Intravital Staining Research Articles

Identifying the role of antigen-specific resident memory CD4 T cells in maintaining disease during Experimental Autoimmune Encephalomyelitis a murine model of Multiple Sclerosis.

Abstract Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) driven by antigen-specific CD4+ T cell mediated destruction of the myelin sheath. Most T cells circulate between lymphoid organs and peripheral tissues, but a fraction can establish tissue residence and do not recirculate. Growing evidence suggests resident T cells exist in the CNS of MS patients, however, their contribution to disease progression and whether they can cause relapses independently of peripheral T cells is unclear. Current disease modifying therapies for MS patients are not curative and none target CNS resident T cells. Using a murine model of MS, Experimental autoimmune encephalomyelitis (EAE), we investigated whether resident T cells form in the CNS of diseased mice and their contribution to disease pathogenesis. Our data show that antigen-specific CNS resident T cells can be detected during EAE by intravital antibody staining and CD69 expression. Following depletion of circulating CD4+ T cells, CNS resident CD69+ CD4+ T cells maintained their numbers, while the number of CD69- T cells in the CNS was significantly depleted. These CNS resident T cells can drive severe disease in the absence of circulating CD4+ T cells and showed memory-like properties including high proliferative capacity and increased cytokine polyfunctionality compared to early effector T cells. Therefore, disruption of resident T cell function in MS patients could represent a novel target for MS therapies.

Сравнительный анализ механического и фемтолазер-ассистированного методов выкраивания лимбальных мини-трансплантатов в эксперименте

Original article Experimental study of manual and femtosecond laser-assisted methods for cutting limbal mini-transplants B.E. Malyugin, S.A. Borzenok, O.N. Nefedova, D.S. Ostrovskii, M.Yu. Gerasymov, A.V. Shatskikh S. Fyodorov Eye Microsurger y Federal State Institution, Moscow, Russian Federation A. Yevdokimov Moscow State University of Medicine and Dentistr y, Moscow, Russian Federation Purpose. To define optimal parameters of femtosecond laser (FSL) (Femto LDV Z8, Ziemer, Switzerland) for cutting limbal mini-transplants to be further used for limbal stem cells (LSC) transplantation and compare the results with manual cutting technique using microsurgical blade. Material and methods. At first step, performed on 3 porcine and 3 cadaveric eyes, the optimal energy parameters of FSL were selected, which ensured the excision of limbal mini-transplants. At the second step, 8 eyes from 4 postmortem donors were used. In the lower and upper parts of the limbus, limbal fragments were cut out into 8 mini-transplants with a FSL (experimental group). Mini-grafts were also formed manually in symmetrical areas of each eye using dosed diamond blade (control group). The structure of the limbal bed after removing mini-transplants was studied by light and scanning electron microscopy. The effect of FSL energy on the viability of LSC was determined by intravital staining using Live & Dead dye. Finally, on the third step, the assessment of reversible and irreversible apoptosis was performed by culturing corneoscleral discs containing mini-transplants not separated from the limbal bed. The obtained samples were cultivated in a medium DMEM/F12 for 7 days followed by immunohistochemical examination. Results. The optimal parameters of FSL operation are determined by energy levels- 100, 110 and 120%. Histological analysis of the limbal residual bed zone showed that the smoothest surface was obtained at energies 110 and 120%. This fact was also confirmed by scanning electron microscopy. When stained with the Live & Dead dye, damage on the lateral and inner sides of limbal mini-transplants was revealed, mainly in the experimental group. After 7 days of cultivation in the cut area in both groups, no expression of apoptosis markers was observed. Conclusion. Cutting out the limbal zone containing LSCs using FSL is safe and allows obtaining limbal mini-grafts. The most optimal parameters of laser operation in the limbal zone were selected – a planar cut at depth of 250 µm using 110% laser energy. Key words: femtosecond laser, limbal stem cells, apoptosis, limbal stem cell deficiency syndrome, scanning electron microscopy, light microscopy, cultivation, histology

Generation of translucent Xenopus tropicalis through triple knockout of pigmentation genes.

Amphibians generally have three types of pigment cells, namely, melanophores (black and brown), xanthophores (yellow and red), and iridophores (iridescent). Single knockout of the tyr, slc2a7, and hps6 genes in Xenopus tropicalis results in the absence of melanophores, xanthophores, and iridophores, respectively. The generation of triple- knockout (3KO) X. tropicalis for these three genes could allow for observation of internal organs without sacrificing the animals, which would be transparent due to the absence of pigments. In this study, we generated 3KO X. tropicalis, which is one of the most widely used model amphibians, through crossing of a slc2a7 single-knockout frog with a tyr and hps6 double-knockout frog, followed by intercrossing of their offspring. The 3KO tadpoles had transparent bodies like the nop mutant and the frogs had translucent bodies. This translucency allowed us to observe the heart, lungs, stomach, liver, and digestive tract through the ventral body skin without surgery. After intravital staining, 3KO X. tropicalis showed much clearer fluorescent signals of mineralized tissues compared with the wild type. These 3KO X. tropicalis provide a useful mutant line for continuous observation of internal organs and fluorescent signals in the body. In particular, such 3KO frogs would revolutionize fluorescence monitoring in transgenic tadpoles and frogs expressing fluorescent proteins.

The role of methylene blue in mitigating surgical morbidity in parotid surgery: a comprehensive review

Parotid surgery is associated with specific surgical morbidity, including temporary facial weakness, permanent facial nerve injury, post-parotidectomy depression, and Frey's syndrome (gustatory sweating). Despite various modifications implemented to reduce surgical morbidity, complications, particularly facial nerve injury, remain a challenge. To overcome this difficulty, the use of an identification method, such as preoperative intravital staining of the gland with methylene blue (MB), has been proposed. This article provides a comprehensive review of the role of MB in parotid surgery, discussing its potential benefits, safety profile, and future directions for research.

Intravital staining to detect mineralization in Xenopus tropicalis during and after metamorphosis.

Observing mineralization is essential for studying skeletal development, maintenance, and regeneration. Calcein and alizarin red have long been used to visualize mineralization in fixed specimens, but this requires the target animals to be sacrificed. However, several intravital bone-staining methods have been developed to visualize mineralized tissues in living animals. These methods have been applied to study fin rays and transparent fishes. Xenopus tropicalis is an excellent experimental animal model for studying bone formation and regeneration because skeletal mineralization begins during the free-living tadpole period, and its regenerative ability changes during metamorphosis. However, intravital bone staining of X. tropicalis has only been reported for tadpoles, and no details on its specificity or appropriate experimental conditions are available. Here, we compared the calcein- and alizarin red S (ARS)-staining methods and optimized these methods for tadpoles and juvenile frogs during and after metamorphosis. Staining with 0.01% ARS yielded acceptable signaling for young tadpoles, whereas calcein either at 0.1 or 0.01% occasionally showed artifactual staining of unmineralized tissues. In addition, 0.1% calcein or 0.1% ARS staining showed a higher signal-to-noise ratio with juvenile frogs compared to staining at 0.01%. We propose the use of 0.01% ARS for tadpoles before stage 61 and 0.1% ARS thereafter for staining mineralized tissues. Using this method, we found that ossification of the neural arches occurred at stage 51 in X. tropicalis. This method enables precise staging and manipulation based on the visualized bone structure.

Application of meso-CF3-Fluorophore BODIPY with Phenyl and Pyrazolyl Substituents for Lifetime Visualization of Lysosomes.

A bright far-red emitting unsymmetrical meso-CF3-BODIPY fluorescent dye with phenyl and pyrazolyl substituents was synthesized by condensation of trifluoropyrrolylethanol with pyrazolyl-pyrrole, with subsequent oxidation and complexation of the formed dipyrromethane. This BODIPY dye exhibits optical absorption at λab ≈ 610–620 nm and emission at λem ≈ 640–650 nm. The BODIPY was studied on Ehrlich carcinoma cells as a lysosome-specific fluorescent dye that allows intravital staining of cell structures with subsequent real-time monitoring of changes occurring in the cells. It was also shown that the rate of uptake by cells, the rate of intracellular transport into lysosomes, and the rate of saturation of cells with the dye depend on its concentration in the culture medium. A concentration of 5 μM was chosen as the most suitable BODIPY concentration for fluorescent staining of living cell lysosomes, while a concentration of 100 μM was found to be toxic to Ehrlich carcinoma cells.

Interaction of Ras Binding Domain (RBD) by chemotherapeutic zinc oxide nanoparticles: Progress towards RAS pathway protein interference.

Zinc oxide (ZnO) NP is considered as a nanoscale chemotherapeutic. Thus, the drug delivery of this inorganic NP is of considerable importance. Ras mutations are common in cancer and the activation of this signaling pathway is a hallmark in carcinoma, melanoma and many other aggressive malignancies. Thus, here we examined the binding and delivery of Ras binding domain (RBD), a model cancer-relevant protein and effector of Ras by ZnO NP. Shifts in zeta potential in water, PBS, DMEM and DMEM supplemented with FBS supported NP interaction to RBD. Fluorescence quenching of the NP was concentration-dependent for RBD, Stern–Volmer analysis of this data was used to estimate binding strength which was significant for ZnO-RBD (Kd < 10−5). ZnO NP interaction to RBD was further confirmed by pull-down assay demonstrated by SDS-PAGE analysis. The ability of ZnO NP to inhibit 3-D tumor spheroid was demonstrated in HeLa cell spheroids—the ZnO NP breaking apart these structures revealing a significant (>50%) zone of killing as shown by light and fluorescence microscopy after intra-vital staining. ZnO 100 nm was superior to ZnO 14 nm in terms of anticancer activity. When bound to ZnO NP, the anticancer activity of RBD was enhanced. These data indicate the potential diagnostic application or therapeutic activity of RBD-NP complexes in vivo which demands further investigation.

Antiproliferative and antitumour activity of saponins from Astragalus glycyphyllos on myeloid Graffi tumour

Ethnopharmacological relevanceAstragalus glycyphyllos L. has been extensively used in Bulgarian folk medicine as an antihypertensive, diuretic, anti-inflammatory, anti-tumour, in cases of cardiac insufficiency, renal inflammation, calculosis, etc. Aim of the studyTo evaluate the possible in vitro/in vivo anti-proliferative/anti-tumour activity of a purified saponins’ mixture (PSM) obtained from the plant. Materials and methodsViability and proliferative activity of the Graffi myeloid tumour cells was assessed by MTT test. The morphological alterations were visualized and analysed by fluorescent microscopy after intravital double staining. An in vivo model of Graffi tumour bearing hamsters was used to examine the influence of PSM on transplantability, tumour growth, survival and mortality as well as to observe pathomorphological changes. ResultsGraffi tumour cells were sensitive to purified saponins’ mixture after 24 and 48 h treatment. The treatment induced a statistically significant decrease of the viability/proliferation of the Graffi tumour cells. These effects were concentration- and time-dependent. Fluorescent microscopy studies showed that these antiproliferative effects were connected to the induction of apoptosis. The in vivo study showed the presence of a stromal component, single mononuclear cells in the stroma. Multiple incorrect mitotic figures were observed in the tumour tissue from the control group. Well-formed stroma with accumulation of mononuclear cells and mitotic cells were found in the group, treated with PSM. The tumour weight was decreased in the group, treated with PMS. ConclusionThe results indicate that PSM exhibited in vitro/in vivo antiproliferative/anti-tumour effects.

Imaging of muscle activity-induced morphometric changes in fibril network of myofascia by two-photon microscopy.

Myofascia, deep fascia enveloping skeletal muscles, consists of abundant collagen and elastin fibres that play a key role in the transmission of muscular forces. However, understanding of biomechanical dynamics in myofascia remains very limited due to less quantitative and relevant approaches for in vivo examination. The purpose of this study was to evaluate the myofascial fibril structure by means of a quantitative approach using two-photon microscopy (TPM) imaging in combination with intravital staining of Evans blue dye (EBD), a far-red fluorescence dye, which potentially labels elastin. With focus on myofascia of the tibial anterior (TA) muscle, the fibril structure intravitally stained with EBD was observed at the depth level of collagen fibrous membrane above the muscle belly. The EBD-labelled fibril structure and orientation in myofascia indicated biomechanical responses to muscle activity and ageing. The orientation histograms of EBD-labelled fibrils were significantly modified depending upon the intensity of muscle activity and ageing. Moreover, the density of EBD-labelled fibrils in myofascia decreased with habitual exercise but increased with muscle immobilization or ageing. In particular, the diameter of EBD-labelled fibrils in aged mice was significantly higher. The orientation histograms of EBD-labelled fibrils after habitual exercise, muscle immobilization and ageing showed significant differences compared to control. Indeed, the histograms in bilateral TA myofascia of exercise mice made simple waveforms without multiple sharp peaks, whilst muscular immobilization or ageing significantly shifted a histogram with sustaining multiple sharp peaks. Therefore, the dynamics of fibre network with EBD fluorescence in response to the biomechanical environment possibly indicate functional tissue adaptation in myofascia. Furthermore, on the basis of the knowledge that neutrophil recruitment occurs locally in working muscles, we suggested the unique reconstruction mechanism involving neutrophilic elastase in the myofascial fibril structure. In addition to the elastolytic susceptibility of EBD-labelled fibrils, distinct immunoreactivities and activities of neutrophil elastase in the myofascia were observed after electric pulse stimulation-induced muscle contraction for 15min. Our findings of EBD-labelled fibril dynamics in myofascia through quantitative approach using TPM imaging and intravital fluorescence labelling potentially brings new insights to examine muscle physiology and pathology.

Distribution and morphology of sensory and autonomic fibres in the subendocardial plexus of the rat heart.

Cardiac reflexes originating from sensory receptors in the heart ensure blood supply to vital tissues and organs in the face of constantly changing demands. Atrial volume receptors are mechanically sensitive vagal afferents which relay to the medulla and hypothalamus, affecting vasopressin release and renal sympathetic activity. To date, two anatomically distinct sensory endings have been identified which may subserve cardiac mechanosensation: end‐nets and flower‐spray endings. To map the distribution of atrial receptors in the subendocardial space, we have double‐labelled rat right atrial whole mounts for neurofilament heavy chain (NFH) and synaptic vesicle protein 2 (SV2) and generated high‐resolution maps of the rat subendocardial neural plexus at the cavo‐atrial region. In order to elucidate the nature of these fibres, double labelling with synaptophysin (SYN) and either NFH, calcitonin gene‐related peptide (CGRP), choline acetyltransferase (ChAT) or tyrosine hydroxylase (TH) was performed. The findings show that subendocardial nerve nets are denser at the superior cavo‐atrial junction than the mid‐atrial region. Adluminal plexuses had the finest diameters and stained positively for synaptic vesicles (SV2 and SYN), CGRP and TH. These plexuses may represent sympathetic post‐ganglionic fibres and/or sensory afferents. The latter are candidate substrates for type B volume receptors which are excited by stretch during atrial filling. Deeper nerve fibres appeared coarser and may be cholinergic (positive staining for ChAT). Flower‐spray endings were never observed using immunohistochemistry but were delineated clearly with the intravital stain methylene blue. We suggest that differing nerve fibre structures form the basis by which atrial deformation and hence atrial filling is reflected to the brain.

Intraperitoneal injection of IFN-\u03b3 restores microglial autophagy, promotes amyloid-\u03b2 clearance and improves cognition in APP/PS1 mice

Autophagy is a major self-degradative process that maintains cellular homeostasis and function in mammalian cells. Autophagic dysfunction occurs in the early pathogenesis of Alzheimer’s disease (AD) and directly regulates amyloid-β (Aβ) metabolism. Although it has been proven that the cytokine IFN-γ enhances autophagy in macrophage cell lines, whether the signaling cascade is implicated in Aβ degradation in AD mouse models remains to be elucidated. Here, we found that 9 days of the intraperitoneal administration of IFN-γ significantly increased the LC3II/I ratio and decreased the level of p62 in APP/PS1 mice, an AD mouse model. In vitro, IFN-γ protected BV2 cells from Aβ toxicity by upregulating the expressions of Atg7 and Atg5 and the LC3II/I ratio, whereas these protective effects were ablated by interference with Atg5 expression. Moreover, IFN-γ enhanced autophagic flux, probably through suppressing the AKT/mTOR pathway both in vivo and in vitro. Importantly, using intravital two-photon microscopy and fluorescence staining, we found that microglia interacted with exogenous IFN-γ and Aβ, and surrounded Aβ in APP/PS1;CX3CR1-GFP+/− mice. In addition, IFN-γ treatment decreased the Aβ plaque load in the cortex and hippocampus and rescued cognitive deficits in APP/PS1 mice. Our data suggest a possible mechanism by which the peripheral injection of IFN-γ restores microglial autophagy to induce the phagocytosis of cerebral Aβ, which represents a potential therapeutic approach for the use of exogenous IFN-γ in AD.

Macrophage replacement in Mycobacterium tuberculosis-induced murine lung granulomas

Abstract Monocytes/macrophages are the dominant cell type in mycobacterial granulomas. Macrophages both provide a survival niche for the bacteria and play a central role in bacterial control. Most of them are recruited from the blood with a Ly6C+ phenotype and they differentiate into F4/80+ macrophages in the lung lesions. It is well demonstrated that monocytes have access to pre-formed granulomatous lesions, but the rate of cell recruitment and the fate of the recruited cells are not well understood. Previously, testing BCG-induced liver granulomas using a transplantation model, we detected continuous cell replacement in the preformed lesions. Here, using intravital staining and cytofluorimetric detection of CD45+ blood cells in combination with IHC, we show that CD11b+ cells can access the lung parenchyma and Mtb-induced lesions over the course of 24 hours in Mtb H37Rv infected animals. We measured the level of monocyte/macrophage infiltration in both acute and chronic infection in C57BL/6 and C3HeB/FeJ mice. Although monocytes have a short lifespan in the blood, using intravenous cell transfer of tdTomato-expressing sentinel cells we show that they can live longer than a week in the lung lesions of Mtb-infected animals. Interestingly, a population of adoptively transferred CD11b+tdTomato+ cells can be detected for more than 10 days in the blood vessels in the infected lung. Gene expression profiling of recently or earlier recruited (CD11b+F4/80− or CD11b+F4/80+) cells sorted from the lung shows differential inflammatory gene expression between these populations. The continuous cell replacement that is required to maintain granulomatous lesions offers a novel possibility to manipulate granuloma pathology for a therapeutic advantage.

Repeated respiratory bacterial exposures elicit lung resident memory B cells in the absence of organized tertiary lymphoid tissue

Abstract Recent work has shown that resident memory B cells (BRM) are generated in the lung after flu infection. However, it is unclear whether the existence of lung BRM is unique to influenza, which often elicits iBALT, or alternatively if these cells represent a common feature of the lung immune landscape. We aimed to determine whether bacterial lung exposures experienced by nearly all human children might also elicit lung BRM. To model this scenario, we gave young mice repeated respiratory exposures to low virulence pneumococci. After at least 4 weeks, we then analyzed lung B cells using flow cytometry and an intravital CD45 stain to exclude intravascular leukocytes. Pneumococcus-exposed mice had many more extravascular IgD- lung B cells than controls, and these B cells remained in the lung at least 12 weeks after the last exposure. Although unorganized aggregates of B and T cells were observed in previously exposed lungs, no iBALT structures were generated. Most of the IgD-B cells expressed the memory markers PD-L2, CD80, and CD73; a majority of the B cells expressed two or more of these markers, indicating they are poised to become antibody secreting upon rechallenge. Notably, the hallmark lung TRM phenotype (CD11abrightCD69+CD44brightCD62Llow) was also observed on lung B cells; these markers may represent a common resident memory lymphocyte phenotype. Although all lung B cells were CD20+, they were not affected by αCD20 antibody treatment, which requires circulation of bound cells for depletion to occur. BRM appear to be a common feature of the adaptive immune cell repertoire of experienced lungs. These cells do not require iBALT for their maintenance and represent a previously unrecognized pool of B cells resistant to depletion with αCD20 therapy.

Osseointegrative effect of rhBMP-2 covalently bound on a titan-plasma-spray-surface after modification with chromosulfuric acid in a large animal bone gap-healing model with the G\xf6ttingen minipig

BackgroundBone morphogenetic proteins play an important role as osseointegrative factors. It is used widely in orthopedic research and surgery to enhance the osseointegrative potential of implants, e.g., in spinal fusion or alveolar socket augmentation. The aim of the present study was to investigate the benefit of rhBMP-2 on a titan plasma spray (TPS) layer after a special modification with chromosulfuric acid (CSA) at different postoperative times, regarding osseoconduction and osseoinduction.MethodsWe allocated 27 Göttinger minipigs into three groups consisting of nine animals each. They received four dumbbell-shaped implants in the metaphyseal parts of the femora. The implants had a TPS surface with (CSA group) and without a CSA treatment (TPS group). The former received an additional layer of BMP-2 (BMP-2 group). For the assessment of osseointegration after healing periods of 4, 8, and 12 weeks, histomorphometry was applied to undecalcified specimens after staining according to Masson-Goldner. An intravital labeling with different fluorochromes was used in the gap model. A multivariable analysis with repeated measurement design was performed for statistical evaluation.ResultsWe observed several statistical differences in a three-way ANOVA. The comparison between the BMP-2 and the TPS group (two-way ANOVA) showed statistically significant differences in terms of the osseoinduction (osteoid volume), and pronounced for the osseoconduction (bone and osteoid ongrowth), in favor of the BMP-2 group. In the pairwise comparison between BMP-2 and CSA (two-way ANOVA), no statistical significance occurred. The intravital staining with tetracycline, calcein green, and xylenol orange revealed no considerable differences between the groups.ConclusionBMP-2, covalently bound on a CSA-treated TPS surface, has positive effects on the osseointegration in the large animal bone gap-healing model over the observation period of 12 weeks.

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Gonadotropin surge stimulate ovarian osteopontin expression via epidermal growth factor receptor signaling to enhance corpus luteum function

cific angiocrine factors, creating an instructive vascular niche which promotes organ development, repair and regeneration. Studying the role of primary ovarian ECs in the regulation of physiological processes in the ovary has been hampered by the need for growth factor deprivation during culture, which leads to apoptosis. Here, we aim to isolate, purify, characterize and culture ovarian ECs in serum-free conditions, creating a platform for the exploration of endothelial-follicular communication. DESIGN: Laboratory study. MATERIALS AND METHODS: Mouse ovarian ECs were isolated via magnetic cell sorting using CD-31 antibody. After a culture period of 5-7 days, EC activation was initiated via lentivirus transduction, allowing prolonged culture in serum-free conditions. Purity of cells was confirmed with flow cytometry using CD31, CD45 and VE-Cadherin antibodies (VECAD). Gene expression was assessed with qPCR. In order to obtain adequate numbers of fresh ECs for comparison, a minimal stimulation protocol was applied. Intravital staining using VECAD and Isolectin was performed, allowing ovarian EC isolation using florescence activated cell sorting (FACS). Gene expression of fresh ECs was assessed with qPCR and compared to cultured counterparts. RESULTS: Flow cytometry showed that 98% of cultured activated ovarian ECs were CD31+, VECAD+ and CD45-. Relative expression of dll1, mmp10 and fgf1 was higher in cultured ECs, while expression of Jagged-1, VECAD and PDGF was comparable in both groups. Expression of Jagged-2, Notch14, dll4, VEGFR1, VEGFR2, mmp4, il6, and il8 was lower in activated, cultured ECs in comparison to fresh ECs but fairly high compared to its own housekeeping gene. CONCLUSION: Here, we report successful isolation and durable culture of ovarian ECs with an angiocrine profile matching that of fresh ovarian ECs, thus providing a novel tool with which to study cell-cell communication and vascularization within the ovary. Supported by: Institutional.

Fluorescein Derivatives in Intravital Fluorescence Imaging

Intravital fluorescence microscopy enables the direct imaging of fluorophores in vivo and advanced techniques such as fluorescence lifetime imaging (FLIM) enable the simultaneous detection of multiple fluorophores. Consequently, it is now possible to record distribution and metabolism of a chemical in vivo and to optimise the delivery of fluorophores in vivo. Recent clinical applications with fluorescein and other intravital fluorescent stains have occurred in neurosurgery, dermatology [including photodynamic therapy (PDT)] and endomicroscopy. Potential uses have been identified in periodontal disease, skin graft and cancer surgery. Animal studies have demonstrated that diseased tissue can be specifically stained with fluorophore conjugates. This review focuses on the fluorescein derived fluorophores in common clinical use and provides examples of novel applications from studies in tissue samples.

Bone Healing with or without Platelet‐Rich Plasma around Four Different Dental Implant Surfaces in Beagle Dogs

Surface development is one of the major aims in dental implant engineering. Additive application of substances could possibly improve the new bone formation around dental implants. The present study evaluated the bone reaction on four different implant surfaces with or without platelet-rich plasma (PRP). Four self-tapping titanium screw implants (Brånemark MK III [Nobel Biocare, Göteborg, Sweden], Osseotite [3i, Miami, FL, USA], Xive [Densply Friadent, Mannheim, Germany], and Compress [IGfZ eG, Diez, Germany]) with different surfaces were inserted in each hemimandible of 12 female beagle dogs; the implant positions and the application of PRP were randomized. After intravital fluorochrome staining, sacrifices and biopsies harvesting were performed after 6 weeks (five dogs; one dog died before) and 12 weeks (six dogs) and the respective specimens were analyzed. The only significant difference in bone remodeling was found for the Compress implants with increased bone formation compared with the Brånemark implants at 12 weeks (sign test, p = .03). Comparing the histological and histomorphometric specimens of all other implant surfaces with respect to peri-implant bone remodeling and the resulting bone-implant contact rates (BICRs), no statistically significant differences were seen in the PRP or non-PRP groups (sign test, all p values ≥ .063). This study found no significant differences in the BICR for roughened implant surfaces compared with machined surfaces. In this animal model, the addition of PRP did not demonstrate evidence of faster bone formation or the resulting BICR.

Visualization of mucosal homeostasis via single- and multiphoton intravital fluorescence microscopy

FIVM has provided many insights into the regulation of immunity. We report the validation of an approach for visualizing murine small bowel via single- and multiphoton FIVM. Tissue damage is limited to ∼200 μm, immediately adjacent to the incision, as confirmed by intravital PI staining. Treatment with 10 KDa dextran-FITC and 70 KDa dextran-TR confirms that perfusion is intact. Selective filtration of 10 KDa but not 70 KDa dextran from the blood indicated that kidney function is also intact. Interestingly, lamina propria vasculature is semipermeable to 10 KDa dextran. Next, reporter mice expressing egfp from the CX3CR1 locus, egfp from the FoxP3 locus, or RFP from the IL-17F locus were used to track DC subsets, FoxP3(+) Tregs, or Th17f cells, respectively. Resident cx3cr1(+/egfp) cells were sessile but actively probed the surrounding microenvironment. Both T cell populations patrol the lamina propria, but the Th17f cells migrate more rapidly than Tregs. Together, these data demonstrate intact vascular perfusion, while intravitally visualizing the mucosal surface of the small bowel. Lastly, the cx3cr1(+) DCs and T cells display activity similar to that found in steady-state, secondary lymphoid organs.