Jove-Journal of Visualized Experiments最新文献

Most neurobiological studies are conducted on laboratory rodents. Despite many similarities across mammalian brains, important differences also exist, which can be misleading in translation. Marked interspecies differences have been found in brain plasticity, particularly neurogenesis. Different neurogenic processes can be prevalent because of evolutionary tradeoffs, displaying variation across brain structures and mammals and shifting the potential for plasticity in divergent species, such as mice and humans. Comparing widely different species raises multiple issues: comparative studies encounter technical difficulties when large-sized brains are involved; the use of heterogeneous experimental approaches by different laboratories can limit the comparison of results; heterogeneity may be related to different time courses of neurodevelopmental processes across mammals, thus adding variables to the comparison. To tackle these limitations, an approach to study the interspecies variation of a population of layer II cortical immature neurons in mammals widely differing for brain size, gyrencephaly, socioecological niche, and age was established. Despite some variables that cannot be fully standardized, a method that combines reduced heterogeneity in collecting brains and the establishment of common anatomical structures as reference points for performing the cell counting on corresponding brain levels is proposed. Data obtained (e.g., cell densities) can be mapped onto phylogenetic trees to reveal evolutionary patterns and analyzed for covariance with neuroanatomical features (e.g., brain size, cortical surface area). This approach has demonstrated remarkable variation in the number of cortical immature neurons between phylogenetic groups and uncovered covariation with brain size. The method can also be used to quantify differences through different developmental stages in the same species and can be extended to other diverse mammals and biological processes to map comparable results that allow for more accurate quantification of different cell populations in adult brains to support plasticity.

Diabetes mellitus (DM) is a widespread chronic metabolic illness around the world, and prolonged hyperglycemia not only causes a variety of problems but also affects metabolic and immunological activities, compromising patients' nutritional status. The Listen-Establish-Adopt-Reinforce-Name-Strengthen (LEARNS) nursing paradigm, based on a patient-centered concept, is a new strategy that aims to foster self-directed learning, improve nurse-patient engagement, increase self-care awareness, and encourage two-way communication. This study looked at the effect of LEARNS nursing on the nutritional status and general health indices of older persons with DM. A total of 168 hospitalized patients aged 60 years or older were involved, with 89 in the LEARNS group and 79 receiving standard treatment. Propensity score matching was used to reduce confounding bias, and the intervention lasted four weeks. After the intervention, patients in the LEARNS group had significantly improved glycemic markers, including reduced HbA1c, FPG, and 2hPG values (P < 0.05). Self-management capacity improved, as evidenced by higher scores on the Chinese Diabetes Management Self-Efficacy Scale. Nutritional indicators such as serum albumin (ALB), hemoglobin (HGB), total serum protein (TSP), and transferrin (TRF) increased, whereas the proportion of high-risk patients identified by the PG-SGA malnutrition score dropped. Quality-of-life assessments also showed improvement, with higher Health-Promoting Lifestyle Profile (HPLP-II) and lower Pittsburgh Sleep Quality Index (PSQI) ratings. The LEARNS group had a considerably higher patient satisfaction rate (92.13%) compared to the control group (P < 0.05). Overall, the data demonstrate that the LEARNS nursing model improves nutritional status, self-management efficacy, and quality of life in senior diabetic patients while also improving glucose control. The study's shortcomings include a single-center design and a short follow-up time. More studies with larger samples and longer periods of observation are needed to confirm long-term effects.

HER2-positive breast cancer is clinically aggressive and frequently disseminates to regional lymph nodes, yet the transcriptomic programs associated with nodal spread remain incompletely defined. Here, we profiled matched tumor-draining lymph nodes (TDLN) and metastatic tumor-involved lymph nodes (TMLN) from six patients with HER2-positive breast cancer using RNA sequencing (RNA-seq). Differential expression analysis was performed with DESeq2, applying Benjamini-Hochberg correction (FDR < 0.05) and an absolute log2 fold-change threshold (|log2FC| ≥ 1). We identified 237 differentially expressed genes (182 upregulated and 55 downregulated in TMLN vs. TDLN). Functional enrichment analyses highlighted extracellular matrix (ECM)-receptor interaction, PI3K-AKT signaling, focal adhesion, and cholesterol metabolism as prominent pathways associated with metastatic lymph nodes, suggesting coordinated ECM remodeling and signaling activation during nodal dissemination. Among the most upregulated genes, POSTN emerged as a key candidate and was further linked to adverse clinicopathologic characteristics. In an independent validation cohort (n = 120), higher POSTN expression was associated with inferior disease-free survival. Collectively, these results nominate POSTN as a potential prognostic biomarker and support ECM-centered and PI3K-AKT-related mechanisms as actionable biological features of lymph-node metastasis in HER2-positive breast cancer.

Surgical intervention has demonstrated established efficacy for benign, borderline, and low-grade malignant tumors of the pancreatic head, as well as for chronic pancreatitis. While conventional surgical approaches are associated with significant trauma and considerable loss of digestive function, the current focus in managing these conditions has shifted toward preserving functional organs through minimally invasive techniques. Through an in-depth understanding of the peripancreatic vascular arches, our team has modified the classic Beger procedure to achieve complete en bloc resection of pancreatic head tissue. By leveraging 3D laparoscopic technology, we have successfully integrated the advantages of minimal invasiveness and precise resection. This refined technique preserves duodenal and biliary integrity while ensuring complete removal of pancreatic head tissue, effectively reducing surgical complications, minimizing operative trauma, and decreasing postoperative gastrointestinal dysfunction. Consequently, patient outcomes are substantially improved. This article aims to elucidate the key technical details of this procedure, enabling more surgeons to master and enhance its success rate, thereby providing better treatment options for eligible patients.

Sepsis is characterized by a dysregulated host immune response and remains a leading cause of mortality worldwide. Long non-coding RNA NEAT1 has been implicated in inflammatory diseases, but its specific role in macrophage polarization during sepsis has not been fully defined. Here, we systematically examine the NEAT1/miR-181a-5p/HMGB1 axis across clinical samples, cultured macrophages, and a CLP mouse model. Quantitative PCR, western blotting, dual-luciferase reporter assays, and RNA pull-down experiments are used to confirm the competitive endogenous RNA (ceRNA) interaction among NEAT1, miR-181a-5p, and HMGB1. Functional assays, including immunofluorescence, transwell migration, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, are applied to assess macrophage polarization, migration, and apoptosis. In vivo, the CLP model combined with ELISA and histopathology validates the impact of NEAT1 knockdown on cytokine profiles and organ injury. NEAT1 and HMGB1 are upregulated, whereas miR-181a-5p is downregulated, in patients with sepsis and in lipopolysaccharide-stimulated macrophages. Silencing NEAT1 promotes M2 macrophage polarization, reduces pro-inflammatory cytokines, impairs macrophage migration, and alleviates tissue damage in septic mice via the miR-181a-5p/HMGB1 axis. To our knowledge, this is the first integrated protocol to characterize the lncRNA-microRNA-HMGB1 regulatory circuit in sepsis using harmonized clinical, in vitro, and in vivo approaches. It provides a methodological framework for targeting NEAT1-related ceRNA networks as potential therapeutic strategies.

Stable isotope methods for estimating vitamin A total body stores (TBS) quantitatively are based on the principle of isotope dilution. Briefly, a single oral dose ²H- or ¹³C-labeled vitamin A is administered to an individual, and plasma concentrations of labeled and unlabeled retinol are measured by mass spectrometry at a pre-specified time after dosing, usually 14-21 days. TBS is calculated using the retinol isotope dilution (RID) equation or the mass balance equation. The RID and mass balance equations require information on retinol specific activity in plasma (SAp; i.e., tracer-to-tracee ratio), which is obtained from mass spectrometry measurements. Both equations require values for coefficients to account for absorption and storage of the oral dose of stable isotope-labeled retinol at the time of TBS estimation. Published values for the coefficients can be used in the equations. Alternatively, TBS can be determined by model-based compartmental analysis of plasma retinol kinetic data using WinSAAM Simulation, Analysis, and Modeling software. Briefly, a super-subject study design with model-based compartmental analysis of plasma retinol kinetic data can be used to determine group TBS and population-specific values for the composite coefficient (FaS), which is subsequently used in the RID equation to determine individual TBS. The super-subject design requires an estimate of mean dietary vitamin A intake for the group of participants, and blood sampling at ~11-16 time points over ~28-91 d, with 5-7 participants/time point, but each participant provides only 2-3 blood samples. Liver vitamin A concentration can be estimated from TBS, using an assumption for the proportion of TBS found in liver and an estimate of liver weight. Vitamin A status is assessed by comparing estimated liver vitamin A concentration with proposed cutoff values for categorizing status across the full continuum, from deficient to excess vitamin A stores.

Brain metastases are a common and devastating complication of advanced solid tumors, frequently associated with poor prognosis, neurological decline, and reduced quality of life. The incidence of central nervous system (CNS) failure and neurological death is rapidly increasing, yet the mechanisms driving the final stages of brain metastasis, such as secondary dissemination, re-colonization, and the contribution of the histological growth pattern (HGP) as a potential surrogate parameter, remain poorly understood. The standardized stereotactic intracortical injection model enables precise and reproducible implantation of tumor cells, or mixed populations including stromal or immune components, directly into the mouse cerebral cortex. This protocol also supports the creation of a preclinical tissue archive, offering a robust platform for investigating essential aspects of CNS colonization, such as: metastatic outgrowth, HGP-specific growth dynamics, and pathophysiological mechanisms contributing to neurological failure. Additionally, this model supports pharmacological testing in a reproducible clinically relevant context. Unlike systemic injection methods (e.g., tail vein or intracardiac), which are optimized for studying early metastatic steps but result in variable and often low rates of brain colonization, the stereotactic model ensures consistent, brain-specific metastatic growth and enables the investigation of late stages of CNS metastasis. Compared to ex vivo systems such as organoids or brain slice cultures, the in vivo stereotactic model preserves vascularization, systemic signaling, and the full complexity of the brain's immune landscape, supporting long-term studies of tumor progression and therapeutic response. By providing a reproducible and clinically relevant platform, our model advances the field's ability to identify prognostic markers, explore therapeutic strategies, and understand the mechanisms of late-stage brain metastasis.

This protocol presents a Gaussian Process Regression (GPR) optimization framework for human-powered electricity generation systems to address technological unemployment and renewable energy demands simultaneously. Seven participants performed 112 trials across 16 configurations combining four battery voltages (12 V, 24 V, 36 V, 48 V) with four electrical loads (10 W, 30 W, 50 W, 70 W) using a modified stationary bicycle generator. The GPR model incorporated automatic relevance determination (ARD) squared exponential kernels to map pedaling speed, pressure, voltage, and load parameters to generation efficiency while quantifying prediction uncertainty. Real-time data acquisition at 2 Hz captured mechanical and electrical parameters through Hall-effect sensors, strain gauges, and power analyzers with Modbus communication protocols. The optimized system achieved 80%-90% theoretical maximum efficiency with coefficient of variation (CoV) below 15%, compared to 60%-70% efficiency and 25%-35% variability in commercial systems. GPR predictions demonstrated R² = 0.713 with sub-10ms latency through sparse approximation techniques, enabling real-time control. Optimal operational parameters varied systematically: low loads required 150-180 revolutions per minute (RPM) at 50-70 N pressure, while higher loads peaked at 100-120 RPM with 100-150 N pressure. The 48 V configuration improved efficiency by 68% over 12 V baseline (measured as relative efficiency index, REI, normalized to baseline = 100%). Phase-specific monitoring identified fatigue onset at 35-40 min, triggering risk-aware control adjustments based on posterior uncertainty. The protocol establishes quantitative methods for balancing energy generation with physiological constraints, providing a reproducible framework for deploying human-powered systems in fitness facilities where break-even occurs at $0.18/kWh electricity pricing.

Microglia are the resident macrophages of the central nervous system (CNS) that respond to tissue infection and injury. In addition to their role in inflammation, microglia play a developmental role in circuit refinement through synaptic pruning. However, the mechanisms of synaptic pruning in neuroinflammation and neurodegeneration remain unknown. In this protocol, we use a mouse retina explant model to study microglia dynamics ex vivo. To examine microglia motility and their interactions with postsynaptic proteins, we label synapses with AAV-PSD95-RFP and record timelapse videos of motile microglia colocalized with postsynaptic proteins using spinning disk confocal microscopy. We then create surface and spot reconstructions of microglia and PSD95 using image analysis software. Data such as microglia displacement length, process speed, and contact with postsynaptic puncta can then be extracted from these surfaces to understand microglia behavior both in homeostatic states and after neuronal injury. This protocol can be useful in examining the role of microglia in synaptic pruning in retinal neurodegenerative diseases.

Subscapularis tendon injuries often pose substantial challenges in arthroscopic repair, with traditional techniques frequently limited by prolonged surgical time, uneven tension distribution, and increased complication risk. Although advanced suture-passing and suturing methods can improve biomechanical outcomes, they generally require complex instrumentation. A hybrid approach is presented here that integrates a continuous sewing machine-like suture technique with a single-portal spinal needle method for subscapularis repair. The sewing machine-like technique facilitates efficient loop formation, which ensures even tension distribution and allows precise suture placement through flexible puncture points. The single-portal approach minimizes invasiveness, reduces tissue trauma, and streamlines the surgical process. Collectively, these techniques enhance the biomechanical strength, shorten operative time, and align with the principles of modern minimally invasive surgery. This improved and innovative strategy represents a promising alternative to conventional methods, with the potential to improve clinical outcomes. Further clinical validation and long-term follow-up are required to confirm its efficacy.