Intravital Microscopy Research Articles

Effects of physical exercise associated with a diet enriched with natural antioxidants on cerebral hypoperfusion and reperfusion injury in spontaneously hypertensive rats.

Oxidative stress is implicated in the pathogenesis of arterial hypertension. The reduction in the bioavailability of nitric oxide (NO) causes endothelial dysfunction, altering the functions of cerebral blood vessels. Physical exercise and intake of antioxidants improve the redox state, increasing the vascular NO production and/or the decrease in NO scavenging by reactive oxygen species (ROS). The present study was aimed at assessing the effects of physical exercise associated with a diet enriched with antioxidants from the Annurca apple in preventing the microvascular damage due to cerebral hypoperfusion and reperfusion injury in spontaneously hypertensive rats (SHRs). The rat pial microcirculation was investigated by intravital fluorescence microscopy through a parietal closed cranial window. As expected, SHRs subjected to physical exercise or an antioxidants-enriched diet showed a reduction of microvascular permeability, ROS formation, and leukocyte adhesion to venular walls, with a major effect of the antioxidants-enriched diet, when compared to untreated SHRs. Moreover, capillary perfusion was preserved by both treatments in comparison with untreated SHRs. Unexpectedly, the combined treatments did not induce higher effects than the single treatment. In conclusion, our results support the efficacy of physical activity or antioxidant supplement in reducing the microvascular alterations due to hypertension and ascribe to an antioxidants-enriched diet effective microvascular protection in SHRs.

Defining the Intravital Renal Disposition of Fluorescence-Quenched Exenatide.

Despite the understanding that renal clearance is pivotal for driving the pharmacokinetics of numerous therapeutic proteins and peptides, the specific processes that occur following glomerular filtration remain poorly defined. For instance, sites of catabolism within the proximal tubule can occur at the brush border, within lysosomes following endocytosis, or even within the tubule lumen itself. The objective of the current study was to address these limitations and develop methodology to study the kidney disposition of a model therapeutic protein. Exenatide is a peptide used to treat type 2 diabetes mellitus. Glomerular filtration and ensuing renal catabolism have been shown to be its principal clearance pathway. Here, we designed and validated a Förster resonance energy transfer-quenched exenatide derivative to provide critical information on the renal handling of exenatide. A combination of in vitro techniques was used to confirm substantial fluorescence quenching of intact peptide that was released upon proteolytic cleavage. This evaluation was then followed by an assessment of the in vivo disposition of quenched exenatide directly within kidneys of living rats via intravital two-photon microscopy. Live imaging demonstrated rapid glomerular filtration and identified exenatide metabolism occurred within the subapical regions of the proximal tubule epithelia, with subsequent intracellular trafficking of cleaved fragments. These results provide a novel examination into the real-time, intravital disposition of a protein therapeutic within the kidney and offer a platform to build upon for future work.

A Novel Window into Angiogenesis-Intravital Microscopy in the AV-Loop-Model.

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¯.

Guidelines for visualization and analysis of DC in tissues using multiparameter fluorescence microscopy imaging methods.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Here, we provide detailed procedures for a variety of multiparameter fluorescence microscopy imaging methods to explore the spatial organization of DC in tissues and to dissect how DC migrate, communicate, and mediate their multiple functional roles in immunity in a variety of tissue settings. The protocols presented here entail approaches to study DC dynamics and T cell cross-talk by intravital microscopy, large-scale visualization, identification, and quantitative analysis of DC subsets and their functions by multiparameter fluorescence microscopy of fixed tissue sections, and an approach to study DC interactions with tissue cells in a 3D cell culture model. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists.

Analysis of Monocyte Recruitment During Inflammation by Intravital Imaging.

Monocytes play essential roles in the inflammatory and anti-inflammatory processes that take place during an immune response, acting both within the vascular network and interstitially. Monocytes are activated, mobilized, and recruited in response to an inflammatory stimulus or different forms of tissue injury. The recruitment of circulating monocytes to the inflamed tissue is essential to resolving the injury.Monocyte recruitment is a multistep process that begins with a decrease in rolling velocity, is followed by adhesion to the endothelium and crawling over the luminal vessel surface, and culminates in monocyte transmigration into the surrounding tissue. Intravital microscopy is a powerful visualization tool for the study of leukocyte behavior and function, intercellular interactions, cell trafficking, and recruitment in pathological and physiological conditions. This modality is therefore widely used for the detailed analysis of the immune response to multiple insults and the molecular mechanisms underlying monocyte interactions within the vascular system in vivo. This chapter describes a protocol for the use of intravital microscopy to analyze monocyte recruitment from the blood vessel to the inflammatory site.

Size-advantage of monovalent nanobodies against the macrophage mannose receptor for deep tumor penetration and tumor-associated macrophage targeting.

Rationale: Nanobodies (Nbs) have emerged as an elegant alternative to the use of conventional monoclonal antibodies in cancer therapy, but a detailed microscopic insight into the in vivo pharmacokinetics of different Nb formats in tumor-bearers is lacking. This is especially relevant for the recognition and targeting of pro-tumoral tumor-associated macrophages (TAMs), which may be located in less penetrable tumor regions. Methods: We employed anti-Macrophage Mannose Receptor (MMR) Nbs, in a monovalent (m) or bivalent (biv) format, to assess in vivo TAM targeting. Intravital and confocal microscopy were used to analyse the blood clearance rate and targeting kinetics of anti-MMR Nbs in tumor tissue, healthy muscle tissue and liver. Fluorescence Molecular Tomography was applied to confirm anti-MMR Nb accumulation in the primary tumor and in metastatic lesions. Results: Intravital microscopy demonstrated significant differences in the blood clearance rate and macrophage targeting kinetics of (m) and (biv)anti-MMR Nbs, both in tumoral and extra-tumoral tissue. Importantly, (m)anti-MMR Nbs are superior in reaching tissue macrophages, an advantage that is especially prominent in tumor tissue. The administration of a molar excess of unlabelled (biv)anti-MMR Nbs increased the (m)anti-MMR Nb bioavailability and impacted on its macrophage targeting kinetics, preventing their accumulation in extra-tumoral tissue (especially in the liver) but only partially influencing their interaction with TAMs. Finally, anti-MMR Nb administration not only allowed the visualization of TAMs in primary tumors, but also at a distant metastatic site. Conclusions: These data describe, for the first time, a microscopic analysis of (m) and (biv)anti-MMR Nb pharmacokinetics in tumor and healthy tissues. The concepts proposed in this study provide important knowledge for the future use of Nbs as diagnostic and therapeutic agents, especially for the targeting of tumor-infiltrating immune cells.

Intravital Imaging of Intestinal Intraepithelial Lymphocytes.

Intestinal intraepithelial lymphocytes (IEL) are a numerous population of T cells located within the epithelium of the small and large intestines, being more numerous in the small intestine (SI). They surveil this tissue by interacting with epithelial cells. Intravital microscopy is an important tool for visualizing the patrolling activity of IEL in the SI of live mice. Most IEL express CD8α; therefore, here we describe an established protocol ofintravital imaging that tracks lymphocytes labeled with a CD8α-specific monoclonal antibody in the SI epithelium of live mice. We also describe data acquisition and quantification of the movement metrics, including mean speed, track length, displacement length, and paths for each CD8α+ IEL using the available software. The intravital imaging technique for measuring IEL movement will provide a better understanding of the role of IEL in homeostasis and protection from injury or infection in vivo.

S-07-2: DISCOVERY OF NEUROENDOTHELIAL CELLS AND THEIR ROLE IN HYPERTENSION AND AGING

Objective: Vascular endothelial cells (ECs) play important roles in the physiological maintenance of organ blood flow and in the development of renal and cardiovascular diseases. Tissue-specific EC dysfunction can contribute to several different diseases including hypertension. Recent transcriptomic studies identified many EC subtypes in multiple organs including the brain and the kidneys, however, their functions are incompletely understood. The present study aimed to explore physiological functional significance and cardiovascular disease and hypertension relevance of a newly discovered minority subtype of scattered ECs expressing neuronal nitric oxide synthase (Nos1). Design and method: A comprehensive research toolbox was applied in this study including transgenic mouse models (Nos1-GFP, GCaMP6, mTORgof/lof), intravital multiphoton imaging of calcium dynamics of Nos1+ endothelial cells in the brain and the kidney, genetic cell fate tracking, two-kidney one-clip (2K1C) model of renovascular (Goldblatt) hypertension (RVHT), whole mount organ imaging for 3D vascular density measurements, and single-cell transcriptomic analysis. Results: Our studies identified and characterized, for the first time, a new endothelial cell type expressing both endothelial and neuron-like functional and gene transcriptomic signatures, therefore we named them neuroendothelial cells (NECs). NECs exhibit a well-defined arteriovenous zonal localization exclusively to small resistance arterioles. NECs are found only in the three organs that exhibit the best blood flow autoregulation capacity, with the highest density in the brain>kidney>heart (NEC/EC (%) 8.42+/-0.79, 3.21+/−0.33, 1.73+/−0.07, respectively). NEC density is reduced with aging in the brain and the kidney (NEC/EC (%) 4.675, and 1.850, respectively, p < 0.01, 2.5 years old compared to 2-month-old). The number of NECs increased significantly in the hypo-perfused, hypoxic clipped (CK) kidney, but reduced in the hyperperfused non-clipped (NCK) kidney in RVHT. Intravital multiphoton microscopy (MPM) of intact brain and kidney arterioles in vivo revealed regular, autonomous NEC calcium transients with blood pressure-dependent frequency alterations. Newly established NEC gain-of-function mouse models exhibited increased endothelium-dependent vasodilation and diminished agonist-induced vascular contractility in brain and kidney resistance arterioles compared to controls. In addition, preliminary single-cell RNA sequencing and transcriptomic analyses showed that, in contrast to other ECs, NECs highly express several traditional (e.g., Nos1, Klotho) and novel (e.g., Aard) tissue trophic factors that are known to play important roles in angiogenesis, aging, vascular (dys)function, and chronic vascular diseases. Conclusions: These new vascular anatomy and hemodynamic findings strongly suggest sensory, blood flow and/or baroreceptor functions of NECs as well as a role in the autoregulation of organ blood flow and hypertension pathogenesis.

Monitoring the Interaction Between Dendritic Cells and T Cells In Vivo with LIPSTIC.

Interactions between different cell types are key for immune function. Traditionally, interactions have been investigated in vivo by intravital two-photon microscopy, but the molecular characterization of the cells participating in a specific interaction is limited by the inability to retrieve the cells for downstream analysis. We recently developed an approach to label cells undergoing specific interactions in vivo, which we called LIPSTIC (Labeling Immune Partnership by Sortagging Intercellular Contacts). Here, we provide detailed instructions on how to track CD40-CD40L interactions between dendritic cells (DCs) and CD4+ T cells using genetically engineered LIPSTIC mice. This protocol requires expertise in animal experimentation and multicolor flow cytometry. Once mouse crossing has been achieved, it takes 3days or more to complete, depending on the kinetics of the interactions that the researcher wishes to investigate.

Intravital imaging and cavitation monitoring of antivascular ultrasound in tumor microvasculature.

Rationale: Focused ultrasound-stimulated microbubbles have been shown to be capable of inducing blood flow shutdown and necrosis in a range of tissue types in an approach termed antivascular ultrasound or nonthermal ablation. In oncology, this approach has demonstrated tumor growth inhibition, and profound synergistic antitumor effects when combined with traditional platforms of chemo-, radiation- and immune-therapies. However, the exposure schemes employed have been broad and underlying mechanisms remain unclear with fundamental questions about exposures, vessel types and sizes involved, and the nature of bubble behaviors and their acoustic emissions resulting in vascular damage - impeding the establishment of standard protocols. Methods: Here, ultrasound transmitters and receivers are integrated into a murine dorsal window chamber tumor model for intravital microscopy studies capable of real-time visual and acoustic monitoring during antivascular ultrasound. Vessel type (normal and tumor-affected), caliber, and viability are assessed under higher pressure conditions (1, 2, and 3 MPa), and cavitation signatures are linked to the biological effects. Results: Vascular events occurred preferentially in tumor-affected vessels with greater incidence in smaller vessels and with more severity as a function of increasing pressure. Vascular blood flow shutdown was found to be due to a combination of focal disruption events and network-related flow changes. Acoustic emissions displayed elevated broadband noise and distinct sub- and ultra-harmonics and their associated third-order peaks with increasing pressure. Conclusions: The observed vascular events taken collectively with identified cavitation signatures provide an improved mechanistic understanding of antivascular ultrasound at the microscale, with implications for establishing a specific treatment protocol and control platform.

Polymeric microneedles enable simultaneous delivery of cancer immunomodulatory drugs and detection of skin biomarkers.

Background: Immune-modulating therapies impart positive outcomes in a subpopulation of cancer patients. Improved delivery strategies and non-invasive monitoring of anti-tumor effects can help enhance those outcomes and understand the mechanisms associated with the generation of anti-tumor immune responses following immunotherapy. Methods: We report on the design of a microneedle (MN) platform capable of simultaneous delivery of immune activators and collection of interstitial skin fluid (ISF) to monitor therapeutic responses. While either approach has shown promise, the integration of the therapy and diagnostic arms into one MN platform has hardly been explored before. MNs were synthesized out of crosslinked hyaluronic acid (HA) and loaded with a model immunomodulatory nanoparticle-containing drug, CpG oligodinucleotides (TLR9 agonist), for cancer therapy in melanoma and colon cancer models. The therapeutic response was monitored by longitudinal analysis of entrapped immune cells in the MNs following patch retrieval and digestion. Results: Transdermal delivery of CpG-containing NPs with MNs induced anti-tumor immune responses in multiple syngeneic mouse cancer models. CpG-loaded MNs stimulated innate immune cells and reduced tumor growth. Intravital microscopy showed deposition and spatiotemporal co-localization of CpG-NPs within the tumor microenvironment when delivered with MNs. Analysis of MN-sampled ISF revealed similar immune signatures to those seen in the bulk tumor homogenate, such as increased populations of macrophages and effector T cells following treatment. Conclusions: Our hydrogel-based MNs enable effective transdermal drug delivery into immune cells in the tumor microenvironment, and upon retrieval, enable studying the immune response to the therapy over time. This platform has the theranostic potential to deliver a range of combination therapies while detecting biomarkers.

Regulating Neutrophil PAD4/NOX-Dependent Cerebrovasular Thromboinflammation.

Background: Neutrophil extracellular trap (NET) production has been implicated in the pathogenesis of thromboinflammatory conditions such as Sickle Cell Disease (SCD), contributing to heightened risk for ischemic stroke. NETs are catalyzed by the enzyme Peptidyl Arginine Deiminase 4 (PAD4) and neutrophil derived reactive oxygen species (ROS), especially NADPH oxidase (NOX) which interacts with PAD4 and is therefore critical for neutrophil function. However, the role that NOX-dependent ROS and NETs play in the accelerated cerebral microvascular thrombosis associated with thromboinflammatory conditions, such as SCD, has not been fully elucidated and is the aim of this study. Methods: The in-vitro effects of targeting PAD4 and NOX were examined using physiologically relevant NET assays with neutrophils isolated from healthy volunteers (control) and SCD patients. In addition, in-vivo intravascular effects of targeting PAD4 and NOX in the cerebral microcirculation of C57BL/6 and sickle transgenic mice (STM) were assessed using a photoactivation thrombosis model (light/dye) coupled with real-time fluorescence intravital microscopy. Results: We found that targeting PAD4 and NOX in human neutrophils significantly inhibited ionomycin dependent H3cit+ neutrophils. Targeting PAD4 and NOX in-vivo resulted in prolonged blood flow cessation in cerebrovascular arterioles as well as venules. Moreover, we were able to replicate the effects of PAD4 and NOX targeting in a clinical model of accelerated thromboinflammation by increasing blood flow cessation times in cerebral microvessels in STM. These findings concurred with the clinical setting i.e. neutrophils isolated from SCD patients, which possessed an attenuation of H3cit+ neutrophil production on targeting PAD4 and NOX. Conclusions: Taken together, our compelling data suggests that PAD4 and NOX play a significant role in neutrophil driven thromboinflammation. Targeting PAD4 and NOX limits pathological H3cit+ neutrophils, which may further explain attenuation of cerebral thrombosis. Overall, this study presents a viable pre-clinical model of prevention and management of thromboinflammatory complications such as ischemic stroke.

Intravital Microscopy of the Metastatic Pulmonary Environment.

Real-time in vivo imaging has become an integral tool for the investigation and understanding of cellular processes in health and disease at single-cell resolution. This includes the dynamic and complex cellular interactions that occur during cancer progression and the subsequent metastatic dissemination of tumor cells to sites distant from the primary tumor. Herein we outline the methodology for the establishment and intravital imaging of the pulmonary metastatic niche, a preferred site of metastasis for many cancers, and describe the implementation of a lung window to visualize and dissect the intricate behaviour of multiple cell types within this environment. We also address the advantages and limitations of this high-resolution technology.

Intravital Imaging of Regulatory T Cells in Inflamed Skin.

Regulatory T cells play key roles in skin homeostasis and inflammation and in regulating antitumor responses. Understanding of the biology of this cell type has been improved by the use of intravital microscopy for their visualization in various organs. Here we describe a multiphoton microscopy-based technique for intravital imaging of regulatory T cells in the skin. We provide a protocol for a model of antigen-dependent inflammation that induces robust regulatory T cell recruitment to the skin and describe the use of a regulatory T cell reporter mouse for visualization of these cells in inflamed skin.

Supermagnetic Human Serum Albumin (HSA) Nanoparticles and PLGA-Based Doxorubicin Nanoformulation: A Duet for Selective Nanotherapy

Predicting the ability of nanoparticles (NP) to access the tumor is key to the success of chemotherapy using nanotherapeutics. In the present study, the ability of the dual NP-based theranostic system to accumulate in the tumor was evaluated in vivo using intravital microscopy (IVM) and MRI. The system consisted of model therapeutic doxorubicin-loaded poly(lactide-co-glycolide) NP (Dox-PLGA NP) and novel hybrid Ce3/4+-doped maghemite NP encapsulated within the HSA matrix (hMNP) as a supermagnetic MRI contrasting agent. Both NP types had similar sizes of ~100 nm and negative surface potentials. The level of the hMNP and PLGA NP co-distribution in the same regions of interest (ROI, ~2500 µm2) was assessed by IVM in mice bearing the 4T1-mScarlet murine mammary carcinoma at different intervals between the NP injections. In all cases, both NP types penetrated into the same tumoral/peritumoral regions by neutrophil-assisted extravasation through vascular micro- and macroleakages. The maximum tumor contrasting in MRI scans was obtained 5 h after hMNP injection/1 h after PLGA NP injection; the co-distribution level at this time reached 78%. Together with high contrasting properties of the hMNP, these data indicate that the hMNP and PLGA NPs are suitable theranostic companions. Thus, analysis of the co-distribution level appears to be a useful tool for evaluation of the dual nanoparticle theranostics, whereas assessment of the leakage areas helps to reveal the tumors potentially responsive to nanotherapeutics.

Platelets play a dual role in the pathophysiology of transfusion-related acute lung injury

Platelets are increasingly recognized as key regulators of inflammatory and immune responses, through their interaction with endothelium and immune cells. Therefore they might have a role in transfusion-related acute lung injury (TRALI), in which endothelial cells and neutrophils are the key players. In this study, by a classic TRALI animal model, combining a custom-designed system for intravital confocal microscopy of pulmonary microvasculature and a platelet tracking technique, we found that thrombin-activated platelets transfusion aggravated TRALI while resting platelets transfusion alleviated TRALI. Promoting endogenous platelets activation also aggravated TRALI while inhibiting endogenous platelets activation alleviated TRALI. Activated platelets interfered with the stability of endothelial barrier function while resting platelets modulated the activation of neutrophils. Anti-thrombin could alleviate TRALI, which was not reproduced upon anti-GPIIbIIIa or anti-P-selectin In conclusion, platelets might play a dual role (protective and pathogenic) in TRALI, the balance between the two roles is highly dependent on whether platelets are activated by thrombin or not. This might explain the conflicting results of previous researches studying the contribution of platelets in TRALI by platelet depletion technology, in which the induction of TRALI and the condition of animals were different, hence the state of platelets during TRALI was different. Moreover, anti-platelet-activation (such as anti-thrombin) might be a better approach than anti-activated-platelets (such as anti-P-selectin) to search for potential therapies in TRALI. Considering the involvement of thrombin-activated platelets in TRALI, anti-thrombin might be needed when blood component transfusion is performed.

BAP31 regulates the expression of ICAM-1/VCAM-1 via MyD88/NF-κB pathway in acute lung injury mice model

AimsThe cell adhesion molecules (CAMs) that mediate neutrophil-endothelium cell adhesion are deeply involved in the pathogenesis of acute lung injury (ALI). B-cell receptor associated protein 31 (BAP31) has been reported to engage in the expression of some CAMs. This study was undertaken to explore whether BAP31 in endotheliocyte affects the pathological process of ALI by regulating CAMs, and its possible mechanism. Main methodsOur study used the shBAP31 endothelium cell lines and endothelial-specific BAP31 conditional knockdown mice constructed via Cre/loxP system. Hematoxylin and eosin staining was used to observe the histopathological manifestations. The adhesion of neutrophils to vascular wall was examined by intravital microscopy. The nuclear translocation of NF-κB was observed by immunofluorescence staining assay. Flow cytometric, real-time polymerase chain reaction and Western blot assay were performed to determine the expression of CAMs and key proteins in MyD88/NF-κB-related signaling pathway. Luciferase reporter and chromatin immunoprecipitation assay were analyzed for transcriptional activity of ICAM-1 and VCAM-1. Key findingsMechanistic investigations indicated that endothelium-specific BAP31 depletion dramatically reduced the capacity of neutrophils adherence to endothelial cells (ECs), which was mainly attributed to the significant downregulation of ICAM-1 (p < 0.05) and VCAM-1 (p < 0.05) expression. Interestingly, BAP31 knockdown apparently deactivated MyD88/TRAF6-mediated TAK1/NF-κB and PI3K/Akt signaling cascades, resulting in the inhibition of NF-κB activation and nuclear translocation. SignificanceOur data furnished convincing evidence that BAP31 deficiency performs a mitigative effect on ALI by decreasing neutrophils-ECs adhesion. These findings identified BAP31 as a promising protein for regulating the pathogenesis process of ALI.

Perioperative Intermittent Fasting Protects Ischemic Musculocutaneous Flap Tissue from Necrosis.

Dietary restriction, such as intermittent fasting (IF), has previously been shown to protect various tissues from ischemia-induced necrosis. Based on this finding, the authors herein evaluated for the first time the tissue-protective effects of IF in musculocutaneous flaps. Male C57BL/6N mice were randomly assigned to an IF group ( n = 8) and a control group with unrestricted access to standard diet ( n = 8). IF animals were put on a perioperative feeding schedule with 8 hours of unrestricted access to standard diet per day starting 7 days before flap elevation up to 3 days after surgery. Random pattern musculocutaneous flaps were raised and mounted into a dorsal skinfold chamber. Intravital fluorescence microscopy was performed on days 1, 3, 5, 7, and 10 after surgery for the quantitative assessment of angiogenesis, nutritive blood perfusion, and flap necrosis. After the in vivo observation period, the flaps were harvested for additional histologic and immunohistochemical analyses. The authors found that the IF group exhibited more newly formed microvessels and a higher functional capillary density within the flap tissue when compared with controls. This was associated with a significantly lower rate of tissue necrosis. Immunohistochemical detection of different inflammatory cell subtypes revealed a markedly reduced number of invading myeloperoxidase-positive neutrophilic granulocytes in the musculocutaneous tissue of IF-treated animals. Considered together, these findings indicate that IF prevents flap necrosis by maintaining nutritive tissue perfusion and suppressing ischemia-induced inflammation. Given the frequent use of flaps in plastic surgery and the high rates of ischemic complications, any new approach to decrease such complications is clinically relevant.

Repeated dosing improves oncolytic rhabdovirus therapy in mice via interactions with intravascular monocytes

There is debate in the field of oncolytic virus (OV) therapy, whether a single viral dose, or multiple administrations, is better for tumor control. Using intravital microscopy, we describe the fate of vesicular stomatitis virus (VSV) delivered systemically as a first or a second dose. Following primary administration, VSV binds to the endothelium, initiates tumor infection and activates a proinflammatory response. This initial OV dose induces neutrophil migration into the tumor and limits viral replication. OV administered as a second dose fails to infect the tumor and is captured by intravascular monocytes. Despite a lack of direct infection, this second viral dose, in a monocyte-dependent fashion, enhances and sustains infection by the first viral dose, promotes CD8 T cell recruitment, delays tumor growth and improves survival in multi-dosing OV therapy. Thus, repeated VSV dosing engages monocytes to post-condition the tumor microenvironment for improved infection and anticancer T cell responses. Understanding the complex interactions between the subsequent viral doses is crucial for improving the efficiency of OV therapy and virus-based vaccines.

Alveolus analysis: a web browser-based tool to analyze lung intravital microscopy

BackgroundAcute lung injury and the acute respiratory distress syndrome are characterized by pulmonary inflammation, reduced endothelial barrier integrity and filling of the alveolar space with protein rich edema fluid and infiltrating leukocytes. Animal models are critical to uncovering the pathologic mechanisms of this devastating syndrome. Intravital imaging of the intact lung via two-photon intravital microscopy has proven a valuable method to investigate lung injury in small rodent models through characterization of inflammatory cells and vascular changes in real time. However, respiratory motion complicates the analysis of these time series images and requires selective data extraction to stabilize the image. Consequently, analysis of individual alveoli may not provide a complete picture of the integrated mechanical, vascular and inflammatory processes occurring simultaneously in the intact lung. To address these challenges, we developed a web browser-based visualization application named Alveolus Analysis to process, analyze and graphically display intravital lung microscopy data.ResultsThe designed tool takes raw temporal image data as input, performs image preprocessing and feature extraction offline, and visualizes the extracted information in a web browser-based interface. The interface allows users to explore multiple experiments in three panels corresponding to different levels of detail: summary statistics of alveolar/neutrophil behavior, characterization of alveolar dynamics including lung edema and inflammatory cells at specific time points, and cross-experiment analysis. We performed a case study on the utility of the visualization with two members or our research team and they found the tool useful because of its ability to preprocess data consistently and visualize information in a digestible and informative format.ConclusionsApplication of our software tool, Alveolus Analysis, to intravital lung microscopy data has the potential to enhance the information gained from these experiments and provide new insights into the pathologic mechanisms of inflammatory lung injury.