to functional magnetic resonance imaging (fMRI). Thus, one highly experienced practitioner underwent an fMRI scanning session to guide the source localization more precisely. The fMRI data helped guide the selection of EEG source localization, making the calculation of K-means of the EEG source localization in the group of 20 intermediate practitioners more precise and reliable. This method enhanced EEG's ability to identify the brain regions specifically engaged during religious chanting, particularly the cardinal role of the posterior cingulate cortex (PCC). The PCC is a brain area related to focus and self-referential processing. These multimodal neuroimaging and neurophysiological results reveal that repetitive religious chanting can induce lower centrality and higher delta-wave power compared to non-religious chanting and resting state conditions. The combination of fMRI and EEG source analysis provides a more detailed understanding of the brain's response to repetitive religious chanting. The protocol contributes significantly to the research on the neural mechanisms involved in religious and meditative practices, which is becoming more prominent nowadays. The results of this study could have significant implications for developing future neurofeedback techniques and psychological interventions.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141332307","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}
Oriel Ratzon, Mor Schlesinger, Keren Ben-Yaakov, Ortal Zaks, Ziv Rotfogel, Arie L Marcovich, Avital Eisenberg-Lerner
Intracameral injection is a standard administration routine in ophthalmology. The application of intracameral injection in rodents for research is challenging due to the limiting dimensions and anatomy of the eye, including the small aqueous humor volume, the lens curvature, and lens thickness. Potential damage during intracameral injections introduces adverse effects and experimental variability. This protocol describes a procedure for intracameral injection in rats, allowing precision and reproducibility. Sprague-Dawley rats were used as experimental models. Since the lens position in rats protrudes into the anterior chamber, injecting from the periphery, as done in humans, is unfavorable. Therefore, an incision is created in the central corneal region using a 31 gauge 0.8 mm stiletto blade to form a self-sealing tunnel into the anterior chamber. An incision at an angle close to the flat allows to create a long tunnel, which minimizes the loss of aqueous humor and shallowing of the anterior chamber. A 34 gauge nanoneedle is inserted into the tunnel for injection. This enables penetration with minimal friction resistance and avoids touching the lens. Injection of trypan-blue allows visualization by slit microscopy the presence of the dye in the anterior chamber and exclude leakage. Bioavailability to the corneal endothelial layer is demonstrated by injection of Hoechst dye, which stained the nuclei of corneal endothelial cells after injection. In conclusion, this protocol implements a procedure for accurate intracameral injection in rats. This procedure may be used for intracameral delivery of various drugs and compounds in experimental rat models, increasing the efficiency and reproducibility of ophthalmic research.
{"title":"Intracameral Injection in Rats with Low Risk of Adverse Effects.","authors":"Oriel Ratzon, Mor Schlesinger, Keren Ben-Yaakov, Ortal Zaks, Ziv Rotfogel, Arie L Marcovich, Avital Eisenberg-Lerner","doi":"10.3791/66662","DOIUrl":"https://doi.org/10.3791/66662","url":null,"abstract":"<p><p>Intracameral injection is a standard administration routine in ophthalmology. The application of intracameral injection in rodents for research is challenging due to the limiting dimensions and anatomy of the eye, including the small aqueous humor volume, the lens curvature, and lens thickness. Potential damage during intracameral injections introduces adverse effects and experimental variability. This protocol describes a procedure for intracameral injection in rats, allowing precision and reproducibility. Sprague-Dawley rats were used as experimental models. Since the lens position in rats protrudes into the anterior chamber, injecting from the periphery, as done in humans, is unfavorable. Therefore, an incision is created in the central corneal region using a 31 gauge 0.8 mm stiletto blade to form a self-sealing tunnel into the anterior chamber. An incision at an angle close to the flat allows to create a long tunnel, which minimizes the loss of aqueous humor and shallowing of the anterior chamber. A 34 gauge nanoneedle is inserted into the tunnel for injection. This enables penetration with minimal friction resistance and avoids touching the lens. Injection of trypan-blue allows visualization by slit microscopy the presence of the dye in the anterior chamber and exclude leakage. Bioavailability to the corneal endothelial layer is demonstrated by injection of Hoechst dye, which stained the nuclei of corneal endothelial cells after injection. In conclusion, this protocol implements a procedure for accurate intracameral injection in rats. This procedure may be used for intracameral delivery of various drugs and compounds in experimental rat models, increasing the efficiency and reproducibility of ophthalmic research.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141332302","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}
Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and sideline products. Therefore, the selection of B. licheniformis with high production of bacitracin is of great importance. In this experimental protocol, Bacillus with a high yield of bacitracin was isolated, purified, and identified from the fresh feces of healthy pigs. The inhibitory effect of secondary metabolite bacitracin on Micrococcus luteus was also tested. Thin-layer chromatography and high-performance liquid chromatography were used for the qualitative and quantitative detection of bacitracin. The physiological and biochemical characteristics of B. licheniformis were determined by relevant kits. The phylogenetic relationships of B. licheniformis were determined and constructed using gene sequence detection. This protocol describes and introduces the standard isolation, purification, and identification process of B. licheniformis from animal fresh feces from multiple perspectives, providing a method for the large-scale utilization of B. licheniformis and bacitracin in factories.
{"title":"Isolation, Purification, and Identification of Bacitracin-producing Bacillus licheniformis from Fresh Feces of Healthy Pigs.","authors":"Yushu Tang, Ning Hao","doi":"10.3791/66777","DOIUrl":"10.3791/66777","url":null,"abstract":"<p><p>Bacillus licheniformis and bacitracin have a huge application market and value in the fields of medicine, chemistry, aquaculture, agricultural, and sideline products. Therefore, the selection of B. licheniformis with high production of bacitracin is of great importance. In this experimental protocol, Bacillus with a high yield of bacitracin was isolated, purified, and identified from the fresh feces of healthy pigs. The inhibitory effect of secondary metabolite bacitracin on Micrococcus luteus was also tested. Thin-layer chromatography and high-performance liquid chromatography were used for the qualitative and quantitative detection of bacitracin. The physiological and biochemical characteristics of B. licheniformis were determined by relevant kits. The phylogenetic relationships of B. licheniformis were determined and constructed using gene sequence detection. This protocol describes and introduces the standard isolation, purification, and identification process of B. licheniformis from animal fresh feces from multiple perspectives, providing a method for the large-scale utilization of B. licheniformis and bacitracin in factories.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-05-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141332304","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}
Pan Wang, Bin Xia, Zhiyuan Chen, Jiyu Xie, Xinli Sun, Qingyun Tian, Chun-Hui Gao, Weibin Xun, Nan Zhang, Ruifu Zhang, Qirong Shen, Zhihui Xu
The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current understanding of it is limited. Here, we provide an approach to rapidly establish synergistic multispecies biofilm communities. The first step is to extract cells from rhizosphere soil using the differential centrifugation method. Afterward, these soil cells are inoculated into the culture medium to form pellicle biofilm. After 36 h of incubation, the bacterial composition of the biofilm and the solution underneath are determined using the 16S rRNA gene amplicon sequencing method. Meanwhile, high-throughput bacterial isolation from pellicle biofilm is conducted using the limiting dilution method. Then, the top 5 bacterial taxa are selected with the highest abundance in the 16S rRNA gene amplicon sequencing data (pellicle biofilm samples) for further use in constructing multispecies biofilm communities. All combinations of the 5 bacterial taxa were quickly established using a 24-well plate, selected for the strongest biofilm formation ability by the crystal violet staining assay, and quantified by qPCR. Finally, the most robust synthetic bacterial multispecies biofilm communities were obtained through the methods above. This methodology provides informative guidance for conducting research on rhizosphere multispecies biofilm and identifying representative communities for studying the principles governing interactions among these species.
{"title":"An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil.","authors":"Pan Wang, Bin Xia, Zhiyuan Chen, Jiyu Xie, Xinli Sun, Qingyun Tian, Chun-Hui Gao, Weibin Xun, Nan Zhang, Ruifu Zhang, Qirong Shen, Zhihui Xu","doi":"10.3791/66926","DOIUrl":"https://doi.org/10.3791/66926","url":null,"abstract":"<p><p>The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current understanding of it is limited. Here, we provide an approach to rapidly establish synergistic multispecies biofilm communities. The first step is to extract cells from rhizosphere soil using the differential centrifugation method. Afterward, these soil cells are inoculated into the culture medium to form pellicle biofilm. After 36 h of incubation, the bacterial composition of the biofilm and the solution underneath are determined using the 16S rRNA gene amplicon sequencing method. Meanwhile, high-throughput bacterial isolation from pellicle biofilm is conducted using the limiting dilution method. Then, the top 5 bacterial taxa are selected with the highest abundance in the 16S rRNA gene amplicon sequencing data (pellicle biofilm samples) for further use in constructing multispecies biofilm communities. All combinations of the 5 bacterial taxa were quickly established using a 24-well plate, selected for the strongest biofilm formation ability by the crystal violet staining assay, and quantified by qPCR. Finally, the most robust synthetic bacterial multispecies biofilm communities were obtained through the methods above. This methodology provides informative guidance for conducting research on rhizosphere multispecies biofilm and identifying representative communities for studying the principles governing interactions among these species.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141297094","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}
Adam Crabtree, Cory McEvoy, Peter Muench, Rebecca A Ivory, Josh Rodriguez, Mohamed Omer, Trinity Charles, James S Meabon
Exposure to explosive blasts is a significant risk factor for brain trauma among exposed persons. Although the effects of large blasts on the brain are well understood, the effects of smaller blasts such as those that occur during military training are less understood. This small, low-level blast exposure also varies highly according to military occupation and training tempo, with some units experiencing few exposures over the course of several years whereas others experience hundreds within a few weeks. Animal models are an important tool in identifying both the injury mechanisms and long-term clinical health risks following low-level blast exposure. Models capable of recapitulating this wide range of exposures are necessary to inform acute and chronic injury outcomes across these disparate risk profiles. Although outcomes following a few low-level blast exposures are easily modeled for mechanistic study, chronic exposures that occur over a career may be better modeled by blast injury paradigms with repeated exposures that occur frequently over weeks and months. Shown here are methods for modeling highly repetitive low-level blast exposure in mice. The procedures are based on established and widely used pneumatic shocktube models of open-field blast exposure that can be scaled to adjust the overpressure parameters and the number or interval of the exposures. These methods can then be used to either enable mechanistic investigations or recapitulate the routine blast exposures of clinical groups under study.
{"title":"Modeling Highly Repetitive Low-level Blast Exposure in Mice.","authors":"Adam Crabtree, Cory McEvoy, Peter Muench, Rebecca A Ivory, Josh Rodriguez, Mohamed Omer, Trinity Charles, James S Meabon","doi":"10.3791/66592","DOIUrl":"https://doi.org/10.3791/66592","url":null,"abstract":"<p><p>Exposure to explosive blasts is a significant risk factor for brain trauma among exposed persons. Although the effects of large blasts on the brain are well understood, the effects of smaller blasts such as those that occur during military training are less understood. This small, low-level blast exposure also varies highly according to military occupation and training tempo, with some units experiencing few exposures over the course of several years whereas others experience hundreds within a few weeks. Animal models are an important tool in identifying both the injury mechanisms and long-term clinical health risks following low-level blast exposure. Models capable of recapitulating this wide range of exposures are necessary to inform acute and chronic injury outcomes across these disparate risk profiles. Although outcomes following a few low-level blast exposures are easily modeled for mechanistic study, chronic exposures that occur over a career may be better modeled by blast injury paradigms with repeated exposures that occur frequently over weeks and months. Shown here are methods for modeling highly repetitive low-level blast exposure in mice. The procedures are based on established and widely used pneumatic shocktube models of open-field blast exposure that can be scaled to adjust the overpressure parameters and the number or interval of the exposures. These methods can then be used to either enable mechanistic investigations or recapitulate the routine blast exposures of clinical groups under study.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141297102","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}
Henrik Frisk, Gustav Burström, Juliane Weinzierl, Linda Westernhagen, Florentin Tranchant, Erik Edström, Adrian Elmi-Terander
This protocol helps assess the accuracy and workflow of an augmented reality (AR) hybrid navigation system using the Magic Leap head-mounted display (HMD) for minimally invasive pedicle screw placement. The cadaveric porcine specimens were placed on a surgical table and draped with sterile covers. The levels of interest were identified using fluoroscopy, and a dynamic reference frame was attached to the spinous process of a vertebra in the region of interest. Cone beam computerized tomography (CBCT) was performed, and a 3D rendering was automatically generated, which was used for the subsequent planning of the pedicle screw placements. Each surgeon was fitted with an HMD that was individually eye-calibrated and connected to the spinal navigation system. Navigated instruments, tracked by the navigation system and displayed in 2D and 3D in the HMD, were used for 33 pedicle cannulations, each with a diameter of 4.5 mm. Postprocedural CBCT scans were assessed by an independent reviewer to measure the technical (deviation from the planned path) and clinical (Gertzbein grade) accuracy of each cannulation. The navigation time for each cannulation was measured. The technical accuracy was 1.0 mm ± 0.5 mm at the entry point and 0.8 mm ± 0.1 mm at the target. The angular deviation was 1.5° ± 0.6°, and the mean insertion time per cannulation was 141 s ± 71 s. The clinical accuracy was 100% according to the Gertzbein grading scale (32 grade 0; 1 grade 1). When used for minimally invasive pedicle cannulations in a porcine model, submillimeter technical accuracy and 100% clinical accuracy could be achieved with this protocol.
{"title":"Pedicle Screw Placement Using an Augmented Reality Head-Mounted Display in a Porcine Model.","authors":"Henrik Frisk, Gustav Burström, Juliane Weinzierl, Linda Westernhagen, Florentin Tranchant, Erik Edström, Adrian Elmi-Terander","doi":"10.3791/64474","DOIUrl":"https://doi.org/10.3791/64474","url":null,"abstract":"<p><p>This protocol helps assess the accuracy and workflow of an augmented reality (AR) hybrid navigation system using the Magic Leap head-mounted display (HMD) for minimally invasive pedicle screw placement. The cadaveric porcine specimens were placed on a surgical table and draped with sterile covers. The levels of interest were identified using fluoroscopy, and a dynamic reference frame was attached to the spinous process of a vertebra in the region of interest. Cone beam computerized tomography (CBCT) was performed, and a 3D rendering was automatically generated, which was used for the subsequent planning of the pedicle screw placements. Each surgeon was fitted with an HMD that was individually eye-calibrated and connected to the spinal navigation system. Navigated instruments, tracked by the navigation system and displayed in 2D and 3D in the HMD, were used for 33 pedicle cannulations, each with a diameter of 4.5 mm. Postprocedural CBCT scans were assessed by an independent reviewer to measure the technical (deviation from the planned path) and clinical (Gertzbein grade) accuracy of each cannulation. The navigation time for each cannulation was measured. The technical accuracy was 1.0 mm ± 0.5 mm at the entry point and 0.8 mm ± 0.1 mm at the target. The angular deviation was 1.5° ± 0.6°, and the mean insertion time per cannulation was 141 s ± 71 s. The clinical accuracy was 100% according to the Gertzbein grading scale (32 grade 0; 1 grade 1). When used for minimally invasive pedicle cannulations in a porcine model, submillimeter technical accuracy and 100% clinical accuracy could be achieved with this protocol.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141297104","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}
Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside. Detailed characterization of this structure could lead to distinctions between hemostasis and thrombosis, a case of excessive platelet aggregation leading to occlusive clotting. An imaging-based approach to puncture wound thrombus structure is presented here that draws upon the ability of thin-section electron microscopy to visualize the interior of hemostatic thrombi. The most basic step in any imaging-based experimental protocol is good sample preparation. The protocol provides detailed procedures for preparing puncture wounds and platelet-rich thrombi in mice for subsequent electron microscopy. A detailed procedure is given for in situ fixation of the forming puncture wound thrombus and its subsequent processing for staining and embedding for electron microscopy. Electron microscopy is presented as the end imaging technique because of its ability, when combined with sequential sectioning, to visualize the details of the thrombus interior at high resolution. As an imaging method, electron microscopy gives unbiased sampling and an experimental output that scales from nanometer to millimeters in 2 or 3 dimensions. Appropriate freeware electron microscopy software is cited that will support wide-area electron microscopy in which hundreds of frames can be blended to give nanometer-scale imaging of entire puncture wound thrombi cross-sections. Hence, any subregion of the image file can be placed easily into the context of the full cross-section.
{"title":"Puncture Wound Hemostasis and Preparation of Samples for Montaged Wide-Area Electron Microscopy Analysis.","authors":"Kelly Ball, Irina Pokrovskaya, Brian Storrie","doi":"10.3791/66479","DOIUrl":"https://doi.org/10.3791/66479","url":null,"abstract":"<p><p>Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside. Detailed characterization of this structure could lead to distinctions between hemostasis and thrombosis, a case of excessive platelet aggregation leading to occlusive clotting. An imaging-based approach to puncture wound thrombus structure is presented here that draws upon the ability of thin-section electron microscopy to visualize the interior of hemostatic thrombi. The most basic step in any imaging-based experimental protocol is good sample preparation. The protocol provides detailed procedures for preparing puncture wounds and platelet-rich thrombi in mice for subsequent electron microscopy. A detailed procedure is given for in situ fixation of the forming puncture wound thrombus and its subsequent processing for staining and embedding for electron microscopy. Electron microscopy is presented as the end imaging technique because of its ability, when combined with sequential sectioning, to visualize the details of the thrombus interior at high resolution. As an imaging method, electron microscopy gives unbiased sampling and an experimental output that scales from nanometer to millimeters in 2 or 3 dimensions. Appropriate freeware electron microscopy software is cited that will support wide-area electron microscopy in which hundreds of frames can be blended to give nanometer-scale imaging of entire puncture wound thrombi cross-sections. Hence, any subregion of the image file can be placed easily into the context of the full cross-section.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141297106","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}
Ana P Valencia, Gavin Pharaoh, Arthur F Brandao, David J Marcinek
Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.
外周单核细胞(PBMC)的线粒体呼吸能力会随着健康和疾病的变化而发生显著变化。虽然这些变化并不总能反映骨骼肌等其他组织的变化,但这些细胞是人体中可获得的有活力线粒体的宝贵来源。PBMC 暴露于影响其生物能状态的系统信号。因此,扩大我们的工具范围,对这一群体的线粒体代谢进行检测,将有助于阐明与疾病进展相关的机制。线粒体的功能测试通常仅限于使用最大底物、抑制剂和解偶联剂浓度下的呼吸输出来确定全部呼吸能力,而这在体内可能无法实现。ATP 合成酶将二磷酸腺苷(ADP)转化为三磷酸腺苷(ATP)会导致线粒体膜电位(mMP)降低和耗氧量增加。为了对线粒体动力学进行更全面的分析,本文介绍了利用高分辨率荧光呼吸测定法同时测量耗氧量和线粒体膜电位(mMP)对生理相关浓度的 ADP 的响应。该技术使用四甲基罗丹明甲酯(TMRM)测量线粒体膜电位极化对 ADP 滴定的响应,ADP 滴定后线粒体膜电位随复合体 I 和 II 底物的最大超极化而极化。这项技术可用于量化健康状况的变化(如衰老和代谢性疾病)如何影响线粒体对人类受试者的 PBMC、T 细胞和单核细胞的能量需求反应的敏感性。
{"title":"High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells.","authors":"Ana P Valencia, Gavin Pharaoh, Arthur F Brandao, David J Marcinek","doi":"10.3791/66863","DOIUrl":"10.3791/66863","url":null,"abstract":"<p><p>Peripheral mononuclear cells (PBMCs) exhibit robust changes in mitochondrial respiratory capacity in response to health and disease. While these changes do not always reflect what occurs in other tissues, such as skeletal muscle, these cells are an accessible and valuable source of viable mitochondria from human subjects. PBMCs are exposed to systemic signals that impact their bioenergetic state. Thus, expanding our tools to interrogate mitochondrial metabolism in this population will elucidate mechanisms related to disease progression. Functional assays of mitochondria are often limited to using respiratory outputs following maximal substrate, inhibitor, and uncoupler concentrations to determine the full range of respiratory capacity, which may not be achievable in vivo. The conversion of adenosine diphosphate (ADP) to adenosine triphosphate (ATP) by ATP-synthase results in a decrease in mitochondrial membrane potential (mMP) and an increase in oxygen consumption. To provide a more integrated analysis of mitochondrial dynamics, this article describes the use of high-resolution fluorespirometry to measure the simultaneous response of oxygen consumption and mitochondrial membrane potential (mMP) to physiologically relevant concentrations of ADP. This technique uses tetramethylrhodamine methylester (TMRM) to measure mMP polarization in response to ADP titrations following maximal hyperpolarization with complex I and II substrates. This technique can be used to quantify how changes in health status, such as aging and metabolic disease, affect the sensitivity of mitochondrial response to energy demand in PBMCs, T-cells, and monocytes from human subjects.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11257029/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141297100","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}
Oscar Moreno, Catherine E Luke, Amber Clay, Laura Durham, Sabrina Rocco, Kiran Kumar, Nathaniel Parchment, David E Babcock, Daniel D Myers, Thomas W Wakefield, Peter Henke, Andrea T Obi
Balloon venoplasty is a commonly used clinical technique to treat deep vein stenosis and occlusion as a consequence of trauma, congenital anatomic abnormalities, acute deep vein thrombosis (DVT), or stenting. Chronic deep venous obstruction is histopathologically characterized by thrombosis, fibrosis, or both. Currently, no direct treatment is available to target these pathological processes. Therefore, a reliable in vivo animal model to test novel interventions is necessary. The rodent survival inferior vena cava (IVC) venoplasty balloon model (VBM) allows the study of balloon venoplasty in non-thrombotic and post-thrombotic conditions across multiple time points. The local and systemic effect of coated and uncoated venoplasty balloons can be quantified via tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent assay (ELISA), zymography, vein wall and thrombus cellular analysis, whole blood and plasma assays, and histological analysis. The VBM is reproducible, replicates surgical human interventions, can identify local vein wall-thrombi protein changes, and allows multiple analyses from the same sample, decreasing the number of animals required per group.
{"title":"Rodent Inferior Vena Cava Venoplasty Balloon Model.","authors":"Oscar Moreno, Catherine E Luke, Amber Clay, Laura Durham, Sabrina Rocco, Kiran Kumar, Nathaniel Parchment, David E Babcock, Daniel D Myers, Thomas W Wakefield, Peter Henke, Andrea T Obi","doi":"10.3791/66820","DOIUrl":"https://doi.org/10.3791/66820","url":null,"abstract":"<p><p>Balloon venoplasty is a commonly used clinical technique to treat deep vein stenosis and occlusion as a consequence of trauma, congenital anatomic abnormalities, acute deep vein thrombosis (DVT), or stenting. Chronic deep venous obstruction is histopathologically characterized by thrombosis, fibrosis, or both. Currently, no direct treatment is available to target these pathological processes. Therefore, a reliable in vivo animal model to test novel interventions is necessary. The rodent survival inferior vena cava (IVC) venoplasty balloon model (VBM) allows the study of balloon venoplasty in non-thrombotic and post-thrombotic conditions across multiple time points. The local and systemic effect of coated and uncoated venoplasty balloons can be quantified via tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent assay (ELISA), zymography, vein wall and thrombus cellular analysis, whole blood and plasma assays, and histological analysis. The VBM is reproducible, replicates surgical human interventions, can identify local vein wall-thrombi protein changes, and allows multiple analyses from the same sample, decreasing the number of animals required per group.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":null,"pages":null},"PeriodicalIF":1.2,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141297072","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}