Jove-Journal of Visualized Experiments最新文献

The therapeutic effectiveness of acupuncture relies on both safety and stability, making these factors essential in acupuncture manipulation research. However, manual manipulation introduces unavoidable inaccuracies, which can impact the reliability of research findings. To address this challenge, a unique lifting and thrusting manipulation control cannula was designed in this study, offering flexible adjustment of movement amplitude. The cannula was created using 3D printing technology, and its effectiveness in maintaining stability was verified by recording the acupuncture needle's movement range with optical sensor technology. The study's results show that the control cannula significantly enhances the stability of acupuncture manipulation, reducing human error. This innovation suggests that the cannula could serve as a valuable auxiliary tool for ensuring both the precision and safety of acupuncture-related experimental research. Its adoption could also contribute to the standardization of acupuncture practices, ensuring more consistent and accurate research outcomes, which is essential for future advancements in acupuncture research and clinical applications.

For noninvasive light-based physiological monitoring, optimal wavelengths of individual tissue components can be identified using absorption spectroscopy. However, because of the lack of sensitivity of hardware at longer wavelengths, absorption spectroscopy has typically been applied for wavelengths in the visible (VIS) and near-infrared (NIR) range from 400 to 1,000 nm. Hardware advancements in the short-wave infrared (SWIR) range have enabled investigators to explore wavelengths in the ~1,000 nm to 3,000 nm range in which fall characteristic absorption peaks for lipid, protein, and water. These molecules are difficult to visualize in the VIS-NIR and can provide label-free sources of biological contrast. Furthermore, lower SWIR absorption has been observed for melanin, the primary chromophore responsible for skin pigmentation. In vivo optical devices like clinically standard pulse oximeters have been found to have reduced accuracy in people with darkly pigmented skin, possibly because of the stronger melanin absorption in the VIS range. Thus, error associated with skin pigmentation could be reduced by using devices operating in the SWIR. Optical instrument design is facilitated by the understanding of the absorption properties of core tissue components from the VIS to the SWIR range. This article describes protocols and instrumentation for obtaining VIS-SWIR absorption spectra of common tissue absorbers: oxygenated hemoglobin, deoxygenated hemoglobin, melanin, water, and lipid.

Umbilical cord-derived mesenchymal stromal/stem cells (UC-MSCs) present low immunogenicity and potent immunomodulatory effects for treating various diseases. Human UC-MSCs are a heterogeneous population consisting of three main subpopulations with different cell shapes, proliferation rates, differentiation abilities, and immune regulatory functions. Previously, BAMBI^highMFGE8^high UC-MSCs, the first subgroup successfully isolated from UC-MSCs were found to fail to alleviate lupus nephritis. Hence, the function and underlying mechanism of this subgroup in MSC therapy for diseases remains unknown. It is necessary to isolate and further investigate BAMBI^highMFGE8^high UC-MSCs in terms of their phenotype, metabolism, and function to completely understand the nature of this MSC subgroup. In this protocol, we describe a detailed method for isolating the BAMBI^highMFGE8^high subpopulation from human UC-MSCs. The subpopulation of UC-MSCs is labeled with two surface markers, BAMBI and MFGE8, by flow cytometry sorting. The isolated cells are cultured and verified by flow cytometry analysis. The specific genes expressed in the BAMBI^highMFGE8^high UC-MSCs are identified by RT-qPCR. This protocol results in highly efficient and pure cell sorting and describes the marker profiles of the BAMBI^highMFGE8^high UC-MSCs.

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Chronic wounds, due to their high prevalence, are a serious global health concern. Effective therapeutic strategies can significantly accelerate healing, thereby reducing the risk of complications and alleviating the economic burden on healthcare systems. Although numerous experimental studies have investigated wound healing, most rely on qualitative observations or quantitative direct measurements. The objective of this study was to standardize an indirect wound measurement method using digital planimetry, incorporating digital scaling and segmentation. This approach addresses the lack of detailed, step-by-step methodologies for accurate wound assessment. A photodocumentation booth was designed and constructed, and computer-assisted digital planimetry tools were employed to minimize variability in measurements of the wound area, perimeter, and the distance from the wound center to its edges. A circular traumatic wound (5 mm in diameter) was created on the dorsal midline at the shoulder blade level of male CD1 mice (n = 4, 10 weeks old, 30-35 g). Wound evolution was photodocumented for 14 days using the custom-designed photo booth, which controlled lighting conditions, focal distance, and subject positioning. Scaling and wound measurements were performed using segmentation in ImageJ software, and statistical analysis was conducted using statistical analysis software. The kinetics of wound closure showed a slight increase in wound size and perimeter between day 0 and day 2, followed by a gradual decrease until complete closure by day 14. The photodocumentation booth and computer-assisted digital planimetry enabled quantitative measurements with minimal variability. In conclusion, these tools provide a reliable and reproducible method for evaluating wound closure kinetics in pre-clinical models.

Transgender (TG) people are individuals whose gender identity and sex assigned at birth do not match. They often undergo gender-affirming hormone therapy (GAHT), a medical intervention that allows the acquisition of secondary sex characteristics more aligned with their individual gender identity, providing consistent results in the improvement of numerous socio-psychological variables. However, GAHT targets different body systems, and some side effects are recorded, although not yet fully identified and characterized. Therefore, TG people undergoing GAHT may be considered as a susceptible sub-group of population and specific attention should be paid in the frame of risk assessment, e.g., through the use of targeted animal models. The present work describes the procedures set to implement two rat models mimicking GAHT: the demasculinizing-feminizing model (dMF) mimicking the GAHT for TG women and the defeminizing-masculinizing model (dFM) mimicking the GAHT for TG men. The models have been implemented through the administration of the same hormones used for human GAHT, namely, β-estradiol plus cyproterone acetate for dMF and testosterone for dFM, by the same routes of exposure for a 2 week period. Rats are checked daily during the treatment to evaluate health status and potentially aggressive behaviors. At sacrifice, blood and target tissues have been sampled and stored for biochemical, molecular, and histopathological analysis. Sex-specific parameters, namely, sperm count and clitoral dimensions, have also been evaluated. In addition, CYP450 isoforms, exclusively and/or preferentially expressed in male and female rat liver, are identified and characterized as novel biomarkers to verify the success of GAHT and to set the model. Thyroid involvement has also been explored as a key target in the endocrine system.

Cardiovascular disease (CVD) is the leading cause of death in the United States. Damage in the cardiovascular system can be due to environmental exposure, trauma, drug toxicity, or numerous other factors. As a result, cardiac tissue and vasculature undergo structural changes and display diminished function. The damage and the resulting remodeling can be detected and quantified with ultrasound (US) imaging at the organ level and mass spectrometry imaging (MSI) at the molecular level. This manuscript describes an innovative methodology for studying murine cardiac pathophysiology, coupling in vivo four-dimensional (4D) ultrasound imaging and analysis with ex vivo matrix-assisted laser desorption/ionization (MADLI) MSI of the heart. 4D ultrasound can provide dynamic volumetric measurements, including radial displacement, surface area strain, and longitudinal strain throughout an entire cardiac cycle. In the vasculature, MSI and ultrasound are used to assess vessel wall compositions, hemodynamics, and vessel wall dynamics. The methodology can be tailored to study a myriad of CV diseases by adjusting functional metrics of interest and/or varying MALDI MSI protocol to target specific molecules. MALDI MSI can be used to study lipids, small metabolites, peptides, and glycans. This protocol outlines the use of MALDI MSI for untargeted lipidomic analysis and the use of ultrasound imaging for cardiovascular hemodynamics and biomechanics.

Platelets are blood cells that play an integral role in hemostasis and the innate immune response. Platelet hyper- and hypoactivity have been implicated in metabolic disorders, increasing risk for both thrombosis and bleeding. Platelet activation and metabolism are tightly linked, with the numerous methods to measure the former but relatively few for the latter. To study platelet metabolism without the interference of other blood cells and plasma components, platelets must be isolated, a process that is not trivial because of platelets shear sensitivity and ability to irreversibly activate. Presented here is a protocol for platelet isolation (washing) that produces quiescent platelets that are sensitive to stimulation by platelet agonists. Successive centrifugation steps are used with the addition of platelet inhibitors to isolate platelets from whole blood and resuspend them in a controlled, isosmotic buffer. This method reproducibly produces 30%-40% recovery of platelets from whole blood with low activation as measured by markers of granule secretion and integrin activity. Platelet count and fuel concentration can be precisely controlled to allow the user to probe a variety of metabolic situations.

The objective of this study was to investigate the cardioprotective effects of Munziq on abnormal body fluid myocardial ischemia-reperfusion injury (MIRI) and its underlying mechanism.Normal rats and rats with abnormal body fluid (ABF) were pre-treated with Munziq for 21 days. Following this, MIRI models were established. Histopathological changes and myocardial ultrastructure changes were observed by Hematoxylin and Eosin (HE)staining and transmission electron microscopy to observe pathological manifestations of myocardial injury. Serum CK-MB, cTn-T, and ICAM-1 levels were detected by Enzyme-Linked Immunosorbent Assay (ELISA) to observe myocardial injury-related markers. The levels of IL-1β, IL-6, and TNF-α in serum and myocardial tissue were also detected by ELISA to observe the anti-inflammatory effect. The expression levels of NF-κB signaling pathway-related proteins NIK, IKKα, Pikα, and p65 were detected by Western blot analysis. The results showed that myocardial injury in the ABF MIRI group was more severe compared to the control MIRI group. Munziq pretreatment has the potential to mitigate the pathological changes induced by ischemia-reperfusion injury and could protect cardiac function. Protein levels of the NF-κB pathway and downstream effectors IL-1β, IL-6, and TNF-α were significantly up-regulated in the MIRI group while down-regulated in the Munziq group. Interestingly, there was more activation of the NF-κB signaling pathway and higher levels of downstream inflammatory cytokines in the ABF MIRI group. The results suggest that MIRI was more severe in ABF. Munziq has cardioprotective effects in ischemia and reperfusion injury. This protective effect may be acted by suppressing the NF-κB signaling pathway.

The iChip isolation technique uses an in-situ isolation device that increases the cultivability of previously unculturable microorganisms. Microorganisms are an important source of novel chemistries and potentially bioactive molecules. However, only 1% of environmental microorganisms can be cultured using conventional laboratory methods. With the rise in antimicrobial resistance, finding new drugs to combat infections and diseases is of foremost importance, and a critical method to finding new molecules is the discovery of new microorganisms. By incubating colonies of soil microorganisms in the wells of a 96-well plate, sealed with a semipermeable membrane and incubated on top of soil, the microbes are in contact with water and growth factors from the soil, allowing for the isolation of novel microbes in a laboratory setting. After a period of domestication in an iChip, microorganisms can potentially be subcultured onto conventional media and used for further study. This device is valuable to bioactive molecule discovery and soil microbiome research and has been used previously in both applications.