Intravital Analysis Research Articles

Features of the parotid gland anatomical parameters in men depending on the shape of the skull

BACKGROUND:Intravital analysis of parotid gland morphometric parameters in the context of various skull shapes is promi­sing, since it allows systematizing the available information on the parotid gland anatomy. AIM:To identify the features of the parotid gland anatomical parameters in men with different skull shapes according to computed tomography and magnetic resonance imaging. MATERIAL AND METHODS:We conducted a retrospective study based on the examination of computer and magnetic resonance tomograms of the heads of 92men. Depending on the skull shape, the study material was divided into groups: dolichocranes (n=30), mesocranes (n=32), brachycranes (n=30); chamecranes (n=19), orthocranes (n=47), hypsicranes (n=26); taipenocranes (n=33), metriocranes (n=30), acrocranes (n=29). Depending on the face shape, three groups were distinguished: euryenes (n=29), mesenes (n=33), leptens (n=40). A morphometric study of the parotid gland was performed with measurement of the sizes in three planes, as well as the volume of the organ using the technique of measuring internal organs’ volume during ultrasound examination. Statistical analysis was performed using nonparametric methods, such as the Kruskal–Wallis test, Mann–Whitney Utest with Bonferroni correction, and Spearman. RESULTS:In men, the sagittal size of the parotid gland significantly increased from brachycranes (4.22cm on the right, 3.99cm on the left) to dolichocranes [4.77cm on the right (p=0.0001), 4.57cm on the left (p=0.0001)]. A decrease in the frontal size of the gland was revealed from brachycranes ([3.43cm on the right, 3.47cm on the left) to dolichocranes [2.49cm on the right (p=0.0001), 2.47cm on the left (p=0.0001)]. With regard to the shapes of the facial skull, a significant difference in the vertical size of the gland was found [in euryenes 6.12cm on the right, 6.18cm on the left; in leptenes 6.98cm on the right (p0.0001), 6.95cm on the left (p0.0001)]. The frontal size value decreased significantly from euryens (3.42cm on the right, 3.53cm on the left) to leptenes [2.51cm on the right (p0.0001), 2.58cm on the left (p0.0001)]. CONCLUSION:In men with different forms of the cerebral skull, determined by the transverse-longitudinal index, the sagittal size of the parotid gland varies significantly; in men, depending on the shape of both the cerebral and facial skull, the frontal size of the gland changes.

Integrated data from intravital imaging and HPLC-MS/MS analysis reveal large interspecies differences in AFB1 metabolism in mice and rats.

Large interspecies differences between rats and mice concerning the hepatotoxicity and carcinogenicity of aflatoxin B1 (AFB1) are known, with mice being more resistant. However, a comprehensive interspecies comparison including subcellular liver tissue compartments has not yet been performed. In this study, we performed spatio-temporal intravital analysis of AFB1 kinetics in the livers of anesthetized mice and rats. This was supported by time-dependent analysis of the parent compound as well as metabolites and adducts in blood, urine, and bile of both species by HPLC-MS/MS. The integrated data from intravital imaging and HPLC-MS/MS analysis revealed major interspecies differences between rats and mice: (1) AFB1-associated fluorescence persisted much longer in the nuclei of rat than mouse hepatocytes; (2) in the sinusoidal blood, AFB1-associated fluorescence was rapidly cleared in mice, while a time-dependent increase was observed in rats in the first three hours after injection followed by a plateau that lasted until the end of the observation period of six hours; (3) this coincided with a far stronger increase of AFB1-lysine adducts in the blood of rats compared to mice; (4) the AFB1-guanine adduct was detected at much higher concentrations in bile and urine of rats than mice. In both species, the AFB1-glutathione conjugate was efficiently excreted via bile, where it reached concentrations at least three orders of magnitude higher compared to blood. In conclusion, major differences between mice and rats were observed, concerning the nuclear persistence, formation of AFB1-lysine adducts, and the AFB1-guanine adducts.

Validation of NBD-coupled taurocholic acid for intravital analysis of bile acid transport in liver and kidney of mice.

Fluorophore-coupled bile acids (BA) represent an important tool for intravital analysis of BA flux in animal models of cholestatic diseases. However, addition of a fluorophore to a BA may alter transport properties. We developed and validated a 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole-coupled taurocholic acid (3β-NBD-TCA) as a probe for intravital analysis of BA homeostasis. We compared transport of 3β-NBD-TCA to [3H]-TCA in HEK293 cells stably expressing the mouse hepatic or renal BA carriers mNtcp or mAsbt, respectively. We also studied distribution kinetics intravitally in livers and kidneys of anesthetized wildtype and mOatp1a/1b cluster knockout mice (OatpKO) with and without administration of the Ntcp inhibitor Myrcludex B and the ASBT inhibitor AS0369. In vitro, 3β-NBD-TCA and [3H]-TCA showed comparable concentration- and time-dependent transport via mNtcp and mAsbt as well as similar inhibition kinetics for Myrcludex B and AS0369. Intravital analysis in the livers of wildtype and OatpKO mice revealed contribution of both mNtcp and mOatp1a/1b in the 3β-NBD-TCA uptake from the sinusoidal blood into hepatocytes. Combined deletion of mOatp1a/1b and inhibition of mNtcp by Myrcludex B blocked the uptake of 3β-NBD-TCA from sinusoidal blood into hepatocytes. This led to an increase of 3β-NBD-TCA signal in the systemic circulation including renal capillaries, followed by strong enrichment in a subpopulation of proximal renal tubular epithelial cells (TEC). The enrichment of 3β-NBD-TCA in TEC was strongly reduced by the systemic ASBT inhibitor AS0369. NBD-coupled TCA has similar transport kinetics as [3H]-TCA and can be used as a tool to study hepatorenal BA transport. See also the graphical abstract(Fig. 1).

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets. The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

Live Intravital Intestine with Blood Flow Visualization in Neonatal Mice Using Two-photon Laser Scanning Microscopy

This protocol describes a novel technique to investigate the microcirculation dynamics underlying the pathology in the small intestine of neonatal mice using two-photon laser-scanning microscopy (TPLSM). Recent technological advances in multi-photon microscopy allow intravital analysis of different organs such as the liver, brain and intestine. Despite these advances, live visualization and analysis of the small intestine in neonatal rodents remain technically challenging. We herein provide a detailed description of a novel method to capture high resolution and stable images of the small intestine in neonatal mice as early as postnatal day 0. This imaging technique allows a comprehensive understanding of the development and blood flow dynamics in small intestine microcirculation.

2053-P: Integrated Blood Flow between Endocrine and Exocrine Pancreas Is Conserved among Multiple Species

Pancreatic islets compose the endocrine pancreas and are considered distinct from the exocrine pancreas in terms of both function and regulation of blood flow. Indeed, enzymatically isolated islets directly respond to glucose and other secretagogues in vitro. The islet has been regarded as an enclosed micro-organ, in which blood flows uni-directionally from an afferent arteriole to an efferent venule. This blood circulation pattern has been termed as “the insulo-acinar portal system.” Historically, the endocrine and exocrine pancreas have been studied separately by different fields of investigators. Using intravital analysis of pancreas blood flow in mice, we have recently reported that islet capillaries are continuously integrated with those in the exocrine pancreas, which makes the islet circulation open, not self-contained. Furthermore, by tracking fluorescently-labeled red blood cells revealed bi-directional blood flow between the endocrine and exocrine pancreas. In the present study, in order to verify whether this integrated vascular network is conserved among different species, we carried out three-dimensional morphological analysis of pancreatic capillary networks using thick pancreatic tissue slices (600-800 µm) in ferrets, rabbits, pigs, dogs and monkeys, besides mice and humans. We have confirmed that the integrated blood flow between endocrine and exocrine pancreas is conserved among multiple species that we studied. Here we propose that the open circulation model physically links both endocrine and exocrine parts of the pancreas as a single organ through the integrated vascular network. This new paradigm may provide revived or contrasting insights into the findings of past studies, as well as a novel approach for prospective research projects. Disclosure J.K. Butterfield: None. M. Hara: None. Funding National Institutes of Health (DK117192, DK020595)

Integrated Pancreatic Blood Flow: Bidirectional Microcirculation Between Endocrine and Exocrine Pancreas.

The pancreatic islet is a highly vascularized endocrine micro-organ. The unique architecture of rodent islets, a so-called core-mantle arrangement seen in two-dimensional images, led researchers to seek functional implications for islet hormone secretion. Three models of islet blood flow were previously proposed, all based on the assumption that islet microcirculation occurs in an enclosed structure. Recent electrophysiological and molecular biological studies using isolated islets also presumed unidirectional flow. Using intravital analysis of the islet microcirculation in mice, we found that islet capillaries were continuously integrated to those in the exocrine pancreas, which made the islet circulation rather open, not self-contained. Similarly in human islets, the capillary structure was integrated with pancreatic microvasculature in its entirety. Thus, islet microcirculation has no relation to islet cytoarchitecture, which explains its well-known variability throughout species. Furthermore, tracking fluorescent-labeled red blood cells at the endocrine-exocrine interface revealed bidirectional blood flow, with similar variability in blood flow speed in both the intra- and extra-islet vasculature. To date, the endocrine and exocrine pancreas have been studied separately by different fields of investigators. We propose that the open circulation model physically links both endocrine and exocrine parts of the pancreas as a single organ through the integrated vascular network.

Brief update on endocytosis of nanomedicines.

The complexity of nanoscale interactions between biomaterials and cells has limited the realization of the ultimate vision of nanotechnology in diagnostics and therapeutics. As such, significant effort has been devoted to advancing our understanding of the biophysical interactions of the myriad nanoparticles. Endocytosis of nanomedicine has drawn tremendous interest in the last decade. Here, we highlight the ever-present barriers to efficient intracellular delivery of nanoparticles as well as the current advances and strategies deployed to breach these barriers. We also introduce new barriers that have been largely overlooked such as the glycocalyx and macromolecular crowding. Additionally, we draw attention to the potential complications arising from the disruption of the newly discovered functions of the lysosomes. Novel strategies of exploiting the inherent intracellular defects in disease states to enhance delivery and the use of exosomes for bioanalytics and drug delivery are explored. Furthermore, we discuss the advances in imaging techniques like electron microscopy, super resolution fluorescence microscopy, and single particle tracking which have been instrumental in our growing understanding of intracellular pathways and nanoparticle trafficking. Finally, we advocate for the push towards more intravital analysis of nanoparticle transport phenomena using the multitude of techniques available to us. Unraveling the underlying mechanisms governing the cellular barriers to delivery and biological interactions of nanoparticles will guide the innovations capable of breaching these barriers.

A Zebrafish Embryo Model for In Vivo Visualization and Intravital Analysis of Biomaterial-associated Staphylococcus aureus Infection.

Biomaterial-associated infection (BAI) is a major cause of the failure of biomaterials/medical devices. Staphylococcus aureus is one of the major pathogens in BAI. Current experimental BAI mammalian animal models such as mouse models are costly and time-consuming, and therefore not suitable for high throughput analysis. Thus, novel animal models as complementary systems for investigating BAI in vivo are desired. In the present study, we aimed to develop a zebrafish embryo model for in vivo visualization and intravital analysis of bacterial infection in the presence of biomaterials based on fluorescence microscopy. In addition, the provoked macrophage response was studied. To this end, we used fluorescent protein-expressing S. aureus and transgenic zebrafish embryos expressing fluorescent proteins in their macrophages and developed a procedure to inject bacteria alone or together with microspheres into the muscle tissue of embryos. To monitor bacterial infection progression in live embryos over time, we devised a simple but reliable method of microscopic scoring of fluorescent bacteria. The results from microscopic scoring showed that all embryos with more than 20 colony-forming units (CFU) of bacteria yielded a positive fluorescent signal of bacteria. To study the potential effects of biomaterials on infection, we determined the CFU numbers of S. aureus with and without 10 µm polystyrene microspheres (PS10) as model biomaterials in the embryos. Moreover, we used the ObjectJ project file "Zebrafish-Immunotest" operating in ImageJ to quantify the fluorescence intensity of S. aureus infection with and without PS10 over time. Results from both methods showed higher numbers of S. aureus in infected embryos with microspheres than in embryos without microspheres, indicating an increased infection susceptibility in the presence of the biomaterial. Thus, the present study shows the potential of the zebrafish embryo model to study BAI with the methods developed here.

THE PROBLEMS OF ANGIOGENESIS IN THE EYE OF PREMATURE CHILD

The purpose of study is to investigate pathogenesis of the first pre-clinical stage of retinopathy in premature children. Materials and methods. The sampling for examination included 642 premature born children. At the moment of birth, the gestation age was uз to 30 weeks, body mass up to 1500 g (very low body mass). The study included: indirect ophthalmoscopy; digital retinoscopy using wild-field retinal pediatric camera; calibermetry of retinal vessels. The intravital analysis of biochemical composition of vitreous body in 74 children with retinopathy and 45 animals (young rabbits chinchilla). The intra-vitreous injection of inhibitors VEGF was applied to more than 300 premature born children. The results. The retinopathy of premature born children is a leading cause of blindness. The article is devoted to mainly studying pathogenesis of only pre-clinical phase I of retinopathy of premature born children that is factually explored insufficiently. It is established that pathological angiogenesis of retina triggers an area of retina with vessels that stimulates functioning of system of struggle with hyper oxygenation and delays a normal development of angiogenesis. The immature auto-regulation of vessels of retina inadequately reacts to oxygen causing expressed angiospasm and developed circulatory disorders in vascularized retina aggravating total hypoxia. These processes delay angiogenesis almost for a month. The hypoxia-induced factors HIF1.2,3 began with significant delay both to be cumulated and to stimulate production of vascular endothelial growth factor. Conclusion. The study of pathogenesis phase I of retinopathy of premature born children open opportunity to effect pathogenic sections of disease and to prevent blindness and visual impairedness in premature born children.

Local inflammation exacerbates cutaneous manifestations in a murine autoimmune pemphigus model

Local inflammation exacerbates cutaneous manifestations in a murine autoimmune pemphigus model

Statins prevent cognitive impairment after sepsis by reverting neuroinflammation, and microcirculatory/endothelial dysfunction

Acute brain dysfunction is a frequent condition in sepsis patients and is associated with increased mortality and long-term neurocognitive consequences. Impaired memory and executive function are common findings in sepsis survivors. Although neuroinflammation and blood-brain barrier dysfunction have been associated with acute brain dysfunction and its consequences, no specific treatments are available that prevent cognitive impairment after sepsis. Experimental sepsis was induced in Swiss Webster mice by intraperitoneal injection of cecal material (5mg/kg, 500μL). Control groups (n=5/group each experiment) received 500μL of saline. Support therapy recover (saline 0.9%, 1mL and imipenem 30mg/kg) were applied (6, 24 and 48h post injection, n=5–10/group, each experiment), together or not with additive orally treatment with statins (atorvastatin/simvastatin 20mg/kg b.w.). Survival rate was monitored at 6, 24 and 48h. In a setting of experiments, animals were euthanized at 6 and 24h after induction for biochemical, immunohistochemistry and intravital analysis. Statins did not prevented mortality in septic mice, however survivors presented lower clinical score. At another setting of experiments, after 15days, mice survivors from fecal supernatant peritoneal sepsis presented cognitive dysfunction for contextual hippocampal and aversive amygdala-dependent memories, which was prevented by atorvastatin/simvastatin treatment. Systemic and brain tissue levels of proinflammatory cytokines/chemokines and activation of microglial were lower in septic mice treated with statins. Brain lipid peroxidation and myeloperoxidase levels were also reduced by statins treatment. Intravital examination of the brain vessels of septic animals revealed decreased functional capillary density and increased rolling and adhesion of leukocytes, and blood flow impairment, which were reversed by treatment with statins. In addition, treatment with statins restored the cholinergic vasodilator response due to sepsis. Taken together, these data demonstrated that statins reverse microvascular dysfunction and reduce neuroinflammation during sepsis, preventing the development of long-term cognitive decline.

A Non-Anticoagulant Fraction of Heparin Inhibits Leukocyte Diapedesis into the Lung by an Effect on Platelets.

We have investigated whether the mechanism by which the non-anticoagulant N-acetyl-de-O-sulfated-heparin (NSH) inhibits leukocyte infiltration is mediated by an effect on platelet function. We show that oral treatment with two doses of NSH significantly inhibits eosinophil and neutrophil recruitment into the lungs. Intravital microscopy analysis shows that NSH inhibits leukocyte and platelet diapedesis in the microcirculation of the cremaster muscle and in the trachea. More importantly, there were significantly lower numbers of leukocytes recruited into the lung in response to LPS in thrombocytopenic mice when transfused with platelets pretreated with NSH in vitro when compared with mice transfused with untreated platelets. Using intravital analysis of the microvasculature of the cremaster muscle, we have demonstrated that the reinfusion of activated platelets significantly re-established leukocyte diapedesis in response to LPS but that this effect was not observed when platelets were pretreated in vitro with NSH. Finally, we investigated whether the effect of NSH altered the expression of adhesion molecules on the surface of platelets and leukocytes in blood samples collected from mice treated orally with NSH. Our results demonstrate that NSH significantly inhibited the detection of P-selectin as evaluated by flow cytometry, confirming that part of the antiinflammatory action of NSH is via an effect on platelet function.

The role of dendritic cells and macrophages in the skin immunity.

The skin is one of the largest organs in the human body, which acts as the primary interface with the external world. In view of its protective role, mammalian skin consists of physical and immunological barriers. The water-impermeable stratum corneum and the tight junctions in the granular layer work at the epidermal level work as the most important first and second "physical" barriers. Upon antigen invasion to the skin, the integrated innate and acquired immune systems in both the epidermis and dermis are activated in a coordinated manner to neutralize the external intruder as the strong third "immunological" barriers. Dendritic cells and macrophages are known to play pivotal roles in such immunological barriers. Intra-vital analysis of the murine skin by two-photon microscopy enabled us to assess the habituate and the direct interactions of various cells in the skin in situ, which reside or infiltrate upon inflammation. We introduce the recent works how dendritic cells and macrophages orchestrate the skin immunity, highlighting the importance of sequential leucocyte cluster formation in the efficient activation of memory T cells in the skin, which can be attributed as 'inducible skin-associated lymphoid tissue (iSALT)'.

Intravital analysis of vascular permeability in mice using two-photon microscopy

Blood vessel endothelium forms a semi-permeable barrier and its permeability controls the traffics of plasma contents. Here we report an intravital evaluation system for vascular permeability in mice using two-photon microscopy. We used various sizes of fluorescein-conjugated dextran as a tracer and its efflux was quantified by measuring the changes of fluorescent intensity both on the blood vessel area and the interstitial space. Using this system, we demonstrated that skin blood vessels limited the passage of dextran larger than 70 kDa under homeostatic conditions. We evaluated the kinetics of vascular permeability in histamine- or IgE-induced type I allergic models and a hapten-induced type IV allergic model. In such inflammatory conditions, the hyperpermeability was selectively induced in the postcapillary venules and dextran as large as 2000-kDa leaked from the bloods. Taken together, our study provides a convenient method to characterize the skin blood vessels as a traffic barrier in physiological conditions.

The Use of Spinning-Disk Confocal Microscopy for the Intravital Analysis of Platelet Dynamics in Response to Systemic and Local Inflammation

Platelets are central players in inflammation and are an important component of the innate immune response. The ability to visualize platelets within the live host is essential to understanding their role in these processes. Past approaches have involved adoptive transfer of labelled platelets, non-specific dyes, or the use of fluorescent antibodies to tag platelets in vivo. Often, these techniques result in either the activation of the platelet, or blockade of specific platelet receptors. In this report, we describe two new methods for intravital visualization of platelet biology, intravenous administration of labelled anti-CD49b, which labels all platelets, and CD41-YFP transgenic mice, in which a percentage of platelets express YFP. Both approaches label endogenous platelets and allow for their visualization using spinning-disk confocal fluorescent microscopy. Following LPS-induced inflammation, we were able to measure a significant increase in both the number and size of platelet aggregates observed within the vasculature of a number of different tissues. Real-time observation of these platelet aggregates reveals them to be large, dynamic structures that are continually expanding and sloughing-off into circulation. Using these techniques, we describe for the first time, platelet recruitment to, and behaviour within numerous tissues of the mouse, both under control conditions and following LPS induced inflammation.

The impact of the extracellular matrix on inflammation

The advent of in situ immunology and intravital analyses of leukocyte movement in tissues has drawn attention to the previously neglected extracellular matrix (ECM) and its role in modulating immune cell behaviour in inflamed tissues. The ECM exists in different biochemical and structural forms; both their individual components and three-dimensional ultrastructure impart specific signals to cells that modulate basic functions that are important for the early steps in inflammation, such as immune cell migration into inflamed tissues and immune cell differentiation. In chronically inflamed tissues, aberrant ECM expression and fragments of the ECM that are derived from tissue-remodelling processes can influence immune cell activation and survival, thereby actively contributing to immune responses at these sites.

Individualized ventriculostomy in hydrocephalus: An intravital anatomical study

Ventriculostomy is a common neuroendoscopic operation but one with disastrous complications in rare cases. The aim of this study was to perform an intravital analysis of the configuration at the floor of the third ventricle as a possible basis for selection of the ventriculostomy site. The study population consisted of 32 patients who underwent ventriculostomy for the treatment of hydrocephalus. Perforation of the floor of the third ventricle was carried out on an individual basis following evaluation of the anatomic situation. Video material and magnetic resonance images (MRI) were analyzed. A classification system including three major groups was developed using the inner distance of the mamillary bodies as the key criterion. It was defined as narrow for values between 0 and 1 mm (observed range: 0-0.5 mm), medium for values between 1.1 and 3.4 mm (range 1.1-3.4 mm) and large for values greater than 3.4 mm (range: 3.8-6.9 mm). Statistical analysis of MR and video measurements revealed a good correlation. The ventriculostomy site was rostral of the mamillary bodies in 23 of the patients (n=27) and slightly occipital in four. The ventriculostomy site was located more to the left in 22 patients and more to the right in five. As a conclusion the ventriculostomy site has to be chosen in each case following a careful review of all available information. A classification system for the anatomical variations as well as the exact size and site of ventriculostomy should be introduced.

Analysis methods of human cell migration

The autonomous migration of specialized cells is an essential characteristic in both physiological and pathological functions in the adult human organism. Leukocytes, fibroblasts, and stem cells, but also tumor cells, are thus the subject of intense investigation in a broad range of research fields. A wide spectrum of methods have therefore been established to analyze chemokinetic and chemotactic cell migration, ranging from easy-to-handle two-dimensional surface migration assays to highly specialized three-dimensional and intravital analysis methods. It is now manifest that the results obtained with these various migration assays substantially differ. This review therefore gives an overview of the migration assays which are currently in use, describes the methods, and critically enlightens the particular advantages and disadvantages of each method.

The role of hepatic stellate cells in liver regeneration

The role of hepatic stellate cells in liver regeneration