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Exposure to nanoparticles (NPs) is frequently associated with adverse cardiovascular effects. In contrast, NPs in nanomedicine hold great promise for precise lung‐specific drug delivery, especially considering the extensive pulmonary capillary network that facilitates interactions with bloodstream‐suspended particles. Therefore, exact knowledge about effects of engineered NPs within the pulmonary microcirculation are instrumental for future application of this technology in patients. To unravel the real‐time dynamics of intravenously delivered NPs and their effects in the pulmonary microvasculature, we employed intravital microscopy of the mouse lung. Only PEG‐amine‐QDs, but not carboxyl‐QDs triggered rapid neutrophil recruitment in microvessels and their subsequent recruitment to the alveolar space and was linked to cellular degranulation, TNF‐α, and DAMP release into the circulation, particularly eATP. Stimulation of the ATP‐gated receptor P2X7R induced expression of E‐selectin on microvascular endothelium thereby mediating the neutrophilic immune response. Leukocyte integrins LFA‐1 and MAC‐1 facilitated adhesion and decelerated neutrophil crawling on the vascular surface. In summary, this study unravels the complex cascade of neutrophil recruitment during NP‐induced sterile inflammation. Thereby we demonstrate novel adverse effects for NPs in the pulmonary microcirculation and provide critical insights for optimizing NP‐based drug delivery and therapeutic intervention strategies, to ensure their efficacy and safety in clinical applications. [ABSTRACT FROM AUTHOR]

A major shortcoming in the treatment of mesangial cell-associated diseases such as IgA nephropathy, diabetic nephropathy, or lupus nephritis, which frequently progress to end-stage renal disease, is poor drug availability in the glomerular mesangium. Drug delivery via active targeting of nanoparticles, using ligands attached to the particle surface for target cell recognition to increase the biodistribution to the mesangium, is a promising strategy to overcome this hurdle. However, although several glomerular tissue targeting approaches have been described, so far no study has demonstrated the particles' ability to deliver sufficient drug amounts combined with an appropriate nanoparticle target retention time to trigger relevant biological effects in the mesangium. In our study, we encapsulated erastin, a ferroptosis-inducing model compound, into adenovirus-mimetic, mesangial cell-targeting nanoparticles, enabling the direct visualisation of biological effects through ferroptosis-dependent histological changes. By intravital microscopy and analysis of histological sections, we were not only able to localise the injected particles over 10 days within the target cells but also to demonstrate biological activity in the renal glomeruli. In conclusion, we have characterised adenovirus-mimetic nanoparticles as a highly suitable drug delivery platform for the treatment of mesangial cell-associated diseases and additionally provided the basis for a potential renal disease model. [ABSTRACT FROM AUTHOR]

Intravital microscopy enables direct observation of cell biology and physiology at subcellular resolution in real time in living animals. Implanted windows extend the scope of intravital microscopy to processes extending for weeks or even months, such as disease progression or tumor development. However, a question that must be addressed in such studies is whether the imaging window, like any foreign body, triggers an inflammatory response, and whether that response alters the biological process under investigation. To directly evaluate this question, we conducted large-scale intravital microscopy of the kidney of LysM-EGFP mice over time after implantation of abdominal imaging windows. These studies demonstrate that windows stimulated a variety of changes consistent with a foreign body response. Within a few days of implantation, leukocytes were recruited to the window and the region between the window and kidney where, over the next 16 days, they increased in number in an expanding volume that developed a new vascular network. These changes were accompanied by a dramatic increase in glomerular albumin permeability within 2–5 days of implantation. Similar results were obtained from mice implanted with windows coated with poly(l -lysine)-graft-polyethylene glycol (PLL-g-PEG), but not from immune-deficient mice. These studies demonstrate the importance of evaluating whether implanted windows induce an inflammatory response, and whether that response impacts the processes under evaluation in longitudinal intravital microscopy studies. NEW & NOTEWORTHY: Intravital microscopy studies of LysM-EGFP mice demonstrate that abdominal imaging windows placed over the kidney stimulated a variety of changes consistent with a foreign body response. Within a day of implantation, leukocytes were recruited to the window where, over the next 16 days, they increased in number in an expanding volume that developed a new vascular network. These changes were accompanied by a dramatic increase in glomerular permeability to albumin. [ABSTRACT FROM AUTHOR]

Shock is a life-threatening condition. This study evaluated if sublingual microcirculatory perfusion on admission is associated with 30-day mortality in older intensive care unit (ICU) shock patients. This trial prospectively recruited ICU patients (≥ 80 years old) with arterial lactate above 2 mmol/L, requiring vasopressors despite adequate fluid resuscitation, regardless of shock cause. All patients received sequential sublingual measurements on ICU admission (± 4 h) and 24 (± 4) hours later. The primary endpoint was 30-day mortality. From September 4th, 2022, to May 30th, 2023, 271 patients were screened, and 44 included. Patients were categorized based on the median percentage of perfused small vessels (sPPV) into those with impaired and sustained microcirculation. 71% of videos were of good or acceptable quality without safety issues. Patients with impaired microcirculation had significantly shorter ICU and hospital stays (p = 0.015 and p = 0.019) and higher 30-day mortality (90.0% vs. 62.5%, p = 0.036). Cox regression confirmed the independent association of impaired microcirculation with 30-day mortality (adjusted hazard ratio 3.245 (95% CI 1.178 to 8.943, p = 0.023). Measuring sublingual microcirculation in critically ill older patients with shock on ICU admission is safe, feasible, and provides independent prognostic information about outcomes. Trial registration NCT04169204. [ABSTRACT FROM AUTHOR]

A comprehensive understanding of physio-pathological processes necessitates non-invasive intravital three-dimensional (3D) imaging over varying spatial and temporal scales. However, huge data throughput, optical heterogeneity, surface irregularity, and phototoxicity pose great challenges, leading to an inevitable trade-off between volume size, resolution, speed, sample health, and system complexity. Here, we introduce a compact real-time, ultra-large-scale, high-resolution 3D mesoscope (RUSH3D), achieving uniform resolutions of 2.6 × 2.6 × 6 μm3 across a volume of 8,000 × 6,000 × 400 μm3 at 20 Hz with low phototoxicity. Through the integration of multiple computational imaging techniques, RUSH3D facilitates a 13-fold improvement in data throughput and an orders-of-magnitude reduction in system size and cost. With these advantages, we observed premovement neural activity and cross-day visual representational drift across the mouse cortex, the formation and progression of multiple germinal centers in mouse inguinal lymph nodes, and heterogeneous immune responses following traumatic brain injury—all at single-cell resolution, opening up a horizon for intravital mesoscale study of large-scale intercellular interactions at the organ level. [Display omitted] • RUSH3D allows long-term, fast 3D imaging at centimeter-scale and single-cell resolution • Compact, high-fidelity mesoscale imaging is achieved in native intravital environments • Intercellular interactions across multiple mouse germinal centers are observed in vivo • Cortex-wide multisensory, locomotion neural activities and immune responses are explored in mice RUSH3D, with the integration of multiple computational imaging methods based on a scanning light-field framework, facilitates long-term, high-speed, centimeter-wide mesoscale 3D imaging at single-cell resolution in a compact system for broad practical applications in understanding large-scale intercellular dynamics at the mammalian organ level. We have demonstrated cortex-wide large-population neural recording with cross-day registration during multisensory input, the formation and progression of multiple germinal centers in mouse inguinal lymph nodes, and cortex-wide heterogeneous immune responses after traumatic brain injury. [ABSTRACT FROM AUTHOR]

In the lower respiratory tract, the alveolar spaces are divided from the bloodstream and the external environment by only a few microns of interstitial tissue. Alveolar macrophages (AMs) defend this delicate mucosal surface from invading infections by regularly patrolling the site. AMs have three behavior modalities to achieve this goal: extending cell protrusions to probe and sample surrounding areas, squeezing the whole cell body between alveoli, and patrolling by moving the cell body around each alveolus. In this study, we found Rho GTPase, cell division control protein 42 (CDC42) expression significantly decreased after berry-flavored e-cigarette (e-cig) exposure. This shifted AM behavior from squeezing to probing. Changes in AM behavior led to a reduction in the clearance of inhaled bacteria, Pseudomonas aeruginosa. These findings shed light on pathways involved in AM migration and highlight the harmful impact of e-cig vaping on AM function. [ABSTRACT FROM AUTHOR]

Abstract Shock is a life-threatening condition. This study evaluated if sublingual microcirculatory perfusion on admission is associated with 30-day mortality in older intensive care unit (ICU) shock patients. This trial prospectively recruited ICU patients (≥ 80 years old) with arterial lactate above 2 mmol/L, requiring vasopressors despite adequate fluid resuscitation, regardless of shock cause. All patients received sequential sublingual measurements on ICU admission (± 4 h) and 24 (± 4) hours later. The primary endpoint was 30-day mortality. From September 4th, 2022, to May 30th, 2023, 271 patients were screened, and 44 included. Patients were categorized based on the median percentage of perfused small vessels (sPPV) into those with impaired and sustained microcirculation. 71% of videos were of good or acceptable quality without safety issues. Patients with impaired microcirculation had significantly shorter ICU and hospital stays (p = 0.015 and p = 0.019) and higher 30-day mortality (90.0% vs. 62.5%, p = 0.036). Cox regression confirmed the independent association of impaired microcirculation with 30-day mortality (adjusted hazard ratio 3.245 (95% CI 1.178 to 8.943, p = 0.023). Measuring sublingual microcirculation in critically ill older patients with shock on ICU admission is safe, feasible, and provides independent prognostic information about outcomes. Trial registration NCT04169204.

Abstract Exposure to nanoparticles (NPs) is frequently associated with adverse cardiovascular effects. In contrast, NPs in nanomedicine hold great promise for precise lung‐specific drug delivery, especially considering the extensive pulmonary capillary network that facilitates interactions with bloodstream‐suspended particles. Therefore, exact knowledge about effects of engineered NPs within the pulmonary microcirculation are instrumental for future application of this technology in patients. To unravel the real‐time dynamics of intravenously delivered NPs and their effects in the pulmonary microvasculature, we employed intravital microscopy of the mouse lung. Only PEG‐amine‐QDs, but not carboxyl‐QDs triggered rapid neutrophil recruitment in microvessels and their subsequent recruitment to the alveolar space and was linked to cellular degranulation, TNF‐α, and DAMP release into the circulation, particularly eATP. Stimulation of the ATP‐gated receptor P2X7R induced expression of E‐selectin on microvascular endothelium thereby mediating the neutrophilic immune response. Leukocyte integrins LFA‐1 and MAC‐1 facilitated adhesion and decelerated neutrophil crawling on the vascular surface. In summary, this study unravels the complex cascade of neutrophil recruitment during NP‐induced sterile inflammation. Thereby we demonstrate novel adverse effects for NPs in the pulmonary microcirculation and provide critical insights for optimizing NP‐based drug delivery and therapeutic intervention strategies, to ensure their efficacy and safety in clinical applications.

Background: Lymphedema is chronic limb swelling resulting from lymphatic dysfunction. It affects an estimated five million Americans. There is no cure for this disease. Assessing lymphatic growth is essential in developing novel therapeutics. Intravital microscopy (IVM) is a powerful imaging tool for investigating various biological processes in live animals. Tissue nanotransfection technology (TNT) facilitates a direct, transcutaneous nonviral vector gene delivery using a chip with nanochannel poration in a rapid (<100 ms) focused electric field. TNT was used in this study to deliver the genetic cargo in the murine tail lymphedema to assess the lymphangiogenesis. The purpose of this study is to experimentally evaluate the applicability of IVM to visualize and quantify lymphatics in the live mice model. Methods and Results: The murine tail model of lymphedema was utilized. TNT was applied to the murine tail (day 0) directly at the surgical site with genetic cargo loaded into the TNT reservoir: TNTpCMV6 group receives pCMV6 (expression vector backbone alone) (n = 6); TNTProx1 group receives pCMV6-Prox1 (n = 6). Lymphatic vessels (fluorescein isothiocyanate [FITC]-dextran stained) and lymphatic branch points (indicating lymphangiogenesis) were analyzed with the confocal/multiphoton microscope. The experimental group TNTProx1 exhibited reduced postsurgical tail lymphedema and increased lymphatic distribution compared to TNTpCMV6 group. More lymphatic branching points (>3-fold) were observed at the TNT site in TNTProx1 group. Conclusions: This study demonstrates a novel, powerful imaging tool for investigating lymphatic vessels in live murine tail model of lymphedema. IVM can be utilized for functional assessment of lymphatics and visualization of lymphangiogenesis following gene-based therapy. [ABSTRACT FROM AUTHOR]

Mycobacterium abscessus (Mab) is an emerging human pathogen that has a high rate of incidence in immunocompromised individuals. We have found a putative secondary metabolite pathway within Mab, which may be a key factor in its pathogenesis. This novel pathway is encoded in a gene cluster spanning MAB_0284c to 0305 and is related to Streptomyces pathways, producing the secondary metabolites streptonigrin and nybomycin. We constructed an in-frame deletion of the MAB_0295 (phzC) gene and tested it in our Xenopus laevis animal model. We have previously shown that X. laevis tadpoles, which have functional lungs and T cells, can serve as a reliable comparative model for persistent Mab infection and pathogenesis. Here, we report that tadpoles intraperitoneally infected with the ∆phzC mutant exhibit early decreased bacterial loads and significantly increased survival compared with those infected with WT Mab. ∆phzC mutant Mab also induced lower transcript levels of several pro-inflammatory cytokines (IL-1β, TNF-α, iNOS, IFN-γ) than those of WT Mab in the liver and lungs. In addition, there was impaired macrophage recruitment and decreased macrophage infection in tadpoles infected with the ∆phzC mutant, by tail wound inoculation, compared to those infected with the WT bacteria, as assayed by intravital confocal microscopy. These data underline the relevance and usefulness of X. laevis tadpoles as a novel comparative animal model to identify genetic determinants of Mab immunopathogenesis, suggesting a role for this novel and uncharacterized pathway in Mab pathogenesis and macrophage recruitment. [ABSTRACT FROM AUTHOR]

Tissue microenvironments during physiology and pathology are highly complex, meaning dynamic cellular activities and their interactions cannot be accurately modelled ex vivo or in vitro. In particular, tissue-specific resident cells which may function and behave differently after isolation and the heterogenous vascular beds in various organs highlight the importance of observing such processes in real-time in vivo. This challenge gave rise to intravital microscopy (IVM), which was discovered over two centuries ago. From the very early techniques of low-optical resolution brightfield microscopy, limited to transparent tissues, IVM techniques have significantly evolved in recent years. Combined with improved animal surgical preparations, modern IVM technologies have achieved significantly higher speed of image acquisition and enhanced image resolution which allow for the visualisation of biological activities within a wider variety of tissue beds. These advancements have dramatically expanded our understanding in cell migration and function, especially in organs which are not easily accessible, such as the brain. In this review, we will discuss the application of rodent IVM in neurobiology in health and disease. In particular, we will outline the capability and limitations of emerging technologies, including photoacoustic, two- and three-photon imaging for brain IVM. In addition, we will discuss the use of these technologies in the context of neuroinflammation. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to investigate the effect of upper eyelid blepharoplasty with the removal of the skin and a strip of orbicularis oculi muscle on the ocular surface, tear film, and dry eye-related symptoms. Methods: Twenty-two eyes of 22 consecutive patients operated by a single surgeon (21 females; mean age, 61 years; age range, 41-75 years) were included. All subjects completed the Ocular Surface Disease Index questionnaire, underwent in vivo confocal microscopy, tear film breakup time measurements, the Schirmer test with anesthesia, and fluorescein and lissamine green staining measurements before, 1 month, and 6 months after upper blepharoplasty alone with preseptal orbicularis excision. Results: A significant increase in Ocular Surface Disease Index, and corneal fluorescein and lissamine green staining and a significant decrease in tear film breakup time were observed after 1 month (p=0.003, p=0.004, p=0.029, and p=0.024 respectively) and 6 months (p=0.001 for all findings). No significant difference in the Schirmer test score was observed during the follow-up. None of the in vivo confocal microscopy parameters showed significant changes during the study. Conclusions: An increase in dry eye symptoms and a decrease in tear film stability along with ocular surface staining were observed in patients undergoing upper eyelid blepharoplasty. [ABSTRACT FROM AUTHOR]

BACKGROUND: Pentobarbital and isoflurane are commonly used veterinary anesthetics. Due to the dangers of overdose by repeat-bolus regimen of pentobarbital, isoflurane has been recommended. However, literature suggests isoflurane-induced inhibition of cytokine and adhesion molecule release, impacting leukocyte adhesion. OBJECTIVE: This study aims to characterize the impacts of pentobarbital versus isoflurane on leukocyte interactions within the intestinal microcirculation with and without endotoxin challenge. METHODS: Female BALB/c mice were subjected to pentobarbital or isoflurane (N = 20) and challenged with endotoxin or saline by intraperitoneal injection. The mice were kept under anesthesia for 2 hours. Fluorochromes, rhodamine-6 G and fluorescein isothiocyanate, were injected intravenously. To visualize leukocyte adhesion within the intestinal microcirculation, laparotomy and intravital microscopy was performed. Leukocyte rolling and adhesion was quantified offline in a blinded fashion. RESULTS: Within collecting venules, leukocyte rolling and adhesion showed no significant differences between pentobarbital and isoflurane anesthesia under basal conditions. Endotoxin challenge caused a similar response in both anesthetic groups. Within postcapillary venules, no statistical differences between the two anesthetics were found for adhering leukocytes under basal conditions or following endotoxin challenge either. However, leukocyte rolling after LPS-challenge was significantly decreased in postcapillary venules during isoflurane anesthesia compared to pentobarbital anesthesia. CONCLUSIONS: Isoflurane anesthesia showed only minor differences in the immune response to endotoxin within the intestinal microcirculation compared to pentobarbital anesthesia. Due to the superior safety profile of volatile anesthetics, immunological studies may choose isoflurane over pentobarbital as the veterinary anesthetic of choice. [ABSTRACT FROM AUTHOR]

Coupling red blood cell (RBC) supply to O2 demand is an intricate process requiring O2 sensing, generation of a stimulus, and signal transduction that alters upstream arteriolar tone. Although actively debated, this process has been theorized to be induced by hypoxia and to involve activation of endothelial inwardly rectifying K+ channels (KIR) 2.1 by elevated extracellular K+ to trigger conducted hyperpolarization via connexin40 (Cx40) gap junctions to upstream resistors. This concept was tested in resting healthy skeletal muscle of Cx40-/- and endothelial KIR2.1-/- mice using state-of-the-art live animal imaging where the local tissue O2 environment was manipulated using a custom gas chamber. Second-by-second capillary RBC flow responses were recorded as O2 was altered. A stepwise drop in PO2 at the muscle surface increased RBC supply in capillaries of control animals while elevated O2 elicited the opposite response; capillaries were confirmed to express Cx40. The RBC flow responses were rapid and tightly coupled to O2; computer simulations did not support hypoxia as a driving factor. In contrast, RBC flow responses were significantly diminished in Cx40-/- mice. Endothelial KIR2.1-/- mice, on the other hand, reacted normally to O2 changes, even when the O2 challenge was targeted to a smaller area of tissue with fewer capillaries. Conclusively, microvascular O2 responses depend on coordinated electrical signaling via Cx40 gap junctions, and endothelial KIR2.1 channels do not initiate the event. These findings reconceptualize the paradigm of blood flow regulation in skeletal muscle and how O2 triggers this process in capillaries independent of extracellular K+. [ABSTRACT FROM AUTHOR]

Optimising drug delivery to tumours remains an obstacle to effective cancer treatment. A prerequisite for successful chemotherapy is that the drugs reach all tumour cells. The vascular network of tumours, extravasation across the capillary wall and penetration throughout the extracellular matrix limit the delivery of drugs. Ultrasound combined with microbubbles has been shown to improve the therapeutic response in preclinical and clinical studies. Most studies apply microbubbles designed as ultrasound contrast agents. Acoustic Cluster Therapy (ACT®) is a novel approach based on ultrasound-activated microbubbles, which have a diameter 5–10 times larger than regular contrast agent microbubbles. An advantage of using such large microbubbles is that they are in contact with a larger part of the capillary wall, and the oscillating microbubbles exert more effective biomechanical effects on the vessel wall. In accordance with this, ACT® has shown promising therapeutic results in combination with various drugs and drug-loaded nanoparticles. Knowledge of the mechanism and behaviour of drugs and microbubbles is needed to optimise ACT®. Real-time intravital microscopy (IVM) is a useful tool for such studies. This paper presents the experimental setup design for visualising ACT® microbubbles within the vasculature of tumours implanted in dorsal window (DW) chambers. It presents ultrasound setups, the integration and alignment of the ultrasound field with the optical system in live animal experiments, and the methodologies for visualisation and analysing the recordings. Dextran was used as a fluorescent marker to visualise the blood vessels and to trace drug extravasation and penetration into the extracellular matrix. The results reveal that the experimental setup successfully recorded the kinetics of extravasation and penetration distances into the extracellular matrix, offering a deeper understanding of ACT's mechanisms and potential in localised drug delivery. [ABSTRACT FROM AUTHOR]

Tissue microenvironments during physiology and pathology are highly complex, meaning dynamic cellular activities and their interactions cannot be accurately modelled ex vivo or in vitro. In particular, tissue-specific resident cells which may function and behave differently after isolation and the heterogenous vascular beds in various organs highlight the importance of observing such processes in real-time in vivo. This challenge gave rise to intravital microscopy (IVM), which was discovered over two centuries ago. From the very early techniques of low-optical resolution brightfield microscopy, limited to transparent tissues, IVM techniques have significantly evolved in recent years. Combined with improved animal surgical preparations, modern IVM technologies have achieved significantly higher speed of image acquisition and enhanced image resolution which allow for the visualisation of biological activities within a wider variety of tissue beds. These advancements have dramatically expanded our understanding in cell migration and function, especially in organs which are not easily accessible, such as the brain. In this review, we will discuss the application of rodent IVM in neurobiology in health and disease. In particular, we will outline the capability and limitations of emerging technologies, including photoacoustic, two- and three-photon imaging for brain IVM. In addition, we will discuss the use of these technologies in the context of neuroinflammation.

Introduction: Opening occluded coronary arteries in patients with myocardial infarction (MI) damages the delicate coronary microvessels through a process called myocardial ischaemia-reperfusion injury. Although mesenchymal stromal cells (MSCs) have the potential to limit this injury, clinical success remains limited. This may be due to (i) poor MSC homing to the heart (ii) infused MSCs, even if derived from the same site, being a heterogeneous population with varying therapeutic efficacy and (iii) conventional 2D culture of MSCs decreasing their homing and beneficial properties. This study investigated whether 3D culture of two distinctly different bone marrow (BM)-derived MSC sub-populations could improve their homing and coronary vasculoprotective efficacy. Methods: Intravital imaging of the anaesthetised mouse beating heart was used to investigate the trafficking and microvascular protective effects of two clonallyderived BM-derived MSC lines, namely CD317neg MSCs-Y201 and CD317pos MSCs-Y202, cultured using conventional monolayer and 3D hanging drop methods. Results: 3D culture consistently improved the adhesive behaviour of MSCs-Y201 to various substrates in vitro. However, it was their differential ability to reduce neutrophil events within the coronary capillaries and improve ventricular perfusion in vivo that was most remarkable. Moreover, dual therapy combined with heparin further improved the vasculoprotection afforded by 3D cultured MSCs-Y201 by also modifying platelet as well as neutrophil recruitment, which subsequently led to the greatest salvage of viable myocardium. Therapeutic benefit could mechanistically be explained by reductions in coronary endothelial oxidative stress and intercellular adhesion molecule-1 (ICAM-1)/vascular cell adhesion molecule-1 (VCAM-1) expression. However, since this was noted by both 2D and 3D cultured MSCs-Y201, therapeutic benefit is likely explained by the fact that 3D cultured MSCs-Y201 were the most potent sub-population at reducing serum levels of several pro-inflammatory cytokines. Conclusion: This novel study highlights the importance of not only 3D culture, but also of a specific CD317neg MSC sub-population, as being critical to realising their full coronary vasculoprotective potential in the injured heart. Since the smallest coronary blood vessels are increasingly recognised as a primary target of reperfusion injury, therapeutic interventions must be able to protect these delicate structures from inflammatory cells and maintain perfusion in the heart. We propose that relatively feasible technical modifications in a specific BMderived MSC sub-population could achieve this. [ABSTRACT FROM AUTHOR]

Insufficient drug accumulation in tumors is still a major concern for using cancer nanotherapeutics. Here, the neutrophil-based delivery of three nanoparticle types—liposomes, PLGA, and magnetite nanoparticles—was assessed both in vitro and in vivo. Confocal microscopy and a flow cytometry analysis demonstrated that all the studied nanoparticles interacted with neutrophils from the peripheral blood of mice with 4T1 mammary adenocarcinoma without a significant impact on neutrophil viability or activation state. Intravital microscopy of the tumor microenvironment showed that the neutrophils did not engulf the liposomes after intravenous administration, but facilitated nanoparticle extravasation in tumors through micro- and macroleakages. PLGA accumulated along the vessel walls in the form of local clusters. Later, PLGA nanoparticle-loaded neutrophils were found to cross the vascular barrier and migrate towards the tumor core. The magnetite nanoparticles extravasated in tumors both via spontaneous macroleakages and on neutrophils. Overall, the specific type of nanoparticles largely determined their behavior in blood vessels and their neutrophil-mediated delivery to the tumor. Since neutrophils are the first to migrate to the site of inflammation, they can increase nanodrug delivery effectiveness for nanomedicine application. [ABSTRACT FROM AUTHOR]

Whole brain irradiation (WBI), a commonly employed therapy for multiple brain metastases and as a prophylactic measure after cerebral metastasis resection, is associated with a progressive decline in neurocognitive function, significantly impacting the quality of life for approximately half of the surviving patients. Recent preclinical investigations have shed light on the multifaceted cerebrovascular injury mechanisms underlying this side effect of WBI. In this study, we aimed to test the hypothesis that WBI induces endothelial senescence, contributing to chronic disruption of the blood-brain barrier (BBB) and microvascular rarefaction. To accomplish this, we utilized transgenic p16-3MR mice, which enable the identification and selective elimination of senescent cells. These mice were subjected to a clinically relevant fractionated WBI protocol (5 Gy twice weekly for 4 weeks), and cranial windows were applied to both WBI-treated and control mice. Quantitative assessment of BBB permeability and capillary density was performed using two-photon microscopy at the 6-month post-irradiation time point. The presence of senescent microvascular endothelial cells was assessed by imaging flow cytometry, immunolabeling, and single-cell RNA-sequencing (scRNA-seq). WBI induced endothelial senescence, which associated with chronic BBB disruption and a trend for decreased microvascular density in the mouse cortex. In order to investigate the cause-and-effect relationship between WBI-induced senescence and microvascular injury, senescent cells were selectively removed from animals subjected to WBI treatment using Navitoclax/ABT263, a well-known senolytic drug. This intervention was carried out at the 3-month post-WBI time point. In WBI-treated mice, Navitoclax/ABT263 effectively eliminated senescent endothelial cells, which was associated with decreased BBB permeability and a trend for increased cortical capillarization. Our findings provide additional preclinical evidence that senolytic treatment approaches may be developed for prevention of the side effects of WBI. [ABSTRACT FROM AUTHOR]

Mycobacterium abscessus (Mab) is an emerging human pathogen that has a high rate of incidence in immunocompromised individuals. We have found a putative secondary metabolite pathway within Mab, which may be a key factor in its pathogenesis. This novel pathway is encoded in a gene cluster spanning MAB_0284c to 0305 and is related to Streptomyces pathways, producing the secondary metabolites streptonigrin and nybomycin. We constructed an in-frame deletion of the MAB_0295 (phzC) gene and tested it in our Xenopus laevis animal model. We have previously shown that X. laevis tadpoles, which have functional lungs and T cells, can serve as a reliable comparative model for persistent Mab infection and pathogenesis. Here, we report that tadpoles intraperitoneally infected with the ∆phzC mutant exhibit early decreased bacterial loads and significantly increased survival compared with those infected with WT Mab. ∆phzC mutant Mab also induced lower transcript levels of several pro-inflammatory cytokines (IL-1β, TNF-α, iNOS, IFN-γ) than those of WT Mab in the liver and lungs. In addition, there was impaired macrophage recruitment and decreased macrophage infection in tadpoles infected with the ∆phzC mutant, by tail wound inoculation, compared to those infected with the WT bacteria, as assayed by intravital confocal microscopy. These data underline the relevance and usefulness of X. laevis tadpoles as a novel comparative animal model to identify genetic determinants of Mab immunopathogenesis, suggesting a role for this novel and uncharacterized pathway in Mab pathogenesis and macrophage recruitment.

Multiple blood-borne pathogens infecting mammals establish close interactions with the host vascular endothelium as part of their life cycles. In this work, we investigate differences in the interactions of three Trypanosoma species: T. brucei, T. congolense and T. vivax with the blood vasculature. Infection with these species results in vastly different pathologies, including different effects on vascular homeostasis, such as changes in vascular permeability and microhemorrhages. While all three species are extracellular parasites, T. congolense is strictly intravascular, while T. brucei is capable of surviving both extra- and intravascularly. Our knowledge regarding T. vivax tropism and its capacity of migration across the vascular endothelium is unknown. In this work, we show for the first time that T. vivax parasites sequester to the vascular endothelium of most organs, and that, like T. congolense, T. vivax Y486 is largely incapable of extravasation. Infection with this parasite species results in a unique effect on vascular endothelium receptors including general downregulation of ICAM1 and ESAM, and upregulation of VCAM1, CD36 and E-selectin. Our findings on the differences between the two sequestering species (T. congolense and T. vivax) and the non-sequestering, but extravasating, T. brucei raise important questions on the relevance of sequestration to the parasite’s survival in the mammalian host, and the evolutionary relevance of both sequestration and extravasation.

IntroductionOpening occluded coronary arteries in patients with myocardial infarction (MI) damages the delicate coronary microvessels through a process called myocardial ischaemia-reperfusion injury. Although mesenchymal stromal cells (MSCs) have the potential to limit this injury, clinical success remains limited. This may be due to (i) poor MSC homing to the heart (ii) infused MSCs, even if derived from the same site, being a heterogeneous population with varying therapeutic efficacy and (iii) conventional 2D culture of MSCs decreasing their homing and beneficial properties. This study investigated whether 3D culture of two distinctly different bone marrow (BM)-derived MSC sub-populations could improve their homing and coronary vasculoprotective efficacy.MethodsIntravital imaging of the anaesthetised mouse beating heart was used to investigate the trafficking and microvascular protective effects of two clonally-derived BM-derived MSC lines, namely CD317neg MSCs-Y201 and CD317pos MSCs-Y202, cultured using conventional monolayer and 3D hanging drop methods.Results3D culture consistently improved the adhesive behaviour of MSCs-Y201 to various substrates in vitro. However, it was their differential ability to reduce neutrophil events within the coronary capillaries and improve ventricular perfusion in vivo that was most remarkable. Moreover, dual therapy combined with heparin further improved the vasculoprotection afforded by 3D cultured MSCs-Y201 by also modifying platelet as well as neutrophil recruitment, which subsequently led to the greatest salvage of viable myocardium. Therapeutic benefit could mechanistically be explained by reductions in coronary endothelial oxidative stress and intercellular adhesion molecule-1 (ICAM-1)/vascular cell adhesion molecule-1 (VCAM-1) expression. However, since this was noted by both 2D and 3D cultured MSCs-Y201, therapeutic benefit is likely explained by the fact that 3D cultured MSCs-Y201 were the most potent sub-population at reducing serum levels of several pro-inflammatory cytokines.ConclusionThis novel study highlights the importance of not only 3D culture, but also of a specific CD317neg MSC sub-population, as being critical to realising their full coronary vasculoprotective potential in the injured heart. Since the smallest coronary blood vessels are increasingly recognised as a primary target of reperfusion injury, therapeutic interventions must be able to protect these delicate structures from inflammatory cells and maintain perfusion in the heart. We propose that relatively feasible technical modifications in a specific BM-derived MSC sub-population could achieve this.

Abstract Inflammation can contribute to the development and progression of cancer. The inflammatory responses in the tumor microenvironment are shaped by complex sequences of dynamic intercellular cross-talks among diverse types of cells, and recapitulation of these dynamic events in vitro has yet to be achieved. Today, intravital microscopy with two-photon excitation microscopes (2P-IVM) is the mainstay technique for observing intercellular cross-talks in situ, unraveling cellular and molecular mechanisms in the context of their spatiotemporal dynamics. In this review, we summarize the current state of 2P-IVM with fluorescent indicators of signal transduction to reveal the cross-talks between cancer cells and surrounding cells including both immune and non-immune cells. We also discuss the potential application of red-shifted indicators along with optogenetic tools to 2P-IVM. In an era of single-cell transcriptomics and data-driven research, 2P-IVM will remain a key advantage in delivering the missing spatiotemporal context in the field of cancer research.

The exocrine pancreas secretes fluid and digestive enzymes in response to parasympathetic release of acetylcholine (ACh) via the vagus nerve and the gut hormone cholecystokinin (CCK). Both secretion of fluid and exocytosis of secretory granules containing enzymes and zymogens are dependent on an increase in the cytosolic [Ca2+] in acinar cells. It is thought that the specific spatiotemporal characteristics of the Ca2+ signals are fundamental for appropriate secretion and that these properties are disrupted in disease states in the pancreas. While extensive research has been performed to characterize Ca2+ signalling in acinar cells, this has exclusively been achieved in ex vivo preparations of exocrine cells, where it is difficult to mimic physiological conditions. Here we have developed a method to optically observe pancreatic acinar Ca2+ signals in vivo using a genetically expressed Ca2+ indicator and imaged with multi‐photon microscopy in live animals. In vivo, acinar cells exhibited baseline activity in fasted animals, which was dependent on CCK1 receptors (CCK1Rs). Both stimulation of intrinsic nervous input and administration of systemic CCK induced oscillatory activity in a proportion of the cells, but the maximum frequencies were vastly different. Upon feeding, oscillatory activity was also observed, which was dependent on CCK1Rs. No evidence of a vago‐vagal reflex mediating the effects of CCK was observed. Our in vivo method revealed the spatial and temporal profile of physiologically evoked Ca2+ signals, which will provide new insights into future studies of the mechanisms underlying exocrine physiology and that are disrupted in pathological conditions. Key points: In the exocrine pancreas, the spatiotemporal properties of Ca2+ signals are fundamentally important for the appropriate stimulation of secretion by the neurotransmitter acetylcholine and gut hormone cholecystokinin.These characteristics were previously defined in ex vivo studies. Here we report the spatiotemporal characteristics of Ca2+ signals in vivo in response to physiological stimulation in a mouse engineered to express a Ca2+ indicator in acinar cells.Specific Ca2+ 'signatures' probably important for stimulating secretion are evoked in vivo in fasted animals, by feeding, neural stimulation and cholecystokinin administration.The Ca2+ signals are probably the result of the direct action of ACh and CCK on acinar cells and not indirectly through a vago‐vagal reflex. [ABSTRACT FROM AUTHOR]

Despite claims of safety or harm reduction for electronic cigarettes (E-cig) use (also known as vaping), emerging evidence indicates that E-cigs are not likely safe, or necessarily safer than traditional cigarettes, when considering the user's risk of developing vascular dysfunction/disease. E-cigs are different from regular cigarettes in that E-cig devices are highly customizable, and users can change the e-liquid composition (such as the base solution, flavors, and nicotine level). Since the effects of E-cigs on the microvascular responses in skeletal muscle are poorly understood, we used intravital microscopy with an acute (one-time 10 puff) exposure paradigm to evaluate the individual components of e-liquid on vascular tone and endothelial function in the arterioles of the gluteus maximus muscle of anesthetized C57Bl/6 mice. Consistent with the molecular responses seen with endothelial cells, we found that the peripheral vasoconstriction response was similar between mice exposed to E-cig aerosol or cigarette smoke (i.e., 3R4F reference cigarette); this response was not nicotine dependent, and endothelial cell-mediated vasodilation was not altered within this acute exposure paradigm. We also report that, regardless of the base solution component [i.e., vegetable glycerin (VG)-only or propylene glycol (PG)-only], the vasoconstriction responses were the same in mice with inhalation exposure to 3R4F cigarette smoke or E-cig aerosol. Key findings from this work reveal that some component other than nicotine, in inhaled smoke or aerosol, is responsible for triggering peripheral vasoconstriction in skeletal muscle, and that regardless of one's preference for an E-cig base solution composition (i.e., ratio of VG-to-PG), the acute physiological response to blood vessels appears to be the same. The data suggest that vaping is not likely to be 'safer' than smoking towards blood vessels and can be expected to produce and/or result in the same adverse vascular health outcomes associated with smoking cigarettes. [ABSTRACT FROM AUTHOR]

Purpose: Shock is a life-threatening condition characterized by substantial alterations in the microcirculation. This study tests the hypothesis that considering sublingual microcirculatory perfusion variables in the therapeutic management reduces 30-day mortality in patients admitted to the intensive care unit (ICU) with shock. Methods: This randomized, prospective clinical multicenter trial-recruited patients with an arterial lactate value above two mmol/L, requiring vasopressors despite adequate fluid resuscitation, regardless of the cause of shock. All patients received sequential sublingual measurements using a sidestream-dark field (SDF) video microscope at admission to the intensive care unit (± 4 h) and 24 (± 4) hours later that was performed blindly to the treatment team. Patients were randomized to usual routine or to integrating sublingual microcirculatory perfusion variables in the therapy plan. The primary endpoint was 30-day mortality, secondary endpoints were length of stay on the ICU and the hospital, and 6-months mortality. Results: Overall, we included 141 patients with cardiogenic (n = 77), post cardiac surgery (n = 27), or septic shock (n = 22). 69 patients were randomized to the intervention and 72 to routine care. No serious adverse events (SAEs) occurred. In the interventional group, significantly more patients received an adjustment (increase or decrease) in vasoactive drugs or fluids (66.7% vs. 41.8%, p = 0.009) within the next hour. Microcirculatory values 24 h after admission and 30-day mortality did not differ [crude: 32 (47.1%) patients versus 25 (34.7%), relative risk (RR) 1.39 (0.91–1.97); Cox-regression: hazard ratio (HR) 1.54 (95% confidence interval (CI) 0.90–2.66, p = 0.118)]. Conclusion: Integrating sublingual microcirculatory perfusion variables in the therapy plan resulted in treatment changes that do not improve survival at all. [ABSTRACT FROM AUTHOR]

Beta-caryophyllene has demonstrated anti-inflammatory effects in a variety of conditions, including interstitial cystitis. These effects are mediated primarily via the activation of the cannabinoid type 2 receptor. Additional antibacterial properties have recently been suggested, leading to our investigation of the effects of beta-caryophyllene in a murine model of urinary tract infection (UTI). Female BALB/c mice were intravesically inoculated with uropathogenic Escherichia coli CFT073. The mice received either beta-caryophyllene, antibiotic treatment using fosfomycin, or combination therapy. After 6, 24, or 72 h, the mice were evaluated for bacterial burden in the bladder and changes in pain and behavioral responses using von Frey esthesiometry. In the 24 h model, the anti-inflammatory effects of beta-caryophyllene were also assessed using intravital microscopy. The mice established a robust UTI by 24 h. Altered behavioral responses persisted 72 h post infection. Treatment with beta-caryophyllene resulted in a significant reduction in the bacterial burden in urine and bladder tissues 24 h post UTI induction and significant improvements in behavioral responses and intravital microscopy parameters, representing reduced inflammation in the bladder. This study demonstrates the utility of beta-caryophyllene as a new adjunct therapy for the management of UTI. [ABSTRACT FROM AUTHOR]

Th1 and Th17 cell migration into the central nervous system (CNS) is a fundamental process in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Particularly, leptomeningeal vessels of the subarachnoid space (SAS) constitute a central route for T cell entry into the CNS during EAE. Once migrated into the SAS, T cells show an active motility behavior, which is a prerequisite for cell-cell communication, in situ reactivation and neuroinflammation. However, the molecular mechanisms selectively controlling Th1 and Th17 cell trafficking in the inflamed leptomeninges are not well understood. By using epifluorescence intravital microscopy, we obtained results showing that myelin-specific Th1 and Th17 cells have different intravascular adhesion capacity depending on the disease phase, with Th17 cells being more adhesive at disease peak. Inhibition of αLβ2 integrin selectively blocked Th1 cell adhesion, but had no effect on Th17 rolling and arrest capacity during all disease phases, suggesting that distinct adhesion mechanisms control the migration of key T cell populations involved in EAE induction. Blockade of α4 integrins affected myelin-specific Th1 cell rolling and arrest, but only selectively altered intravascular arrest of Th17 cells. Notably, selective α4β7 integrin blockade inhibited Th17 cell arrest without interfering with intravascular Th1 cell adhesion, suggesting that α4β7 integrin is predominantly involved in Th17 cell migration into the inflamed leptomeninges in EAE mice. Two-photon microscopy experiments showed that blockade of α4 integrin chain or α4β7 integrin selectively inhibited the locomotion of extravasated antigen-specific Th17 cells in the SAS, but had no effect on Th1 cell intratissue dynamics, further pointing to α4β7 integrin as key molecule in Th17 cell trafficking during EAE development. Finally, therapeutic inhibition of α4β7 integrin at disease onset by intrathecal injection of a blocking antibody attenuated clinical severity and reduced neuroinflammation, further demonstrating a crucial role for α4β7 integrin in driving Th17 cell-mediated disease pathogenesis. Altogether, our data suggest that a better knowledge of the molecular mechanisms controlling myelin-specific Th1 and Th17 cell trafficking during EAE delevopment may help to identify new therapeutic strategies for CNS inflammatory and demyelinating diseases. [ABSTRACT FROM AUTHOR]

Background: Smallfor- size syndrome ( SFSS) is a major complication following extended liver resection. The role of platelets in the early development of SFSS remains to be cleared. We aimed to investigate the impact of platelets and PAR-4, a receptor for platelet activation, on the acute phase microcirculatory injury after liver resection by in vivo microscopy analyzing the changes in leukocyte recruitment, platelet-neutrophil interaction, and microthrombosis-induced perfusion failure. Methods: Sixty - percent partial hepatectomy ( PH ) models using £ 57BL / 6 mice receiving platelet depiction with anti-GPIbcz, PAR-4 blockade with tcY-NH2, or vehicle treatment with saline were used. Sham-operated animals served as controls. Epifluorescence microscopic analysis was performed 2 h after PH to quantify the leukocyte recruitment and microcirculatory changes. Sinusoidal neutrophil recruitment, platelet-neutrophil interaction, and microthrombosis were evaluated using two-photon microscopy. ICAM-1 expression and liver liver injury were assessed in tissue/blood samples. Results: The increments of leukocyte recruitment in post„sinusoidal venules and sinusoidal perfusion failure, the upregulation of ICAM-1 expression, and the deterioration of liver function 2 h after 60% PH were alleviated in the absence of platelets or by PAR-4 blockade. Intensified platelet-neutrophil interaction and microthrombosis in sinusoids were observed 2 h after 60% PH, which significantly attenuated after PAR-4 blockade. Conclusion: Platelets play a critical role in acute liver injury after extended liver resection within 2 h. The deactivation of platelets via PAR-4 blockade ameliorated liver function deterioration by suppressing early leukocyte recruitment, platelet-neutrophil interaction, and microthrombosis in hepatic sinusoids. [ABSTRACT FROM AUTHOR]

Inflammation is essential in the protection of the organism and wound repair, but in cases of chronic inflammation can also cause microvasculature deterioration. Thus, inflammation monitorization studies are important to test potential therapeutics. The intravital microscopy (IVM) technique monitors leukocyte trafficking in vivo, being a commonly used procedure to report systemic conditions. Although the cremaster muscle, an established protocol for IVM, may affect the hemodynamics because of its surgical preparation, only male animals are used, and longitudinal studies over time are not feasible. Thinking how this impacts future studies, our aim is to understand if the IVM technique can be successfully performed using the ear lobe instead of the cremaster muscle. Elevated IL-1β plasmatic concentrations confirmed the systemic inflammation developed in a diabetic animal model, while the elevated number of adherent and rolling leukocytes in the ear lobe allowed for the same conclusion. Thus, this study demonstrates that albeit its thickness, the ear lobe protocol for IVM is efficient, non-invasive, more reliable, cost-effective and timesaving. [ABSTRACT FROM AUTHOR]

Optimising drug delivery to tumours remains an obstacle to effective cancer treatment. A prerequisite for successful chemotherapy is that the drugs reach all tumour cells. The vascular network of tumours, extravasation across the capillary wall and penetration throughout the extracellular matrix limit the delivery of drugs. Ultrasound combined with microbubbles has been shown to improve the therapeutic response in preclinical and clinical studies. Most studies apply microbubbles designed as ultrasound contrast agents. Acoustic Cluster Therapy (ACT®) is a novel approach based on ultrasound-activated microbubbles, which have a diameter 5–10 times larger than regular contrast agent microbubbles. An advantage of using such large microbubbles is that they are in contact with a larger part of the capillary wall, and the oscillating microbubbles exert more effective biomechanical effects on the vessel wall. In accordance with this, ACT® has shown promising therapeutic results in combination with various drugs and drug-loaded nanoparticles. Knowledge of the mechanism and behaviour of drugs and microbubbles is needed to optimise ACT®. Real-time intravital microscopy (IVM) is a useful tool for such studies. This paper presents the experimental setup design for visualising ACT® microbubbles within the vasculature of tumours implanted in dorsal window (DW) chambers. It presents ultrasound setups, the integration and alignment of the ultrasound field with the optical system in live animal experiments, and the methodologies for visualisation and analysing the recordings. Dextran was used as a fluorescent marker to visualise the blood vessels and to trace drug extravasation and penetration into the extracellular matrix. The results reveal that the experimental setup successfully recorded the kinetics of extravasation and penetration distances into the extracellular matrix, offering a deeper understanding of ACT’s mechanisms and potential in localised drug delivery.

Th1 and Th17 cell migration into the central nervous system (CNS) is a fundamental process in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis (MS). Particularly, leptomeningeal vessels of the subarachnoid space (SAS) constitute a central route for T cell entry into the CNS during EAE. Once migrated into the SAS, T cells show an active motility behavior, which is a prerequisite for cell-cell communication, in situ reactivation and neuroinflammation. However, the molecular mechanisms selectively controlling Th1 and Th17 cell trafficking in the inflamed leptomeninges are not well understood. By using epifluorescence intravital microscopy, we obtained results showing that myelin-specific Th1 and Th17 cells have different intravascular adhesion capacity depending on the disease phase, with Th17 cells being more adhesive at disease peak. Inhibition of αLβ2 integrin selectively blocked Th1 cell adhesion, but had no effect on Th17 rolling and arrest capacity during all disease phases, suggesting that distinct adhesion mechanisms control the migration of key T cell populations involved in EAE induction. Blockade of α4 integrins affected myelin-specific Th1 cell rolling and arrest, but only selectively altered intravascular arrest of Th17 cells. Notably, selective α4β7 integrin blockade inhibited Th17 cell arrest without interfering with intravascular Th1 cell adhesion, suggesting that α4β7 integrin is predominantly involved in Th17 cell migration into the inflamed leptomeninges in EAE mice. Two-photon microscopy experiments showed that blockade of α4 integrin chain or α4β7 integrin selectively inhibited the locomotion of extravasated antigen-specific Th17 cells in the SAS, but had no effect on Th1 cell intratissue dynamics, further pointing to α4β7 integrin as key molecule in Th17 cell trafficking during EAE development. Finally, therapeutic inhibition of α4β7 integrin at disease onset by intrathecal injection of a blocking antibody attenuated clinical severity and reduced neuroinflammation, further demonstrating a crucial role for α4β7 integrin in driving Th17 cell-mediated disease pathogenesis. Altogether, our data suggest that a better knowledge of the molecular mechanisms controlling myelin-specific Th1 and Th17 cell trafficking during EAE delevopment may help to identify new therapeutic strategies for CNS inflammatory and demyelinating diseases.

The principle of enhanced permeability and retention (EPR) effect has been used to design anti-cancer nanomedicines over decades. However, it is being challenged due to the poor clinical outcome of nanoparticles and controversial physiological foundation. Herein, we use a near-infrared-II (1000–1700 nm, NIR-II) fluorescence probe BPBBT to investigate the pathway for the entry of human serum albumin-bound nanoparticles (BPBBT-HSA NPs) into tumor compared with BPBBT micelles with phospholipid-poly (ethylene glycol) of the similar particle size about 110 nm. The plasma elimination half-life of BPBBT micelles was 2.8-fold of that of BPBBT-HSA NPs. However, the area under the BPBBT concentration in tumor-time curve to 48 h post-injection (AUCtumor0→48h) of BPBBT-HSA NPs was 7.2-fold of that of BPBBT micelles. The intravital NIR-II fluorescence microscopy revealed that BPBBT-HSA NPs but not BPBBT micelles were transported from the tumor vasculature into tumor parenchyma with high efficiency, and endocytosed by the tumor cells within 3 h post-injection in vivo. This effect was blocked by cross-linking BPBBT-HSA NPs to denature HSA, resulting in the AUCtumor0→48h decreased to 22% of that of BPBBT-HSA NPs. Our results demonstrated that the active process of endothelial transcytosis is the dominant pathway for albumin-bound nanoparticles' entry into tumor. [ABSTRACT FROM AUTHOR]

Background Vascular abnormalities and endothelial dysfunction are part of the spectrum of autosomal dominant polycystic kidney disease (ADPKD). The mechanisms behind these manifestations, including potential effects on the endothelial surface layer (ESL) and glycocalyx integrity, remain unknown. Methods Forty-five ambulatory adult patients with ADPKD were enrolled in this prospective, observational, cross-sectional, single-centre study. Fifty-one healthy volunteers served as a control group. All participants underwent real-time microvascular perfusion measurements of the sublingual microcirculation using sidestream dark field imaging. After image acquisition, the perfused boundary region (PBR), an inverse parameter for red blood cell (RBC) penetration into the ESL, was automatically calculated. Microvascular perfusion was assessed by RBC filling and capillary density. Concentrations of circulating glycocalyx components were determined by enzyme-linked immunosorbent assay. Results ADPKD patients showed a significantly larger PBR compared with healthy controls (2.09 ± 0.23 µm versus 1.79 ± 0.25 µm; P  < .001). This was accompanied by significantly lower RBC filling (70.4 ± 5.0% versus 77.9 ± 5.4%; P  < .001) as well as a higher valid capillary density {318/mm2 [interquartile range (IQR) 269–380] versus 273/mm2 [230–327]; P  = .007}. Significantly higher plasma concentrations of heparan sulphate (1625 ± 807 ng/ml versus 1329 ± 316 ng/ml; P  = .034), hyaluronan (111 ng/ml [IQR 79–132] versus 92 ng/ml [82–98]; P  = .042) and syndecan-1 were noted in ADPKD patients compared with healthy controls (35 ng/ml [IQR 27–57] versus 29 ng/ml [23–42]; P  = .035). Conclusions Dimensions and integrity of the ESL are impaired in ADPKD patients. Increased capillary density may be a compensatory mechanism for vascular dysfunction to ensure sufficient tissue perfusion and oxygenation. [ABSTRACT FROM AUTHOR]

Due to the limitations of current in vivo experimental designs, our comprehensive knowledge of vascular development and its implications for the development of large-scale engineered tissue constructs is very limited. Therefore, the purpose of this study was to develop unique in vivo imaging chambers that allow the live visualization of cellular processes in the arteriovenous (AV) loop model in rats. We have developed two different types of chambers. Chamber A is installed in the skin using the purse sting fixing method, while chamber B is installed subcutaneously under the skin. Both chambers are filled with modified gelatin hydrogel as a matrix. Intravital microscopy (IVM) was performed after the injection of fluorescein isothiocyanate (FITC)-labeled dextran and rhodamine 6G dye. The AV loop was functional for two weeks in chamber A and allowed visualization of the leukocyte trafficking. In chamber B, microvascular development in the AV loop could be examined for 21 days. Quantification of the microvascular outgrowth was performed using Fiji-ImageJ. Overall, by combining these two IVM chambers, we can comprehensively understand vascular development in the AV loop tissue engineering model¯. [ABSTRACT FROM AUTHOR]

Objective: Post‐hepatectomy liver failure (PHLF) is the main limitation of extended liver resection. The molecular mechanism and the role of leukocytes in the development of PHLF remain to be unveiled. We aimed to address the impact of serine proteases (SPs) on the acute phase after liver resection by intravitally analyzing leukocyte recruitment and changes in hemodynamics and microcirculation of the liver. Methods: C57BL/6 mice undergoing 60% partial hepatectomy were treated with aprotinin (broad‐spectrum SP inhibitor), tranexamic acid (plasmin inhibitor), or vehicle. Sham‐operated animals served as controls. In vivo fluorescence microscopy was used to quantify leukocyte‐endothelial interactions immediately after, as well as 120 min after partial hepatectomy in postsinusoidal venules, along with measurement of sinusoidal perfusion rate and postsinusoidal shear rate. Recruitment of leukocytes, neutrophils, T cells, and parameters of liver injury were assessed in tissue/blood samples. Results: Leukocyte recruitment, sinusoidal perfusion failure rate, and shear rate were significantly increased in mice after 60% partial hepatectomy compared to sham‐operated animals. The inhibition of SPs or plasmin significantly attenuated leukocyte recruitment and improved the perfusion rate in the remnant liver. ICAM‐1 expression and neutrophil recruitment significantly increased after 60% partial hepatectomy and were strongly reduced by plasmin inhibition. Conclusions: Endothelial activation and leukocyte recruitment in the liver in response to the increment of sinusoidal shear rate were hallmarks in the acute phase after liver resection. SPs mediated leukocyte recruitment and contributed to the impairment of sinusoidal perfusion in an ICAM‐1‐dependent manner in the acute phase after liver resection. [ABSTRACT FROM AUTHOR]

Following a myocardial infarction (MI), the prognosis of patients is highly dependent upon the re‐establishment of perfusion not only in the occluded coronary artery, but also within the coronary microcirculation. However, our fundamental understanding of the pathophysiology of the tiniest blood vessels of the heart is limited primarily because no current clinical imaging tools can directly visualise them. Moreover, in vivo experimental studies of the beating heart using intravital imaging have also been hampered due to obvious difficulties related to significant inherent contractile motion, movement of the heart brought about by nearby lungs and its location in an anatomically challenging position for microscopy. However, recent advances in microscopy techniques, and the development of fluorescent reporter mice and fluorescently conjugated antibodies allowing visualisation of vascular structures, thromboinflammatory cells and blood flow, have allowed us to overcome some of these challenges and increase our basic understanding of cardiac microvascular pathophysiology. In this review, the elegant attempts of the pioneers in intravital imaging of the beating heart will be discussed, which focussed on providing new insights into the anatomy and physiology of the healthy heart microvessels. The reviews end with the more recent studies that focussed on disease pathology and increasing our understanding of myocardial thromboinflammatory cell recruitment and flow disturbances, particularly in the setting of diseases such as MI. [ABSTRACT FROM AUTHOR]

Fluorescence intravital microscopy captures large data sets of dynamic multicellular interactions within various organs such as the lungs, liver, and brain of living subjects. In medical imaging, edge detection is used to accurately identify and delineate important structures and boundaries inside the images. To improve edge sharpness, edge detection frequently requires the inclusion of low-level features. Herein, a machine learning approach is needed to automate the edge detection of multicellular aggregates of distinctly labeled blood cells within the microcirculation. In this work, the Structured Adaptive Boosting Trees algorithm (AdaBoost.S) is proposed as a contribution to overcome some of the edge detection challenges related to medical images. Algorithm design is based on the observation that edges over an image mask often exhibit special structures and are interdependent. Such structures can be predicted using the features extracted from a bigger image patch that covers the image edge mask. The proposed AdaBoost.S is applied to detect multicellular aggregates within blood vessels from the fluorescence lung intravital images of mice exposed to e-cigarette vapor. The predictive capabilities of this approach for detecting platelet-neutrophil aggregates within the lung blood vessels are evaluated against three conventional machine learning algorithms: Random Forest, XGBoost and Decision Tree. AdaBoost.S exhibits a mean recall, F-score, and precision of 0.81, 0.79, and 0.78, respectively. Compared to all three existing algorithms, AdaBoost.S has statistically better performance for recall and F-score. Although AdaBoost.S does not outperform Random Forest in precision, it remains superior to the XGBoost and Decision Tree algorithms. The proposed AdaBoost.S is widely applicable to analysis of other fluorescence intravital microscopy applications including cancer, infection, and cardiovascular disease. [Display omitted] • Lung intravital imaging forms large data sets of complex multicellular interactions. • Commercial analysis software detects limited data and requires human input. • A new Structured Adaptive Boosting Trees algorithm for edge detection is developed. • AdaBoost.S achieves fast and accurate detection of dynamic multicellular aggregates. [ABSTRACT FROM AUTHOR]

Insufficient drug accumulation in tumors is still a major concern for using cancer nanotherapeutics. Here, the neutrophil-based delivery of three nanoparticle types—liposomes, PLGA, and magnetite nanoparticles—was assessed both in vitro and in vivo. Confocal microscopy and a flow cytometry analysis demonstrated that all the studied nanoparticles interacted with neutrophils from the peripheral blood of mice with 4T1 mammary adenocarcinoma without a significant impact on neutrophil viability or activation state. Intravital microscopy of the tumor microenvironment showed that the neutrophils did not engulf the liposomes after intravenous administration, but facilitated nanoparticle extravasation in tumors through micro- and macroleakages. PLGA accumulated along the vessel walls in the form of local clusters. Later, PLGA nanoparticle-loaded neutrophils were found to cross the vascular barrier and migrate towards the tumor core. The magnetite nanoparticles extravasated in tumors both via spontaneous macroleakages and on neutrophils. Overall, the specific type of nanoparticles largely determined their behavior in blood vessels and their neutrophil-mediated delivery to the tumor. Since neutrophils are the first to migrate to the site of inflammation, they can increase nanodrug delivery effectiveness for nanomedicine application.

Acute respiratory distress syndrome (ARDS) and sepsis are risk factors contributing to mortality in patients with pneumonia. In ARDS, also termed acute lung injury (ALI), pulmonary immune responses lead to excessive pro-inflammatory cytokine release and aberrant alveolar neutrophil infiltration. Systemic spread of cytokines is associated with systemic complications including sepsis, multi-organ failure, and death. Thus, dampening pro-inflammatory cytokine release is a viable strategy to improve outcome. Activation of cannabinoid type II receptor (CB2) has been shown to reduce cytokine release in various in vivo and in vitro studies. Herein, we investigated the effect of HU-308, a specific CB2 agonist, on systemic and pulmonary inflammation in a model of pneumonia-induced ALI. C57Bl/6 mice received intranasal endotoxin or saline, followed by intravenous HU-308, dexamethasone, or vehicle. ALI was scored by histology and plasma levels of select inflammatory mediators were assessed by Luminex assay. Intravital microscopy (IVM) was performed to assess leukocyte adhesion and capillary perfusion in intestinal and pulmonary microcirculation. HU-308 and dexamethasone attenuated LPS-induced cytokine release and intestinal microcirculatory impairment. HU-308 modestly reduced ALI score, while dexamethasone abolished it. These results suggest administration of HU-308 can reduce systemic inflammation without suppressing pulmonary immune response in pneumonia-induced ALI and systemic inflammation. [ABSTRACT FROM AUTHOR]

Human islet transplantations into rodent models are an essential tool to aid in the development and testing of islet and cellular-based therapies for diabetes prevention and treatment. Through the ability to evaluate human islets in an in vivo setting, these studies allow for experimental approaches to answer questions surrounding normal and disease pathophysiology that cannot be answered using other in vitro and in vivo techniques alone. Intravital microscopy enables imaging of tissues in living organisms with dynamic temporal resolution and can be employed to measure biological processes in transplanted human islets revealing how experimental variables can influence engraftment, and transplant survival and function. A key consideration in experimental design for transplant imaging is the surgical placement site, which is guided by the presence of vasculature to aid in functional engraftment of the islets and promote their survival. Here, we review transplantation sites and mouse models used to study beta cell biology in vivo using intravital microscopy and we highlight fundamental observations made possible using this methodology. [ABSTRACT FROM AUTHOR]

Hematology and bone marrow analysis is central to our understanding of the hematopoietic system and how it responds to insults, and this session presented during the 2022 STP symposium provided a review of current and novel approaches for the evaluation of the hematopoietic system in the context of nonclinical investigations. This publication summarizes the information presented on novel approaches for evaluation of the hematopoietic system using automated hematology analyzers, including details around the quantitative assessment of bone marrow cell suspensions as well as introducing several newly available hematology parameters. It was followed by a discussion on intravital microscopy and live cell imaging and how these methods can assist with de-risking hematopoiesis-associated safety concerns, and a review of recent assays using artificial intelligence for the evaluation of bone marrow. [ABSTRACT FROM AUTHOR]

At the Nanomedicine Innovation Center (NICE) at the Erasmus MC in Rotterdam, we have approached the treatment of cancer by starting with a vision of first establishing a platform that enables us to overcome the low levels of drugs delivered to tumors and the issue of dose-limiting toxicity. Showing that a reduction of the volume of distribution, and a lowering of toxicity and side-effects, accompanied by augmented intratumoral drug delivery, could change outcomes in patients, paved the way to target, not only localized disease, but also systemic and metastasized cancers. In particular, the detailed studies with intravital microscopy we performed at NICE provided us with the necessary insights and affected to a large extent our program on liposome-based cancer therapy. Together with our experience with the loco-regional treatment of cancer, this helped us to develop a program that focused on the subsequent aspects discussed here. We recognized that passive accumulation of nanoparticles was not as effective as previously believed and undertook to improve the local accumulation by changing the tumor pathophysiology and, in particular, the vascular permeability. We added the targeting of liposomes using vascular and tumor directed moieties, to improve cellular drug delivery. To improve payload delivery, we studied the modification of liposomes with phospholipids that help passive drug release and augment cellular accumulation. Second, and importantly, modification of liposomes was undertaken, to enable triggered drug release. The capability for modifying liposomes to respond to a trigger, and the ability to now apply an external trigger (e.g., hyperthermia) and specifically reach the tumor volume, resulted in the current smart drug delivery systems. Our experience at NICE, after a few decades of research on lipid-based nanoparticles, shows that, after the first liposomal formulation registered for clinical application in cancer therapy, further developments quickly followed, while further clinical applications lagged behind. Now we need to focus on and make the next steps towards the clinic, to fulfil the promise that is found there. [ABSTRACT FROM AUTHOR]

Human islet transplantations into rodent models are an essential tool to aid in the development and testing of islet and cellular-based therapies for diabetes prevention and treatment. Through the ability to evaluate human islets in an in vivo setting, these studies allow for experimental approaches to answer questions surrounding normal and disease pathophysiology that cannot be answered using other in vitro and in vivo techniques alone. Intravital microscopy enables imaging of tissues in living organisms with dynamic temporal resolution and can be employed to measure biological processes in transplanted human islets revealing how experimental variables can influence engraftment, and transplant survival and function. A key consideration in experimental design for transplant imaging is the surgical placement site, which is guided by the presence of vasculature to aid in functional engraftment of the islets and promote their survival. Here, we review transplantation sites and mouse models used to study beta cell biology in vivo using intravital microscopy and we highlight fundamental observations made possible using this methodology.

The effect of the lowering of body temperature on the responses of cerebral arteries was studied in anesthetized (urethane, 1000 mg/kg) male Wistar rats in conditions of hemodynamic stability and after pre-induced acute massive blood loss. Changes in the diameter of the pial arteries (initial diameter 10–40 µm) were evaluated via intravital microscopy in four experimental groups: without and with pre-induced blood loss (35% of the circulating blood volume), and in each group, at room temperature (22–23°C) and during gradually developing cold-water immersion hypothermia (12–13°C) until respiratory arrest. In the groups with pre-induced blood loss, respiratory arrest came about at room temperature after an average of 3 h with a decrease in body temperature to 32.4 ± 0.5°C, while during immersion cooling, it occurred after 2.5 h at 15.1 ± 0.8°C. In the groups without blood loss, respiratory arrest did not come about at room temperature for more than 3.5 h of observation, although rectal temperature decreased to 34.4 ± 0.2°C; during immersion cooling, respiratory arrest was observed after 3 h at a body temperature of 13.1 ± 0.3°C. During the lowering of body temperature, the rats of all experimental groups showed vasoconstriction. In the groups with blood loss, the reduction in the diameter of the pial vessels accounted for 10–15% of the initial immediately after blood withdrawal and more than 30% of that during subsequent immersion cooling. In rats without blood loss, the constriction of pial arteries during immersion cooling was significantly less. Thus, systemic hypothermia aggravates microcirculatory dysfunction induced by acute massive blood loss, suggesting an additive effect of hypothermia and hemorrhage on vasoconstrictor responses of the pial arteries in rats. [ABSTRACT FROM AUTHOR]

The arteriovenous (AV) loop model is a key technique to solve one of the major problems of tissue engineering—providing adequate vascular support for a tissue construct of significant size. However, the molecular and cellular mechanisms of vascularization and factors influencing the generation of new tissue in the AV loop are still poorly understood. We previously established a novel intravital microscopy approach to study these events. In this study, we implanted our observation chamber filled with two types of hydrogels such as fibrin and methacrylate gelatin (GelMA) and performed intravital microscopy (IVM) on days 7, 14, and 21. Initial microvessel formation was observed in GelMA on day 14, while the vessel network showed clear indicators of network rearrangement and maturation on day 21. No visible microvessels were observed in fibrin. The chambers were explanted on day 21. Histological examination revealed higher numbers of microvessels in GelMA compared to fibrin, while the AV loop was thrombosed in all fibrin constructs, possibly due to matrix degradation. GelMA proved to be an ideal matrix for IVM studies in the AV loop model due to its slow degradation and transparency. This IVM model can be employed as a novel tool for live and thus faster comprehension of crucial events in the tissue regeneration process, which can improve tissue engineering application. [ABSTRACT FROM AUTHOR]

The ability to observe cells in live organisms is essential for understanding their function in complex in vivo milieus. A major challenge today has been the limited ability to perform higher multiplexing beyond four to six colors to define cell subtypes in vivo. Here, a click chemistry‐based strategy is presented for higher multiplexed in vivo imaging in mouse models. The method uses a scission‐accelerated fluorophore exchange (SAFE), which exploits a highly efficient bioorthogonal mechanism to completely remove fluorescent signal from antibody‐labeled cells in vivo. It is shown that the SAFE‐intravital microscopy imaging method allows 1) in vivo staining of specific cell types in dorsal and cranial window chambers of mice, 2) complete un‐staining in minutes, 3) in vivo click chemistries at lower (µm) and thus non‐toxic concentrations, and 4) the ability to perform in vivo cyclic imaging. The potential utility of the method is demonstrated by 12 color imaging of immune cells in live mice. [ABSTRACT FROM AUTHOR]

The nanometer spatial resolution of electron microscopy imaging remains an advantage over light microscopy, but the restricted field of view that can be inspected and the inability to visualize dynamic cellular events are definitely drawbacks of standard transmission electron microscopy (TEM). Several methods have been developed to overcome these limitations, mainly by correlating the light microscopical image to the electron microscope with correlative light and electron microscopy (CLEM) techniques. Since there is more than one method to obtain the region of interest (ROI), the workflow must be adjusted according to the research question and biological material addressed. Here, we describe in detail the development of a three-dimensional CLEM workflow for mouse skin tissue exposed to an inflammation stimulus and imaged by intravital microscopy (IVM) before fixation. Our aim is to relocate a distinct vessel in the electron microscope, addressing a complex biological question: how do cells interact with each other and the surrounding environment at the ultrastructural level? Retracing the area over several preparation steps did not involve any specific automated instruments but was entirely led by anatomical and artificially introduced landmarks, including blood vessel architecture and carbon-coated grids. Successful retrieval of the ROI by electron microscopy depended on particularly high precision during sample manipulation and extensive documentation. Further modification of the TEM sample preparation protocol for mouse skin tissue even rendered the specimen suitable for serial block-face scanning electron microscopy (SBF-SEM). [ABSTRACT FROM AUTHOR]

Hepatotoxicity remains an as yet unsolved problem for adenovirus (Ad) cancer therapy. The toxic effects originate both from rapid Kupffer cell (KCs) death (early phase) and hepatocyte transduction (late phase). Several host factors and capsid components are known to contribute to hepatotoxicity, however, the complex interplay between Ad and liver cells is not fully understood. Here, by using intravital microscopy, we aimed to follow the infection and immune response in mouse liver from the first minutes up to 72 h post intravenous injection of three Ads carrying delta-24 modification (Ad5-RGD, Ad5/3, and Ad5/35). At 15–30 min following the infusion of Ad5-RGD and Ad5/3 (but not Ad5/35), the virus-bound macrophages demonstrated signs of zeiosis: the formation of long-extended protrusions and dynamic membrane blebbing with the virus release into the blood in the membrane-associated vesicles. Although real-time imaging revealed interactions between the neutrophils and virus-bound KCs within minutes after treatment, and long-term contacts of CD8+ T cells with transduced hepatocytes at 24–72 h, depletion of neutrophils and CD8+ T cells affected neither rate nor dynamics of liver infection. Ad5-RGD failed to complete replicative cycle in hepatocytes, and transduced cells remained impermeable for propidium iodide, with a small fraction undergoing spontaneous apoptosis. In Ad5-RGD-immune mice, the virus neither killed KCs nor transduced hepatocytes, while in the setting of hepatic regeneration, Ad5-RGD enhanced liver transduction. The clinical and biochemical signs of hepatotoxicity correlated well with KC death, but not hepatocyte transduction. Real-time in vivo tracking for dynamic interactions between virus and host cells provides a better understanding of mechanisms underlying Ad-related hepatotoxicity. [ABSTRACT FROM AUTHOR]

Abstract Intravital video microscopy permits the observation of microcirculatory blood flow. This often requires fluorescent probes to visualize structures and dynamic processes that cannot be observed with conventional bright-field microscopy. Conventional light microscopes do not allow for simultaneous bright-field and fluorescent imaging. Moreover, in conventional microscopes, only one type of fluorescent label can be observed. This study introduces multispectral intravital video microscopy, which combines bright-field and fluorescence microscopy in a standard light microscope. The technique enables simultaneous real-time observation of fluorescently-labeled structures in relation to their direct physical surroundings. The advancement provides context for the orientation, movement, and function of labeled structures in the microcirculation.