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Knowledge driven materials and component design is key for improving the performance of Li-ion batteries and solving the remaining hurdles for next-generation battery concepts like all-solid-state batteries. While the spatial and time dependent distribution of Li would help elucidating performance bottlenecks and degradation phenomena, only a few analysis techniques are available, due to the unique nature of Li, especially as Li+-ion. In fact, only two non-destructive techniques with good time resolution can combine spatial information with absolute quantification of Li, one being Neutron Depth Profiling (NDP), the other Ion-Beam-Analysis (IBA). While both exploit nuclear processes, the information gained is complementary. NDP provides high depth resolution, but only limited lateral resolution, whereas IBA has high lateral, but only limited depth resolution. In this study, we benchmark both techniques for the first time using a set of Li-battery test-samples and show the strengths and synergies of both techniques. The derived information regarding the depth dependent Li-concentration is then used to validate a microstructure resolved continuum model of charge, discharge, and relaxation behavior of the cells and electro-chemical analysis. This fundamental work demonstrates a new route to optimize Li batteries on material and component level by combining advanced characterization and digital twin modelling. • LLZO + LCO ASBs analyzed using MeV Ion-Beam-Analysis + Neutron-Depth-Profiling. • Both methods results agree to each other the SoC dependent Li concentrations. • Surface near reactions of LLZO redistribute, but conserve the total Li content. • Digital twin modelling based on 3D cell reconstruction shows relaxation within ∼1h. • Li concentration is ∼20 % above nominal value, reduces by high sintering temperature. [ABSTRACT FROM AUTHOR]

Solid-state batteries based on Li 7 La 3 Zr 2 O 12 (LLZO) garnet electrolyte are a robust and safe alternative to conventional lithium-ion batteries. However, the large-scale implementation of ceramic composite cathodes is still challenging due to a complex multistep manufacturing process. A new one-step route for the direct synthesis of LLZO during the manufacturing of LLZO/LiCoO 2 (LCO) composite cathodes based on cheap precursors and utilizing the industrially established tape casting process is presented. It is shown that Al, Ta:LLZO can be formed directly in the presence of LCO from metal oxide precursors (LiOH, La 2 O 3 , ZrO 2 , Al 2 O 3 , and Ta 2 O 5 ) by heating to 1050 °C, eliminating the time- and energy-consuming synthesis of preformed LLZO powders. In addition, performance-optimized gradient microstructures can be produced by sequential casting of slurries with different compositions, resulting in dense and flat phase-pure cathodes without unwanted ion interdiffusion or secondary phase formation. Freestanding cathodes with a thickness of 85 µm, a relative density of 95%, and an industrial relevant LCO loading of 15 mg show an initial capacity of 82 mAh g -1 (63% of the theoretical capacity of LCO) in a solid-state cell with Li metal anodes, which is comparable to conventional LCO/LLZO cathodes and can be further improved in the future., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Ion Beam Analysis (IBA) utilizing MeV ion beams provides valuable insights into surface elemental composition across the entire periodic table. While ion beam measurements have advanced towards high throughput for mapping applications, data analysis has lagged behind due to the challenges posed by large volumes of data and multiple detectors providing diverse analytical information. Traditional physics-based fitting algorithms for these spectra can be time-consuming and prone to local minima traps, often taking days or weeks to complete. This study presents an approach employing a Mixture Density Network (MDN) to model the posterior distribution of Elemental Depth Profiles (EDP) from input spectra. Our MDN architecture includes an encoder module (EM), leveraging a Convolutional Neural Network-Gated Recurrent Unit (CNN-GRU), and a Mixture Density Head (MDH) employing a Multi-Layer Perceptron (MLP). Validation across three datasets with varying complexities demonstrates that for simple and intermediate cases, the MDN performs comparably to the conventional automatic fitting method (Autofit). However, for more complex datasets, Autofit still outperforms the MDN. Additionally, our integrated approach, combining MDN with the automatic fit method, significantly enhances accuracy while still reducing computational time, offering a promising avenue for improved analysis in IBA., (© 2024. The Author(s).)

In this work, the effects of dopant size and oxidation state on the structure and electrochemical performance of LiNi 0.8 Co 0.1 Mn 0.1 O 2 (NCM811) are investigated. It is shown that doping with boron (B) which has a small ionic radius and an oxidation state of 3+, leads to the formation of a boron oxide-containing surface coating (probably Li 3 BO 3 ), mainly on the outer surface of the secondary particles. Due to this effect, boron only slightly affects the size of the primary particle and the initial capacity, but significantly improves the capacity retention. On the other hand, the dopant ruthenium (Ru) with a larger ionic radius and a higher oxidation state of 5+ can be stabilized within the secondary particles and does not experience a segregation to the outer agglomerate surface. However, the Ru dopant preferentially occupies incoherent grain boundary sites, resulting in smaller primary particle size and initial capacity than for the B-doped and pristine NCM811. This work demonstrates that a small percentage of dopant (2 mol%) cannot significantly affect bulk properties, but it can strongly influence the surface and/or grain boundary properties of microstructure and thus the overall performance of cathode materials., (© 2024 The Authors. Small published by Wiley‐VCH GmbH.)

The phosphate lithium-ion conductor Li 1.5 Al 0.5 Ti 1.5 (PO 4 ) 3 (LATP) is an economically attractive solid electrolyte for the fabrication of safe and robust solid-state batteries, but high sintering temperatures pose a material engineering challenge for the fabrication of cell components. In particular, the high surface roughness of composite cathodes resulting from enhanced crystal growth is detrimental to their integration into cells with practical energy density. In this work, we demonstrate that efficient free-standing ceramic cathodes of LATP and LiFePO 4 (LFP) can be produced by using a scalable tape casting process. This is achieved by adding 5 wt % of Li 2 WO 4 (LWO) to the casting slurry and optimizing the fabrication process. LWO lowers the sintering temperature without affecting the phase composition of the materials, resulting in mechanically stable, electronically conductive, and free-standing cathodes with a smooth, homogeneous surface. The optimized cathode microstructure enables the deposition of a thin polymer separator attached to the Li metal anode to produce a cell with good volumetric and gravimetric energy densities of 289 Wh dm -3 and 180 Wh kg -1 , respectively, on the cell level and Coulombic efficiency above 99% after 30 cycles at 30 °C.

Experimental data suggested activation of yes-associated protein (YAP-1) as a critical regulator of liver regeneration (LR). Serotonin (5-HT) promotes LR in rodent models and has been proposed to act via YAP-1. How 5-HT affects LR is incompletely understood. A possible mechanism how 5-HT affects human LR was explored. Sixty-one patients were included. Tissue samples prior and 2 h after induction of LR were collected. Circulating levels of 5-HT and osteopontin (OPN) were assessed. YAP-1, its phosphorylation states, cytokeratin 19 (CK-19) and OPN were assessed using immunofluorescence. A mouse model of biliary epithelial cells (BECs) specific deletion of YAP/TAZ was developed. YAP-1 increased as early as 2 h after induction of LR (p = 0.025) predominantly in BECs. BEC specific deletion of YAP/TAZ reduced LR after 70% partial hepatectomy in mice (Ki67%, p < 0.001). SSRI treatment, depleting intra-platelet 5-HT, abolished YAP-1 and OPN induction upon LR. Portal vein 5-HT levels correlated with intrahepatic YAP-1 expression upon LR (R = 0.703, p = 0.035). OPN colocalized with YAP-1 in BECs and its circulating levels increased in the liver vein 2 h after induction of LR (p = 0.017). In the context of LR tyrosine-phosphorylated YAP-1 significantly increased (p = 0.042). Stimulating BECs with 5-HT resulted in increased YAP-1 activation via tyrosine-phosphorylation and subsequently increased OPN expression. BECs YAP-1 appears to be critical for LR in mice and humans. Our evidence suggests that 5-HT, at least in part, exerts its pro-regenerative effects via YAP-1 tyrosine-phosphorylation in BECs and subsequent OPN-dependent paracrine immunomodulation., (© 2023 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals LLC.)

All-solid-state lithium batteries are promising candidates for next-generation energy storage systems. Their performance critically depends on the capacity and cycling stability of the cathodic layer. Cells with a garnet Li 7 La 3 Zr 2 O 12 (LLZO) electrolyte can show high areal storage capacity. However, they commonly suffer from performance degradation during cycling. For fully inorganic cells based on LiCoO 2 (LCO) as cathode active material and LLZO, the electrochemically induced interface amorphization has been identified as an origin of the performance degradation. This study shows that the amorphized interface can be recrystallized by thermal recovery (annealing) with nearly full restoration of the cell performance. The structural and chemical changes at the LCO/LLZO heterointerface associated with degradation and recovery were analyzed in detail and justified by thermodynamic modeling. Based on this comprehensive understanding, this work demonstrates a facile way to recover more than 80% of the initial storage capacity through a thermal recovery (annealing) step. The thermal recovery can be potentially used for cost-efficient recycling of ceramic all-solid-state batteries.

The garnet-type Li 7 La 3 Zr 2 O 12 (LLZO) ceramic solid electrolyte combines high Li-ion conductivity at room temperature with high chemical stability. Several all-solid-state Li batteries featuring the LLZO electrolyte and the LiCoO 2 (LCO) or LiCoO 2 -LLZO composite cathode were demonstrated. However, all batteries exhibit rapid capacity fading during cycling, which is often attributed to the formation of cracks due to volume expansion and the contraction of LCO. Excluding the possibility of mechanical failure due to crack formation between the LiCoO 2 /LLZO interface, a detailed investigation of the LiCoO 2 /LLZO interface before and after cycling clearly demonstrated cation diffusion between LiCoO 2 and the LLZO. This electrochemically driven cation diffusion during cycling causes the formation of an amorphous secondary phase interlayer with high impedance, leading to the observed capacity fading. Furthermore, thermodynamic analysis using density functional theory confirms the possibility of low- or non-conducting secondary phases forming during cycling and offers an additional explanation for the observed capacity fading. Understanding the presented degradation paves the way to increase the cycling stability of garnet-based all-solid-state Li batteries.

Solid electrolyte is the key component in all-solid-state batteries (ASBs). It is required in electrodes to enhance Li-conductivity and can be directly used as a separator. With its high Li-conductivity and chemical stability towards metallic lithium, lithium-stuffed garnet material Li 7 La 3 Zr 2 O 12 (LLZO) is considered one of the most promising solid electrolyte materials for high-energy ceramic ASBs. However, in order to obtain high conductivities, rare-earth elements such as tantalum or niobium are used to stabilize the highly conductive cubic phase. This stabilization can also be obtained via high levels of aluminum, reducing the cost of LLZO but also reducing processability and the Li-conductivity. To find the sweet spot for a potential market introduction of garnet-based solid-state batteries, scalable and industrially usable syntheses of LLZO with high processability and good conductivity are indispensable. In this study, four different synthesis methods (solid-state reaction (SSR), solution-assisted solid-state reaction (SASSR), co-precipitation (CP), and spray-drying (SD)) were used and compared for the synthesis of aluminum-substituted LLZO (Al:LLZO, Li 6.4 Al 0.2 La 3 Zr 2 O 12 ), focusing on electrochemical performance on the one hand and scalability and environmental footprint on the other hand. The synthesis was successful via all four methods, resulting in a Li-ion conductivity of 2.0-3.3 × 10 -4 S/cm. By using wet-chemical synthesis methods, the calcination time could be reduced from two calcination steps for 20 h at 850 °C and 1000 °C to only 1 h at 1000 °C for the spray-drying method. We were able to scale the synthesis up to a kg-scale and show the potential of the different synthesis methods for mass production.

Garnet-based Li-ion conductors are one of the most promising oxide-ceramic solid electrolytes for next-generation Li batteries. However, they undergo a Li + /H + exchange (LHX) reaction with most protic solvents used in component manufacturing routes and even with moisture in ambient air. These protonated garnets show a lower Li-ionic conductivity, and even if only the surface is protonated, this degraded layer hinders the Li-ion exchange with, for example, a metallic Li anode. Furthermore, the resulting unstable surface properties during the processing in air lead to challenges with respect to reproducibility of the final component performance, limiting their commercial applicability. However, in recent years, the knowledge about the underlying chemical mechanisms has led to the development of mitigation strategies and enabled a push of this promising material class towards sustainable and scalable fabrication routes. This Minireview covers the following four aspects, which are relevant for a comprehensive understanding of these developments: (1) reports of LHX phenomenon in garnets exposed to air and solvents; (2) recent understandings of the fundamentals and properties of LHX; (3) strategies to prevent LHX and to recover garnets; and (4) sustainable application of LHX for material processing and energy-related devices., (© 2021 The Authors. ChemSusChem published by Wiley-VCH GmbH.)

We apply high-temperature oxide melt solution calorimetry to assess the thermodynamic properties of the material Li 1+ x Al x Ti 2- x (PO 4 ) 3 , which has been broadly recognized as one of the best Li-ion-conducting solid electrolytes of the NASICON family. The experimental results reveal large exothermic enthalpies of formation from binary oxides (Δ H f,ox ° ) and elements (Δ H f,el ° ) for all compositions in the range 0 ≤ x ≤ 0.5. This indicates substantial stability of Li 1+ x Al x Ti 2- x (PO 4 ) 3 , driven by thermodynamics and not just kinetics, during long-term battery operation. The stability increases with increasing Al 3+ content. Furthermore, the dependence of the formation enthalpy on the Al 3+ content shows a change in behavior at x = 0.3, a composition near which the Li + conductivity reaches the highest values. The strong correlation among thermodynamic stability, ionic transport, and clustering is a general phenomenon in ionic conductors that is independent of the crystal structure as well as the type of charge carrier. Therefore, the thermodynamic results can serve as guidelines for the selection of compositions with potentially the highest Li + conductivity among different NASICON-type series with variable dopant contents.

The importance of tetraspanin proteins in regulating migration has been demonstrated in many diverse cellular systems. However, the function of the leukocyte-restricted tetraspanin CD53 remains obscure. We therefore hypothesized that CD53 plays a role in regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflammatory stimuli. Deletion of CD53 significantly reduced neutrophil recruitment to the acutely inflamed peritoneal cavity. Intravital microscopy revealed that in response to several inflammatory and chemotactic stimuli, absence of CD53 had only minor effects on leukocyte rolling and adhesion in postcapillary venules. In contrast, Cd53 -/- mice showed a defect in leukocyte transmigration induced by TNF, CXCL1 and CCL2, and a reduced capacity for leukocyte retention on the endothelial surface under shear flow. Comparison of adhesion molecule expression in wild-type and Cd53 -/- neutrophils revealed no alteration in expression of β 2 integrins, whereas L-selectin was almost completely absent from Cd53 -/- neutrophils. In addition, Cd53 -/- neutrophils showed defects in activation-induced cytoskeletal remodeling and translocation to the cell periphery, responses necessary for efficient transendothelial migration, as well as increased α 3 integrin expression. These alterations were associated with effects on inflammation, so that in Cd53 -/- mice, the onset of neutrophil-dependent serum-induced arthritis was delayed. Together, these findings demonstrate a role for tetraspanin CD53 in promotion of neutrophil transendothelial migration and inflammation, associated with CD53-mediated regulation of L-selectin expression, attachment to the endothelial surface, integrin expression and trafficking, and cytoskeletal function., (Copyright © 2020 by The American Association of Immunologists, Inc.)

There is an urgent need for an easily assessable preoperative test to predict postoperative liver function recovery and thereby determine the optimal time point of liver resection, specifically as current markers are often expensive, time consuming, and invasive. Emerging evidence suggests that microRNA (miRNA) signatures represent potent diagnostic, prognostic, and treatment-response biomarkers for several diseases. Using next-generation sequencing as an unbiased systematic approach, 554 miRNAs were detected in preoperative plasma of 21 patients suffering from postoperative liver dysfunction (LD) after liver resection and 27 matched controls. Subsequently, we identified a miRNA signature-consisting of miRNAs 151a-5p, 192-5p, and 122-5p-that highly correlated with patients developing postoperative LD after liver resection. The predictive potential for postoperative LD was subsequently confirmed using real-time PCR in an independent validation cohort of 98 patients. Ultimately, a regression model of the two miRNA ratios 151a-5p to 192-5p and 122-5p to 151a-5p was found to reliably predict postoperative LD, severe morbidity, prolonged intensive care unit and hospital stays, and even mortality before an operation with a remarkable accuracy, thereby outperforming established markers of postoperative LD. Ultimately, we documented that miRNA ratios closely followed liver function recovery after partial hepatectomy. Conclusion: Our data demonstrate the clinical utility of an miRNA-based biomarker to support the selection of patients undergoing partial hepatectomy. The dynamical changes during liver function recovery indicate a possible role in individualized patient treatment. Thereby, our data might help to tailor surgical strategies to the specific risk profile of patients., (© 2019 The Authors. Hepatology published by Wiley Periodicals, Inc., on behalf of American Association for the Study of Liver Diseases.)

Platelet-leukocyte interactions promote acute glomerulonephritis. However, neither the nature of the interactions between platelets and immune cells nor the capacity of platelets to promote leukocyte activation has been characterized in this condition. We used confocal intravital microscopy to define the interactions of platelets with neutrophils, monocytes, and endothelial cells in glomerular capillaries in mice. In the absence of inflammation, platelets underwent rapid on/off interactions with immune cells. During glomerulonephritis induced by in situ immune complex formation, platelets that interacted with neutrophils or monocytes, but not with other intraglomerular cells, were retained in the glomerulus for prolonged durations. Depletion of platelets inhibited both neutrophil recruitment and activation. Inhibition of platelet activating factor reduced neutrophil recruitment without impacting reactive oxygen species generation, while blocking CXC chemokine ligand 7 (CXCL7) reduced both responses. In contrast, inhibition of the adenosine diphosphate and thromboxane A2 pathways inhibited neutrophil reactive oxygen species generation without affecting neutrophil adhesion. Thus, platelet retention in glomerular capillaries following immune complex deposition stems from prolongation of platelet interactions with immune cells but not other substrates. Pro-inflammatory mediators play divergent roles in promoting neutrophil retention and activation in glomerular capillaries., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Platelets, besides their specialised role in haemostasis and atherothrombosis, actively modulate innate and adaptive immune responses with crucial roles in immune surveillance, inflammation and host defence during infection. An important prerequisite for platelet-mediated changes of immune functions involves direct engagement with different types of leukocytes. Indeed, increased platelet-leukocyte aggregates (PLAs) within the circulation and/or locally at the site of inflammation represent markers of many thrombo-inflammatory diseases, such as cardiovascular diseases, acute lung injury, renal and cerebral inflammation. Therefore, measurement of PLAs could provide an attractive and easily accessible prognostic and/or diagnostic tool for many diseases. To measure PLAs in different (patho-)physiological settings in human and animal models flow cytometric and microscopic approaches have been applied. These techniques represent complementary tools to study different aspects relating to the involvement of leukocyte subtypes and molecules, as well as location of PLAs within tissues, dynamics of their interactions and/or dynamic changes in leukocyte and platelet behaviour. This review summarises various approaches to measure and interpret PLAs and discusses potential experimental factors influencing platelet binding to leukocytes. Furthermore, we summarise insights gained from studies regarding the underlying mechanism of platelet-leukocyte interactions and discuss implications of these interactions in health and disease.

The development of high-capacity, high-performance all-solid-state batteries requires the specific design and optimization of its components, especially on the positive electrode side. For the first time, we were able to produce a completely inorganic mixed positive electrode consisting only of LiCoO 2 and Ta-substituted Li 7 La 3 Zr 2 O 12 (LLZ:Ta) without the use of additional sintering aids or conducting additives, which has a high theoretical capacity density of 1 mAh/cm 2 . A true all-solid-state cell composed of a Li metal negative electrode, a LLZ:Ta garnet electrolyte, and a 25 μm thick LLZ:Ta + LiCoO 2 mixed positive electrode was manufactured and characterized. The cell shows 81% utilization of theoretical capacity upon discharging at elevated temperatures and rather high discharge rates of 0.1 mA (0.1 C). However, even though the room temperature performance is also among the highest reported so far for similar cells, it still falls far short of the theoretical values. Therefore, a 3D reconstruction of the manufactured mixed positive electrode was used for the first time as input for microstructure-resolved continuum simulations. The simulations are able to reproduce the electrochemical behavior at elevated temperature favorably, however fail completely to predict the performance loss at room temperature. Extensive parameter studies were performed to identify the limiting processes, and as a result, interface phenomena occurring at the cathode active material/solid-electrolyte interface were found to be the most probable cause for the low performance at room temperature. Furthermore, the simulations are used for a sound estimation of the optimization potential that can be realized with this type of cell, which provides important guidelines for future oxide based all-solid-state battery research and fabrication.

The severity of cardiac dysfunction predicts mortality in sepsis. Activation of transient receptor potential vanilloid receptor type (TRPV)-1, a predominantly neuronal nonselective cation channel, has been shown to improve outcome in sepsis and endotoxemia. However, the role of TRPV1 and the identity of its endogenous ligands in the cardiac dysfunction caused by sepsis and endotoxemia are unknown. Using TRPV1 -/- and TRPV1 +/+ mice, we showed that endogenous activation of cardiac TRPV1 during sepsis is key to limiting the ensuing cardiac dysfunction. Use of liquid chromatography-tandem mass spectrometry lipid analysis and selective inhibitors of arachidonic metabolism suggest that the arachidonate-derived TRPV1 activator, 20-hydroxyeicosateraenoic acid (20-HETE), underlies a substantial component of TRPV1-mediated cardioprotection in sepsis. Moreover, using selective antagonists for neuropeptide receptors, we show that this effect of TRPV1 relates to the activity of neuronally released cardiac calcitonin gene-related peptide (CGRP) and that, accordingly, administration of CGRP can rescue cardiac dysfunction in severe endotoxemia. In sum activation of TRPV1 by 20-HETE leads to the release of CGRP, which protects the heart against the cardiac dysfunction in endotoxemia and identifies both TRPV1 and CGRP receptors as potential therapeutic targets in endotoxemia.-Chen, J., Hamers, A. J. P., Finsterbusch, M., Massimo, G., Zafar, M., Corder, R., Colas, R. A., Dalli, J., Thiemermann, C., Ahluwalia, A. Endogenously generated arachidonate-derived ligands for TRPV1 induce cardiac protection in sepsis.

Although effector CD4 + T cells readily respond to antigen outside the vasculature, how they respond to intravascular antigens is unknown. Here we show the process of intravascular antigen recognition using intravital multiphoton microscopy of glomeruli. CD4 + T cells undergo intravascular migration within uninflamed glomeruli. Similarly, while MHCII is not expressed by intrinsic glomerular cells, intravascular MHCII-expressing immune cells patrol glomerular capillaries, interacting with CD4 + T cells. Following intravascular deposition of antigen in glomeruli, effector CD4 + T-cell responses, including NFAT1 nuclear translocation and decreased migration, are consistent with antigen recognition. Of the MHCII + immune cells adherent in glomerular capillaries, only monocytes are retained for prolonged durations. These cells can also induce T-cell proliferation in vitro. Moreover, monocyte depletion reduces CD4 + T-cell-dependent glomerular inflammation. These findings indicate that MHCII + monocytes patrolling the glomerular microvasculature can present intravascular antigen to CD4 + T cells within glomerular capillaries, leading to antigen-dependent inflammation.

Our aim was to investigate if deoxyribonuclease (DNase) 1 is a potential therapeutic agent to reduce pathogenic effects of cigarette smoke exposure in the lung. Cigarette smoke causes protease imbalance with excess production of proteases, which is a key process in the pathogenesis of emphysema. The mechanisms responsible for this effect are not well-defined. Our studies demonstrate both in vitro and in vivo that cigarette smoke significantly increases the expression of neutrophil and macrophage extracellular traps with coexpression of the pathogenic proteases, neutrophil elastase and matrix metalloproteinases 9 and 12. This response to cigarette smoke was significantly reduced by the addition of DNase 1, which also significantly decreased macrophage numbers and lung proteolysis. DNase 1, a treatment currently in clinical use, can diminish the pathogenic effects of cigarette smoke.

The kidney can be negatively affected by a range of innate and adaptive immune responses, resulting in alterations in the functions of the kidney and, in some cases, progression to renal failure. In many of these responses, infiltration of blood-borne leukocytes into the kidney is central to the response. In addition, a large population of mononuclear phagocytes resident in the kidney can modulate these responses. A great deal of research has investigated both the mechanisms of leukocyte recruitment to the kidney and the actions of immune cells resident within the kidney. Because of the dynamic nature of the processes whereby leukocytes enter sites of inflammation, in vivo imaging has been one of the key approaches used for understanding leukocyte recruitment as it occurs throughout the body, and this is also true for kidney. However, imaging this organ and its complicated microvasculature during different forms of renal pathology presents a unique set of challenges. In this review, we examine the approaches used for intravital imaging of the kidney and summarize the insights gained from these studies regarding the mechanisms of leukocyte entry into the kidney during inflammation and the actions of immune cells within this organ.

Nonclassical monocytes undergo intravascular patrolling in blood vessels, positioning them ideally to coordinate responses to inflammatory stimuli. Under some circumstances, the actions of monocytes have been shown to involve promotion of neutrophil recruitment. However, the mechanisms whereby patrolling monocytes control the actions of neutrophils in the circulation are unclear. Here, we examined the contributions of monocytes to antibody- and neutrophil-dependent inflammation in a model of in situ immune complex-mediated glomerulonephritis. Multiphoton and spinning disk confocal intravital microscopy revealed that monocytes patrol both uninflamed and inflamed glomeruli using β2 and α4 integrins and CX3CR1. Monocyte depletion reduced glomerular injury, demonstrating that these cells promote inappropriate inflammation in this setting. Monocyte depletion also resulted in reductions in neutrophil recruitment and dwell time in glomerular capillaries and in reactive oxygen species (ROS) generation by neutrophils, suggesting a role for cross-talk between monocytes and neutrophils in induction of glomerulonephritis. Consistent with this hypothesis, patrolling monocytes and neutrophils underwent prolonged interactions in glomerular capillaries, with the duration of these interactions increasing during inflammation. Moreover, neutrophils that interacted with monocytes showed increased retention and a greater propensity for ROS generation in the glomerulus. Also, renal patrolling monocytes, but not neutrophils, produced TNF during inflammation, and TNF inhibition reduced neutrophil dwell time and ROS production, as well as renal injury. These findings show that monocytes and neutrophils undergo interactions within the glomerular microvasculature. Moreover, evidence indicates that, in response to an inflammatory stimulus, these interactions allow monocytes to promote neutrophil recruitment and activation within the glomerular microvasculature, leading to neutrophil-dependent tissue injury., Competing Interests: The authors declare no conflict of interest.

Objective: Reduced antiplatelet drug efficacy occurs in conditions of increased platelet turnover, associated with increased proportions of drug-free, that is, uninhibited, platelets. Here, we detail mechanisms by which drug-free platelets promote platelet aggregation in the face of standard antiplatelet therapy., Approach and Results: To model standard antiplatelet therapy, platelets were treated in vitro with aspirin, the P2Y12 receptor blocker prasugrel active metabolite, or aspirin plus prasugrel active metabolite. Different proportions of uninhibited platelets were then introduced. Light transmission aggregometry analysis demonstrated clear positive associations between proportions of drug-free platelets and percentage platelet aggregation in response to a range of platelet agonists. Using differential platelet labeling coupled with advanced flow cytometry and confocal imaging we found aggregates formed in mixtures of aspirin-inhibited platelets together with drug-free platelets were characterized by intermingled platelet populations. This distribution is in accordance with the ability of drug-free platelets to generate thromboxane A2 and so drive secondary platelet activation. Conversely, aggregates formed in mixtures of prasugrel active metabolite-inhibited or aspirin plus prasugrel active metabolite-inhibited platelets together with drug-free platelets were characterized by distinct cores of drug-free platelets. This distribution is consistent with the ability of drug-free platelets to respond to the secondary activator ADP., Conclusions: These experiments are the first to image the interactions of inhibited and uninhibited platelets in the formation of platelet aggregates. They demonstrate that a general population of platelets can contain subpopulations that respond strikingly differently to overall stimulation of the population and so act as the seed for platelet aggregation., (© 2015 The Authors.)

Testing of platelet function is central to the cardiovascular phenotyping of genetically modified mice. Traditional platelet function tests have been developed primarily for testing human samples and the volumes required make them highly unsuitable for the testing of mouse platelets. This limits research in this area. To address this problem, we have developed a miniaturized whole blood aggregometry assay, based on a readily accessible 96-well plate format coupled with quantification of single platelet depletion by flow cytometric analysis. Using this approach, we observed a concentration-dependent loss of single platelets in blood exposed to arachidonic acid, collagen, U46619 or protease activated receptor 4 activating peptide. This loss was sensitive to well-established antiplatelet agents and genetic manipulation of platelet activation pathways. Observations were more deeply analyzed by flow cytometric imaging, confocal imaging, and measurement of platelet releasates. Phenotypic analysis of the reactivity of platelets taken from mice lacking intercellular adhesion molecule (ICAM)-1 identified a marked decrease in fibrinogen-dependent platelet-monocyte interactions, especially under inflammatory conditions. Such findings exemplify the value of screening platelet phenotypes of genetically modified mice and shed further light upon the roles and interactions of platelets in inflammation., (© 2015 by The American Society of Hematology.)