Journal of Visualized Experiments

This protocol describes manual whole-endolysosome patch-clamp recordings from pharmacologically or genetically enlarged endolysosomes in cultured mammalian cells. Steps include vesicle enlargement, fabrication and fire-polishing of high-resistance pipettes, mechanical dissection and isolation of enlarged vesicles, giga-seal formation, and configuration to whole-endolysosome modes. For complete details on the use and execution of this protocol, please refer to Wang et al1.

Quantitative analysis of action potential (AP) waveforms is central to studies of neuronal excitability, ion channel function, disease mechanisms, and pharmacological modulation. However, AP analysis is still often performed using partially manual workflows, laboratory-specific spreadsheets, or proprietary software environments that can limit reproducibility, transparency, and throughput. Here we present APpar, a freely available, open-source software tool for extracting AP parameters, developed for use with the OriginLab software package Origin/OriginPro. APpar detects APs from membrane voltage recordings using a user-defined derivative criterion and calculates a comprehensive set of excitability parameters, including resting membrane potential, AP threshold, dV/dt at threshold, overshoot, undershoot, AP amplitude, AP half-amplitude, rise time, decay time, AP duration, AP half-width, AP width at 0 mV, AP area above voltage threshold, dV/dtMAX, dV/dtMIN, interspike interval for the respective AP. Because AP threshold is a particularly sensitive and method-dependent measurement, APpar includes a TRUE-threshold validation algorithm. After the initial forward dV/dt threshold crossing is identified, the software finds AP overshoot, searches backward to the closest preceding local dV/dt maximum, then searches backward to the user-defined dV/dt crossing and recalculates AP parameters from this validated threshold point. We validated APpar using APs from dorsal root ganglion neurons current-clamp recordings, including copied identical APs, current-evoked repetitive firing, and long-duration spontaneous firing. The software produced stable measurements from identical copied APs and extracted dynamic changes in AP parameters across repetitive and spontaneous firing sequences. APpar provides a transparent, customizable, and Origin-compatible framework for reproducible AP analysis in neuronal electrophysiology. Significance statementAction potential waveform analysis is essential for interpreting neuronal excitability, but many AP measurements remain vulnerable to user-dependent threshold placement, manual cursor selection, and inconsistent parameter definitions. APpar, a freely available, open-source software tool, addresses this problem by automating AP detection and parameter extraction within the OriginLab environment widely available to electrophysiology laboratories. The software formalizes definitions of AP threshold, amplitude, duration, half-width, afterhyperpolarization, derivative-based parameters, and firing metrics, and introduces a TRUE-threshold validation algorithm that recalculates AP parameters from a derivative-validated threshold point. This workflow reduces operator-dependent variability while preserving user control over physiologically meaningful detection criteria. HighlightsAutomated action potential waveform analysis within OriginLab Origin environments AP threshold validation improves reproducibility of derivative-based threshold detection Extracts action potential kinetics, amplitudes, widths, and dV/dt measurements Open-source workflow supports reproducible neuronal electrophysiology data analysis Validated using repetitive and spontaneous firing in DRG neurons

The patch-clamp experimental technique is widely used to study the electrical properties of ion channels in biological and artificial lipid membranes. The key to the high quality of the experiments is the manufacturing of glass pipettes that provide highly electrically resistant contact between the edge of the pipette tip and the lipid bilayer. Preparation of the pipettes is particularly challenging for studies of the mitochondrial membranes due to the need for very small pipette tip sizes. Here, we present a robust procedure for producing pipettes suitable for experiments with native mitochondrial membranes. This procedure involves a two-step approach: initial fabrication of relatively large glass micropipettes using a standard micropipette puller, followed by tip refinement using a microforger to achieve smooth glass surface and reduced opening size. Pipette tip diameters and surface structure were examined using field emission - scanning electron microscopy (FE-SEM) imaging to assess the effects of variable parameters on pipette geometry and size. The resulting pipettes were validated in patch-clamp recording of the mitochondrial inner membranes. This approach enables the reproducible production of optimized pipettes for mitochondrial patch-clamp experiments, improving the quality and throughput of electrophysiological recordings of the mitochondrial ion channels.

CaMKII promoter is widely used to label and manipulate hippocampal pyramidal neurons via transgenic mouse lines or viral approaches. While it targets most excitatory neurons, a small subset remains unlabeled and often overlooked. We present an AAV-based strategy combined with CaMKII-driven Cre expression to access and study this remaining population. Furthermore, we provide a detailed protocol for in-house AAV production, targeted stereotaxic delivery, and functional validation of targeted neurons through slice electrophysiology and behavior. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/723440v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@3a31ccorg.highwire.dtl.DTLVardef@9b7e90org.highwire.dtl.DTLVardef@92297borg.highwire.dtl.DTLVardef@1e159eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

This protocol describes the workflow for the preparation of tissue samples for proteome and phosphoproteome analyses using mass spectrometry. The tissue samples are cryogenically pulverized and homogenized in a sucrose-based buffer to ensure proper tissue disruption. For depletion of lipid contaminants, proteins are purified using chloroform-methanol precipitation, followed by a resuspension in a urea-based buffer for enzymatic digestion. Peptides are desalted and enriched for phosphopeptides prior LC-MS/MS analysis. The workflow was developed for skin biopsies but is compatible with a broad range of tissue types. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=51 SRC="FIGDIR/small/732915v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@6a44aforg.highwire.dtl.DTLVardef@c34dc9org.highwire.dtl.DTLVardef@27d7ecorg.highwire.dtl.DTLVardef@1d1038e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

IntroductionIntraoperative glycemic dysregulation, including unrecognized hypoglycemia and stress-induced hyperglycemia, is common during elective surgery. Conventional point-of-care (POC) monitoring provides only intermittent measurements, limiting the anesthesiologists ability to detect rapid glucose fluctuations. Continuous glucose monitoring (CGM) enables real-time, trend-based assessment, potentially shifting intraoperative glycemic management from reactive to proactive. ObjectiveTo meta-analyze the analytical accuracy, intraoperative glycemic efficacy, and feasibility of subcutaneous CGM in adults undergoing elective surgery, informing anesthesiology practice. MethodsThis systematic review and meta-analysis followed the PRISMA 2020 statement. Searches were conducted in PubMed, Embase, and Cochrane Central Register of Controlled Trials from January 2010 to May 2025. Eligible studies included randomized controlled trials and prospective cohorts of adults undergoing elective surgery under general or neuraxial anesthesia using subcutaneous CGM. Primary outcomes were pooled mean absolute relative difference (MARD) and time in range (TIR, 70-180 mg/dL). Random-effects models were applied. ResultsTen studies (3 RCTs, 7 cohorts; N=557) were included. Pooled MARD was 14.1% (95% CI 11.3-16.9%; I{superscript 2}=78%), lower in non-cardiac surgery (12.7%) than cardiac procedures with hypothermia (19.2%; p=0.03). CGM improved TIR by +14.9 percentage points (95% CI 7.2-22.6; p<0.001). Clinically significant hypoglycemia was detected in 43% of patients, all missed by POC. Sensor availability exceeded 96%, with no serious device-related events. ConclusionSubcutaneous CGM provides acceptable intraoperative accuracy and improves glycemic control, supporting its integration into anesthetic management.

O_LIThe effect of some bioactive compounds on living organisms is dependent on their concentration and gradients, as is the case of hormones and signalling peptides, determining cell identity, activity and organism development. C_LIO_LIThere are a handful of methods that allow to produce spatially confined peaks of concentration local application of biochemicals on plants, such as agar blocks and microinjection, but they lack in precision, throughput and/or simplicity. C_LIO_LIWe developed the MicroTron, a microfluidics-based method specifically for filamentous organisms or life cycle stages, like the moss plant Physcomitrium patens protonemata, that serves as a platform for the application of chemicals on single cells and study the cell response. C_LIO_LIWe show how chemical applications could be performed on cells, either on the side or apically with dyes and hormones, targeting the cell wall, cell membrane, cytosol and nucleus. C_LIO_LITreatments could be applied on single filaments and with a precision of up to single cells in optimal conditions. C_LIO_LIThis method could be used to study live responses to chemicals with high spatiotemporal resolution. C_LI

Apolipoprotein E (ApoE) is the strongest genetic predictor of Alzheimers disease (AD) risk, with ApoE4 increasing and ApoE2 decreasing risk relative to ApoE3. Using a global LC-MS proteomic approach, we integrated protein abundance and kinetics in Human-APOE knock-in mice for young (3-month) and aged (18-month) cohorts to quantify the changes in steady-state proteostasis. By mapping 6,052 identified proteins and 3,986 associated turnover rates into ontological groups, we observed that vesicle trafficking and mitochondrial dysregulation occur as early as 3 months in ApoE4 mice accompanied by hyperactive metabolism that eventually reduces with age. In contrast, young and old ApoE2 mice retain similar signatures to ApoE3 mice in metabolic, mitochondrial, cellular regulation, and membrane trafficking ontologies. We found that females had more isoform-induced ontological changes relative to ApoE3, providing insight into sex-dependent vulnerabilities. Our global proteomic approach for ApoE proteostasis crucially unifies independent literature observations while providing turnover kinetics to uncover the underlying mechanism behind abundance changes. Data are available via ProteomeXchange with identifier PXD079261. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/735293v1_ufig2.gif" ALT="Figure 1000"> View larger version (45K): org.highwire.dtl.DTLVardef@54c14borg.highwire.dtl.DTLVardef@5e490dorg.highwire.dtl.DTLVardef@df5b1org.highwire.dtl.DTLVardef@7d7717_HPS_FORMAT_FIGEXP M_FIG C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/735293v1_ufig1.gif" ALT="Figure 1001"> View larger version (48K): org.highwire.dtl.DTLVardef@115c4cdorg.highwire.dtl.DTLVardef@2baac8org.highwire.dtl.DTLVardef@d965e6org.highwire.dtl.DTLVardef@b0e49a_HPS_FORMAT_FIGEXP M_FIG C_FIG

This step-by-step protocol provides a cross-species, non-surgical approach that enables prenatal gene delivery to the developing nervous system in rats and marmosets. Under transabdominal ultrasound guidance, intracerebroventricular injection of recombinant adeno-associated virus vectors into the fetal brain achieves robust and long-term transduction from prenatal stages into adulthood. This approach can be adapted to other species and target sites outside nervous system, enabling safe and selective intrauterine manipulation and the generation of diverse experimental models for basic and preclinical research. For complete details on the use and execution of this protocol, please refer to Ribeiro Gomes et al (2026)1. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/737050v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@696364org.highwire.dtl.DTLVardef@fc3c7forg.highwire.dtl.DTLVardef@1e7c7caorg.highwire.dtl.DTLVardef@1edcef0_HPS_FORMAT_FIGEXP M_FIG C_FIG Before you beginExperimental procedures during gestation allow researchers to study developmental processes, including how manipulations of the fetus and its intrauterine environment influence biological outcomes. Ultrasound imaging guidance greatly facilitates such interventions by providing safe and targeted access to fetal compartments, including for prenatal gene delivery to developing neural cell populations. Critically, delivery of recombinant adeno-associated viruses (rAAVs) into the cerebrospinal fluid (CSF) of developing animals enables widespread gene transfer across the brain. The efficiency and distribution of transduction are strongly influenced by developmental stage, making the timing of delivery an important experimental variable. In altricial species such as mice, major developmental processes, including cortical lamination and the establishment of long-range connections, begin prenatally but continue throughout early postnatal life. In primates, however, development is more advanced at birth, and many equivalent developmental events are shifted to the prenatal period. Consequently, developmental stages that can be targeted postnatally in mice require prenatal access in primates. Here, we present a step-by-step protocol for ultrasound-guided fetal intracerebroventricular viral injection (FIVI) of rAAV in marmosets (Callithrix jacchus) and rats (Rattus norvegicus). The procedure was initially developed and optimized in rats before being translated to marmosets, small New World primates that share key developmental, anatomical, and functional characteristics with humans. Together, these models illustrate the cross-species applicability of the approach, while providing gene delivery strategies for both a genetically tractable rodent model and a translationally relevant nonhuman primate. FIVI enables broad gene transfer and stable, long-term transgene expression in wild type animals, facilitating the generation of complementary quasi-transgenic models for research and translational applications from prenatal development through adulthood.

Background. Scanning electron microscopy (SEM) is widely used to determine fibrin fiber structural properties such as fiber diameter and fiber length. However, conventional SEM preparation protocols are time-consuming and typically require conductive sputter coating. The coating process introduces an additional layer onto the sample surface and may influence measurements of nanoscale fiber structure. Furthermore, preparation of purified fibrinogen clots often follows protocols originally developed for plasma clots, resulting in unnecessary processing steps. Objective. To evaluate indium tin oxide (ITO) as a flat, conductive substrate for SEM imaging of fibrin fibers, investigate the effects of sputter coating on measured fiber diameter, and develop a simplified SEM preparation protocol for purified fibrinogen clots. Methods. Platelet-poor plasma clots and purified fibrinogen clots were formed on ITO substrates and imaged by SEM following 0 s, 45 s, or 90 s sputter coating. Fibrin fiber diameters were quantified and compared across coating conditions. For purified fibrinogen clots, an ITO-based simplified preparation protocol, in which clots were formed and imaged directly on the conductive ITO surface, was compared with a previously developed, standardized SEM protocol, in which clots were formed in microtube lids and subsequently transferred onto carbon tape for imaging. Results. Fiber diameter measurements were affected by sputter coating duration, with increasing coating time resulting in larger apparent fiber diameters. Plasma and purified fibrinogen clots exhibited distinct fiber diameter distributions and coating responses. For purified fibrinogen clots, the simplified ITO-based protocol produced fiber diameter measurements that were not significantly different from those obtained using the standardized lid-to-carbon-tape workflow when identical coating times were applied. Conclusions. ITO provides a practical conductive substrate for SEM imaging of fibrin fibers and enables substantial simplification of purified fibrinogen clot preparation. When coating conditions are matched, the simplified ITO-based protocol yields fiber diameter measurements comparable to those obtained using the previously standardized lid-to-carbon-tape workflow. These findings support the use of ITO as an alternative conductive imaging substrate and provide a simplified workflow for SEM analysis of purified fibrinogen clots. By reducing washing and transfer steps, this workflow may also provide a useful platform for future controlled studies of fibrin interactions with added proteins or other associated components.

Blood biomarkers are central to monitoring disease progression and evaluating treatment responses, yet traditional venipuncture captures a single physiological snapshot in time and becomes burdensome with repeated sampling. Remote blood self-sampling offers a path toward longitudinal, decentralized monitoring, but maintaining protein integrity from draw to analysis remains a critical challenge. Here, we optimized pre-analytical blood collection and stabilization parameters to maintain protein levels at the time of collection for use with remote sampling technology. First, we optimized blood collection time with Tasso remote self-sampling devices to minimize interference from clotting, finding that a 2.5 min collection time best reduces clot formation while collecting enough blood. Next, we found that Protein Plus, a commercial protein stabilizer, limited hemolysis (a metric for stabilizer efficacy) in venous blood for up to 5 days at 25{degrees}C-35{degrees}C and for 1 day at 40{degrees}C. In addition, we optimized the stabilizer volume and acceptable blood volume range for self-sampling as the stabilizer efficacy is impacted by the stabilizer to blood ratio and collection volume can vary with remote self-sampling devices. Finally, we incubated stabilized blood samples collected via Tasso device at 25{degrees}C-35{degrees}C for 72 h, mimicking a 2-day shipping period. Using a panel of 21 inflammatory proteins, we found that Protein Plus limited intracellular protein release for various proteins (e.g., VEGF-A, CCL11, and IL-8), inhibited protein degradation for CCL2, and enabled minimal hemolysis. These results support Protein Plus as a viable stabilization strategy for remote blood collection technology targeting longitudinal inflammatory protein monitoring.

Cooperation in social species is shaped by ongoing social relationships, partner choice, and group interactions, demanding experimental systems that preserve the social context in which these behaviors unfold. Here we introduce the e-Seesaw, a wireless system for automated liquid reward delivery designed to support home-enclosure experiments on reward access distribution in common marmosets (Callithrix jacchus) with minimal human intervention. The apparatus combines a modular peristaltic pump with a Bluetooth-controlled trigger, allowing spatial separation between the site of action and the site of reward delivery while preserving group housing. We provide detailed design files, software, and assembly instructions to support reproduction and adaptation. In a proof-of-concept deployment across seven families, animals readily engaged with the device, producing a median of ~87 trigger activations per session. Engagement was concentrated early within sessions and remained largely stable across repeated deployments, including under increased action-reward separation. These results established the e-Seesaw as a flexible and reproducible platform for automated reward-delivery experiments in animals tested within their social groups, while reducing human involvement and avoiding fixed dyadic testing.

Manual analysis of skeletal muscle cross-sections is time-consuming and subject to error and user bias. To overcome these limitations, we developed and validated a semi-automated, quantitative, and reproducible image-analysis pipeline specifically tailored to quantify Pax7+ satellite cells, myonuclei, and cross-sectional area by fiber type. The workflow combines FIJI/ImageJ-based image preprocessing with CellProfiler, Cellpose, and a custom Python script to process and analyze immunohistological images of muscle tissue cross-sections. Outcomes include Pax7+ satellite cells and myonuclei quantified per fiber by fiber type, along with cross-sectional area, perimeter, and fiber type classification. This semi-automated approach provides a robust and efficient platform for high-throughput analysis of muscle tissue cross-sections from large datasets. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/729866v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@a3401dorg.highwire.dtl.DTLVardef@1c63145org.highwire.dtl.DTLVardef@ccbf76org.highwire.dtl.DTLVardef@2e0da0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cryopreservation offers an option for long-term storage and global distribution of complex in vitro models, yet protocols for multicellular microphysiolgocial systems (MPS) such as brain organoids/spheroids remain limited. Here, we systematically compared three commercially available cryopreservation (mFreSR, CryoStorCS10, and 3dGRO) and two freezing time points, and established a robust workflow for freezing and recovering brain organoids. After defrosting, we assessed morphology and metabolic activity. We also evaluated electrophysiology, calcium transients, and neurite outgrowth. In addition, we measured astrocyte migration, apoptosis, mitochondrial integrity, microglia survival, and neural marker expression. We found that organoids require a 4-week recovery period to regain structural and functional stability. Although organoids frozen at week 6 showed higher metabolic activity after recovery, organoids cryopreserved at week 2 had clearly better functional outcomes. They exhibited stronger spontaneous network firing and maintained calcium transients. Finally, incorporated microglia-like cells survived the freezing and displayed comparable morphology to unfrozen controls. Across the endpoints measured here, 3dGRO showed the most favorable overall performance; formal ranking across media awaits harmonized normalization, single-organoid electrophysiology, and prespecified QC thresholds. Together, these results define a practical and reproducible cryopreservation strategy that preserves key physiological features of brain organoids and supports the establishment of ready-to-use organoid banks. The ability to reliably store and distribute complex brain-like tissues represents an essential step toward global standardization, scalable experimentation, and wider adoption of human-relevant microphysiological systems. Together, these results demonstrate recovery of key physiological features in the subset of organoids that remain viable after thaw and support the feasibility of brain organoid banking.

Coenzyme A is an essential cofactor synthesized from pantothenate, cysteine, and ATP, and is involved in numerous processes of cellular metabolism through its ability to carry activated acyl groups. Coenzyme A participates in catabolism of carbohydrate, fat and amino acids; biosynthesis of fatty acids, cholesterol and heme; and protein modification including acetylation and 4-phosphopantetheinylation. Despite CoAs critical functions, the regulation of CoA levels and the rate of CoA synthesis in different cell types and disease states are not well understood. One reason for this gap is that many acyl-CoA species are analytically challenging to measure due to factors including instability, poor ionization, and the wide range of biochemical properties conferred by different acyl chain lengths. In addition, most current methods do not support analysis of CoA isotopic labeling, which is required to quantify CoA synthesis rate or to measure absolute concentration using isotope-labeled internal standards. Here, we describe a method to quantify the concentration and isotopic labeling of total CoA, defined as the sum of CoASH plus all acyl-CoA species. Acyl-CoA species are hydrolyzed using sodium hydroxide to remove acyl chains, then CoA is derivatized on the thiol with N-ethylmaleimide (NEM). Following protein precipitation and solid phase extraction, samples are analyzed by liquid chromatography-mass spectrometry. This method is linear in a wide range that captures mouse tissue CoA levels, with accuracy within 15% error and precision below 15% relative standard deviation for both pure standards and tissue samples. We applied this method to measure total CoA concentration in five tissues from male and female mice, and total CoA synthesis rate in mouse liver via infusion of 13C-15N-pantothenate. Overall, this method offers a tractable approach to measure total CoA concentration and isotopic labeling to enable study of total CoA synthesis rates and concentrations in health and disease.

Chronic conditions such as Type 2 Diabetes Mellitus (T2DM) and Hypertension (HTN) remain underdiagnosed in community settings, particularly in resource-limited populations. Conventional diagnostic approaches rely on episodic measurements and laboratory-based assessments, limiting scalability for large-scale screening. Non-invasive physiological monitoring systems offer a potential pathway for accessible and rapid wellness assessment in real-world environments. This study aimed to evaluate the feasibility, signal acquisition performance, and exploratory physiological signal characteristics of a non-invasive multimodal monitoring system (Hayl) in community-based screening settings. Methods: A prospective, cross-sectional, multicenter observational pilot study was conducted across rural and urban screening camps in south India. A total of 281 adult participants were enrolled, including individuals with known T2DM, HTN, and those without known comorbidities, encompassing both symptomatic and asymptomatic subjects. Physiological data were acquired using the Hayl system, which integrates photoplethysmography (PPG) and temperature sensing. Signal acquisition feasibility, waveform quality, and derived signal characteristics were evaluated. Comparative and exploratory analyses were performed across predefined clinical subgroups. The study was conducted under Institutional Ethics Committee approval in accordance with guidelines from the Indian Council of Medical Research. Conclusion: The Hayl system demonstrated high feasibility for physiological signal acquisition, with successful PPG recordings in 274 participants (97.5%) and temperature signals in 279 participants (99.3%). Most recordings exhibited high waveform quality (74.0%), with observable variations in signal characteristics across clinically relevant subgroups. Reduced pulse variability and increased waveform irregularity were more frequently observed in participants with T2DM and HTN, while symptomatic individuals demonstrated greater signal variability compared to asymptomatic participants. Temperature measurements were stable, with a mean peripheral temperature of 33.4 with a variation of 1.2C degrees. These findings support the potential of Hayl as a non-invasive multimodal platform for community-based wellness screening and exploratory signal-based physiological assessment. Further large-scale and longitudinal studies are required to establish clinical utility.

Proprioception and reflexive control of muscle tone depend on the activity of muscle spindles, specialized sensory receptors embedded deep within skeletal muscle that detect changes in muscle length. Their location and complex three-dimensional architecture have historically limited morphological analysis to techniques such as silver-impregnation, muscle teasing, or serial sectioning followed by volumetric reconstruction. Here, we describe a workflow for three-dimensional, in situ visualization of muscle spindles in the rabbit tenuissimus muscle, a preparation uniquely enriched in spindles and well suited for whole-mount imaging. The protocol combines fluorescent labeling of spindle sensory and motor innervation, including intrafusal {gamma} neuromuscular junctions labeled with -bungarotoxin, with immunolabeling and solvent-based optical clearing. Optically cleared tenuissimus muscles were compatible with both whole-mount confocal and light-sheet microscopy, enabling volumetric imaging of complete spindle structures and detailed visualization of Ia annulospiral endings at the spindle equator. This approach provides access to spindle morphology and connectivity at multiple spatial scales while avoiding physical sectioning and reconstruction. By enabling reproducible three-dimensional imaging of intact muscle spindles, this workflow offers a practical platform for studying spindle structure and plasticity in health and disease.

Pediatric patients with epicardial cardiac implantable electronic devices (CIEDs) are frequently excluded from the Magnetic Resonance Imaging (MRI) primarily due to RF heating safety concerns. In this study we evaluate RF heating of two bipolar epicardial leads during MRI at 0.55 T and 1.5 T under different termination conditions. Our findings showed that the mean RF heating was significantly reduced at 0.55 T MRI compared to that at 1.5 T. Similarly, the RF heating at 0.55 T MRI was highest for full system whereas, during MRI at 1.5 T, the RF heating was highest for the capped abandoned lead, showing dependence of RF heating pattern on MRI field strength. While RF heating at both fields surpassed the safety limit, the capped abandoned leads at 1.5 T MRI showed significantly higher RF heating with temperature rise surpassing 50{degrees}C in some of the cases. These results highlight the difference in RF heating of bipolar epicardial leads compared to the previously reported findings for monopolar epicardial lead which showed smallest heating for capped abandoned lead at both field strengths. These findings emphasize the necessity of device-specific evaluations at each field-strength to inform clinical decision-making and expand MRI access for this vulnerable population.

Digital Pathology (DP) is a fast-emerging branch of pathology focused on digitizing pathology data. A key challenge of DP usage for pathology laboratories, especially mid- to small-sized clinical labs, are the upfront costs associated with instrumentation and the logistical challenges of implementation. In the current project, we built an end-to-end DP solution using low-cost, open-source components that is user-friendly at a small scale. We repurposed readily available microscopy components in a pathology lab to assemble a fully functional DP pipeline for translational research applications. We tested multiple low-cost complementary metal-oxide semiconductor (CMOS) cameras in this project and chose a user-friendly Canon camera for image acquisition. An open-source DP server solution, OMERO v.5.6.4, was used as the image management system (IMS) to host and serve the WSIs on an Ubuntu 22.04 operating system. The server-hosted WSI images were evaluated remotely and asynchronously by multiple pathologists physically situated in Albuquerque, NM; Salt Lake City, UT; and Palo Alto, CA. Each pathologist assessed the quality of the WSI pipeline, image quality, and WSI interaction experience using a 23-question survey. Overall, the custom, low-cost WSI pipeline was noted to be a robust and user-friendly experience by the pathologists. The current DP setup is unlikely to be useful as a commercial, scalable DP pipeline for large-scale clinical applications. However, it demonstrates the feasibility of creating customized, small-scale DP solutions (at a low price point) for asynchronous translational pathology research applications. Additionally, building customized DP pipelines provides excellent educational opportunities for pathology residents to gain in-depth knowledge of the various technical elements of a DP workflow. In summary, we have established a low-cost, end-to-end WSI DP pipeline useful for spatiotemporally asynchronous translational pathology research, in an academic setting.

Intrathecal (IT) drug delivery is a critical technique for bypassing the blood-brain and blood-spinal cord barriers in preclinical CNS research. However, conventional rat catheterization methods suffer from high rates of neurologic complications, poor reliability, and unverified dosing due to epidural reflux and inconsistent supraspinal distribution. Our objective was to develop and validate an improved method for lumbar IT catheterization in rats that ensures distribution to the brain and to confirm supraspinal pharmacodynamic target engagement. We describe a refined microsurgical technique using dural puncture under direct visual control at the L6-S1 interlaminar space, a site chosen for its anatomical safety margin. The method uses a small-bore (0.33 mm OD) polyurethane (PU) catheter to minimize durotomy size, air-bubble tracking to compensate for catheter dead volume, and epidural sealing with Surgifoam(R) to minimize reflux. Visualization using Evans Blue dye confirmed complete neuraxial distribution from a single 30 {micro}L lumbar bolus injection, with dye reaching the ventral/dorsal brain cisterns. Pharmacodynamic validation of cortical exposure was achieved using an S6 kinase 1 (S6K1) inhibitor. Lumbar IT administration over a period of 6 hours via a pump resulted in significant supraspinal S6K1 engagement, demonstrated by a reproducible reduction in S6 phosphorylation in the cerebral cortex. HighlightsO_LIMethod for lumbar intrathecal catheterization in rats under direct visual control, using basic surgical tools C_LIO_LICNS distribution validated by Evans Blue dye reaching ventral and dorsal brain C_LIO_LIPharmacodynamic confirmation of supraspinal target engagement following lumbar intrathecal delivery of a small molecule kinase inhibitor C_LIO_LIServes as a faithful preclinical model for therapeutics intended for clinical intrathecal administration C_LIO_LIProvides a screening route for early-stage compounds not yet optimized for CNS penetrance, supporting efficacy testing prior to medicinal chemistry investment C_LI