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Background: The discrepancy between experimental research and clinical trial outcomes is a persistent challenge in preclinical studies, particularly in stroke research. A possible factor contributing to this issue is the lack of standardization across experimental stroke models, leading to poor reproducibility in multicenter studies. This study addresses this gap by aiming to enhance reproducibility and the efficacy of multicenter studies through the harmonization of protocols and training of involved personnel., Methods: We established a set of standard operating procedures for various stroke models and the Neuroscore. These standard operating procedures were implemented across multiple research centers, followed by specialized, in-person training for all participants. We measured the variability in infarct volume both before and after the implementation of these standardized protocols and training sessions., Results: The standardization process led to a significant reduction in variability of infarct volume across different stroke models (40%-50% reduction), demonstrating the effectiveness of our harmonized protocols and training. Additionally, the implementation of the Neuroscore system across centers showed low variability and consistent results up to 28 days poststroke, underscoring its utility in chronic phase evaluations., Conclusions: The harmonization of protocols and surgeon training significantly reduced variability in experimental outcomes across different centers. This improvement can increase the comparability of data between research groups and enhance the statistical power of multicenter studies. Our findings also establish the Neuroscore as a reliable tool for long-term assessment in stroke research, paving the way for more consistent and impactful multicenter preclinical studies., Competing Interests: None.

Evaluation of microvascular networks was impeded until recently by the need of histological tissue sectioning, which precluded 3D analyses. Using light-sheet microscopy, we investigated microvascular network characteristics in the peri-infarct cortex of mice 3-56 days after transient middle cerebral artery occlusion. In animal subgroups, the sphingosine-1-phosphate analog FTY720 (Fingolimod) was administered starting 24 hours post-ischemia. Light-sheet microscopy revealed a striking pattern of microvascular changes in the peri-infarct cortex, that is, a loss of microvessels, which was most prominent after 7 days and followed by the reappearance of microvessels over 56 days which revealed an increased branching point density and shortened branches. Using a novel AI-based image analysis algorithm we found that the length density of microvessels expressing the arterial specification marker α-smooth muscle actin markedly increased in the peri-infarct cortex already at 7 days post-ischemia. The length and branch density of small microvessels, but not of intermediate or large microvessels increased above pre-ischemic levels within 14-56 days. FTY720 increased the length and branch density of small microvessels. This study demonstrates long-term alterations of microvascular architecture post-ischemia indicative of increased collateralization most notably of small microvessels. Light-sheet microscopy will greatly advance the assessment of microvascular responses to restorative stroke therapies., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The ecosystem of brain tumors is considered immunosuppressed, but our current knowledge may be incomplete. Here we analyzed clinical cell and tissue specimens derived from patients presenting with glioblastoma or nonmalignant intracranial disease to report that the cranial bone (CB) marrow, in juxtaposition to treatment-naive glioblastoma tumors, harbors active lymphoid populations at the time of initial diagnosis. Clinical and anatomical imaging, single-cell molecular and immune cell profiling and quantification of tumor reactivity identified CD8 + T cell clonotypes in the CB that were also found in the tumor. These were characterized by acute and durable antitumor response rooted in the entire T cell developmental spectrum. In contrast to distal bone marrow, the CB niche proximal to the tumor showed increased frequencies of tumor-reactive CD8 + effector types expressing the lymphoid egress marker S1PR1. In line with this, cranial enhancement of CXCR4 radiolabel may serve as a surrogate marker indicating focal association with improved progression-free survival. The data of this study advocate preservation and further exploitation of these cranioencephalic units for the clinical care of glioblastoma., (© 2024. The Author(s).)

3D data from high-resolution volumetric imaging is a central resource for diagnosis and treatment in modern medicine. While the fast development of AI enhances imaging and analysis, commonly used visualization methods lag far behind. Recent research used extended reality (XR) for perceiving 3D images with visual depth perception and touch but used restrictive haptic devices. While unrestricted touch benefits volumetric data examination, implementing natural haptic interaction with XR is challenging. The research question is whether a multisensory XR application with intuitive haptic interaction adds value and should be pursued. In a study, 24 experts for biomedical images in research and medicine explored 3D medical shapes with 3 applications: a multisensory virtual reality (VR) prototype using haptic gloves, a simple VR prototype using controllers, and a standard PC application. Results of standardized questionnaires showed no significant differences between all application types regarding usability and no significant difference between both VR applications regarding presence. Participants agreed to statements that VR visualizations provide better depth information, using the hands instead of controllers simplifies data exploration, the multisensory VR prototype allows intuitive data exploration, and it is beneficial over traditional data examination methods. While most participants mentioned manual interaction as the best aspect, they also found it the most improvable. We conclude that a multisensory XR application with improved manual interaction adds value for volumetric biomedical data examination. We will proceed with our open-source research project ISH3DE (Intuitive Stereoptic Haptic 3D Data Exploration) to serve medical education, therapeutic decisions, surgery preparations, or research data analysis., (© 2024. The Author(s).)

Post-injury dysfunction of humoral immunity accounts for infections and poor outcomes in cardiovascular diseases. Among immunoglobulins (Ig), IgA, the most abundant mucosal antibody, is produced by plasma B cells in intestinal Peyer's patches (PP) and lamina propria. Here we show that patients with stroke and myocardial ischemia (MI) had strongly reduced IgA blood levels. This was phenocopied in experimental mouse models where decreased plasma and fecal IgA were accompanied by rapid loss of IgA-producing plasma cells in PP and lamina propria. Reduced plasma IgG was detectable in patients and experimental mice 3-10 d after injury. Stroke/MI triggered the release of neutrophil extracellular traps (NETs). Depletion of neutrophils, NET degradation or blockade of NET release inhibited the loss of IgA + cells and circulating IgA in experimental stroke and MI and in patients with stroke. Our results unveil how tissue-injury-triggered systemic NET release disrupts physiological Ig secretion and how this can be inhibited in patients., (© 2024. The Author(s).)

Cytotoxic T lymphocyte (CTL) motility is an important feature of effective CTL responses and is impaired when CTLs become exhausted, e.g. during chronic retroviral infections. A prominent T cell exhaustion marker is programmed cell death protein 1 (PD-1) and antibodies against the interaction of PD-1 and PD-ligand 1 (PD-L1) are known to improve CTL functions. However, antibody blockade affects all PD-1/PD-L1-expressing cell types, thus, the observed effects cannot be attributed selectively to CTLs. To overcome this problem, we performed CRISPR/Cas9 based knockout of the PD-1 coding gene PDCD1 in naïve Friend Retrovirus (FV)-specific CTLs. We transferred 1,000 of these cells into mice where they proliferated upon FV-infection. Using intravital two-photon microscopy we visualized CTL motility in the bone marrow and evaluated cytotoxic molecule expression by flow cytometry. Knockout of PDCD1 improved the CTL motility at 14 days post infection and enhanced the expression of cytotoxicity markers. Our data show the potential of genetic tuning of naive antiviral CTLs and might be relevant for future designs of improved T cell-mediated therapies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Mittermüller, Otto, Kilian, Schnormeier, Littwitz-Salomon, Hasenberg, Dittmer and Gunzer.)

Background: Until now, the analysis of microvascular networks in the reperfused ischemic brain has been limited due to tissue transparency challenges., Methods: Using light sheet microscopy, we assessed microvascular network remodeling in the striatum from 3 hours to 56 days post-ischemia in 2 mouse models of transient middle cerebral artery occlusion lasting 20 or 40 minutes, resulting in mild ischemic brain injury or brain infarction, respectively. We also examined the effect of a clinically applicable S1P (sphingosine-1-phosphate) analog, FTY720 (fingolimod), on microvascular network remodeling., Results: Over 56 days, we observed progressive microvascular degeneration in the reperfused striatum, that is, the lesion core, which was followed by robust angiogenesis after mild ischemic injury induced by 20-minute middle cerebral artery occlusion. However, more severe ischemic injury elicited by 40-minute middle cerebral artery occlusion resulted in incomplete microvascular remodeling. In both cases, microvascular networks did not return to their preischemic state but displayed a chronically altered pattern characterized by higher branching point density, shorter branches, higher unconnected branch density, and lower tortuosity, indicating enhanced network connectivity. FTY720 effectively increased microvascular length density, branching point density, and volume density in both models, indicating an angiogenic effect of this drug., Conclusions: Utilizing light sheet microscopy together with automated image analysis, we characterized microvascular remodeling in the ischemic lesion core in unprecedented detail. This technology will significantly advance our understanding of microvascular restorative processes and pave the way for novel treatment developments in the stroke field., Competing Interests: Disclosures None.

Persistent immune activation contributes significantly to left ventricular (LV) dysfunction and adverse remodeling in heart failure (HF). In contrast to their well-known essential role in acute myocardial infarction (MI) as first responders that clear dead cells and facilitate subsequent reparative macrophage polarization, the role of neutrophils in the pathobiology of chronic ischemic HF is poorly defined. To determine the importance of neutrophils in the progression of ischemic cardiomyopathy, we measured their production, levels, and activation in a mouse model of chronic HF 8 weeks after permanent coronary artery ligation and large MI. In HF mice, neutrophils were more abundant both locally in failing myocardium (more in the border zone) and systemically in the blood, spleen, and bone marrow, together with increased BM granulopoiesis. There were heightened stimuli for neutrophil recruitment and trafficking in HF, with increased myocardial expression of the neutrophil chemoattract chemokines CXCL1 and CXCL5, and increased neutrophil chemotactic factors in the circulation. HF neutrophil NETotic activity was increased in vitro with coordinate increases in circulating neutrophil extracellular traps (NETs) in vivo. Neutrophil depletion with either antibody-based or genetic approaches abrogated the progression of LV remodeling and fibrosis at both intermediate and late stages of HF. Moreover, analogous to murine HF, the plasma milieu in human acute decompensated HF strongly promoted neutrophil trafficking. Collectively, these results support a key tissue-injurious role for neutrophils and their associated cytotoxic products in ischemic cardiomyopathy and suggest that neutrophils are potential targets for therapeutic immunomodulation in this disease., Competing Interests: Declaration of competing interest The authors have no conflicts of interest relevant to the contents of this paper to disclose., (Copyright © 2023. Published by Elsevier Ltd.)

Mass spectrometry-based metabolomics with stable isotope labeling (SIL) is an established tool for sensitive and precise analyses of tissue metabolism, its flux, and pathway activities in diverse models of physiology and disease. Despite the simplicity and broad applicability of deuterium ( 2 H)-labeled precursors for tracing metabolic pathways with minimal biological perturbations, they are rarely employed in LC-MS/MS-guided metabolomics. In this study, we have developed a LC-MS/MS-guided workflow to trace deuterium metabolism in mouse organs following 2 H 7 -glucose infusion. The workflow includes isotopically labeled glucose infusion, mouse organ isolation and metabolite extraction, zwitterion-based hydrophilic interaction liquid chromatography (HILIC) coupled to high-resolution tandem mass spectrometry, targeted data acquisition for sensitive detection of deuterated metabolites, a spectral library of over 400 metabolite standards, and multivariate data analysis with pathway mapping. The optimized method was validated for matrix effects, normalization, and quantification to provide both tissue metabolomics and tracking the in-vivo metabolic fate of deuterated glucose through key metabolic pathways. We quantified more than 100 metabolites in five major mouse organ tissues (liver, kidney, brain, brown adipose tissue, and heart). Furthermore, we mapped isotopologues of deuterated metabolites from glycolysis, tricarboxylic acid (TCA) cycle, and amino acid pathways, which are significant for studying both health and various diseases. This study will open new avenues in LC-MS based analysis of 2 H-labeled tissue metabolism research in animal models and clinical settings., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Prasad Phapale reports was provided by ISAS Leibniz Institute for Analytical Sciences. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Autonomous migration is essential for the function of immune cells such as neutrophils and plays an important role in numerous diseases. The ability to routinely measure or target it would offer a wealth of clinical applications. Video microscopy of live cells is ideal for migration analysis, but cannot be performed at sufficiently high-throughput (HT). Here we introduce ComplexEye, an array microscope with 16 independent aberration-corrected glass lenses spaced at the pitch of a 96-well plate to produce high-resolution movies of migrating cells. With the system, we enable HT migration analysis of immune cells in 96- and 384-well plates with very energy-efficient performance. We demonstrate that the system can measure multiple clinical samples simultaneously. Furthermore, we screen 1000 compounds and identify 17 modifiers of migration in human neutrophils in just 4 days, a task that requires 60-times longer with a conventional video microscope. ComplexEye thus opens the field of phenotypic HT migration screens and enables routine migration analysis for the clinical setting., (© 2023. The Author(s).)