Podcasts about intravital

BUFFALO, NY- October 23, 2023 – A new research paper was published in Oncotarget's Volume 14 on October 19, 2023, entitled, “Apoptotic cells may drive cell death in hair follicles during their regression cycle.” Intravital microscopy in live mice has shown that the elimination of epithelial cells during hair follicle regression involves supra-basal cell differentiation and basal cell apoptosis through synergistic action of TGF-β (transforming growth factor) and mesenchymal-epithelial interactions. In this process the basal epithelial cells are not internally committed to death and the mesenchymal dermal papilla (DP) plays an essential role in death induction. Given that DP cells are not necessary for completion of the cycle, only for its initiation, it is still an open question as to the mechanism that leads to the propagation of apoptosis towards the regenerative stem cell population. In their new study, researchers Bradley D. Keister, Kailin R. Mesa and Krastan B. Blagoev from the National Science Foundation, The Jane Coffin Childs Memorial Fund for Medical Research, Yale School of Medicine, Johns Hopkins University, Bulgarian Academy of Sciences, and Sorbonne Université performed a quantitative analysis of the length of hair follicles to investigate their regression cycle. “In this paper we introduced a mathematical model of the hair follicle regression cycle that postulates that the regression is initiated by the dermal papilla, but that this signal affects only the cells adjacent to it.” The data are consistent with a propagation mechanism driven by apoptotic cells inducing apoptosis in their neighboring cells. The observation that the apoptosis slows down as the apoptotic front approaches the stem cells at the end of the follicle is consistent with a gradient of a pro-survival signal sent by these stem cells. An experiment that can falsify this mechanism is proposed. “In conclusion, hair follicle regression may be governed by cell-cell induced programmed cell death, which slows down as the stem cell compartment is approached and does not affect the stem cell compartment from which the growth phase is initiated. The class of models introduced here can be used to describe the renewal kinetics of other stem cell niches like the intestinal stem cell niche [18].” DOI - https://doi.org/10.18632/oncotarget.28529 Correspondence to - Krastan B. Blagoev - kblagoev@nsf.gov Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28529 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - cancer, hair follicle, stem cells, regression cycle, mathematical model, analysis About Oncotarget Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ X - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.01.526724v1?rss=1 Authors: HE, Y., Heng, Y., Qin, Z., Wei, X., Wu, Z., Qu, J. Abstract: Skeletal muscle regeneration requires the highly coordinated cooperation of muscle satellite cells (MuSCs) with other cellular components. However, due to technical limitations, it remains unclear how MuSCs dynamically interact with non-myogenic cells, especially myeloid cells, in live animals. In this work, we developed a dual-laser multimodal nonlinear optical microscope platform to serve as an effective tool for studying the real-time interaction between MuSCs and non-myogenic cells during the early phase of muscle regeneration. Increased cell volume and mitochondrial mass, cell density, and myotube formation are indicative of MuSCs activation/growth, proliferation, and differentiation, respectively. Using 3D time-lapse imaging on live reporter mice containing both red fluorescence protein (RFP)-labeled macrophages and yellow fluorescence protein (YFP)-labeled MuSCs, and taking advantages of the autofluorescence of reduced nicotinamide adenine dinucleotide (NADH), we monitored the real-time spatiotemporal interaction between RFP+ macrophages/RFP- non-myogenic cells and YFP+ muscle stem/progenitor cells during the activation and the proliferation stages of regeneration. Our results indicated that their cell-cell contact was transient in nature. By inhibiting macrophage infiltration, we further showed that direct cell-cell contact between macrophages and MuSCs was not required for early activation of MuSCs before the proliferation stage. However, decreased macrophage infiltration impeded the proliferation and differentiation of MuSCs and also led to intramuscular fibrosis. Besides, neutrophil depletion in the CCR2 deficient mice did not delay the initial growth of MuSCs. These findings provide a new perspective on the role of myeloid cells during muscle regeneration. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

In today's edition of The Oncology Podcast Rachael talks to Dr Michelle McDonald from the Garvan Institute about her research on bone metastasis and intravital imaging.Dr Michelle McDonald is Group Leader of the Bone Microenvironment Group at the Garvan Institute of Medical Research. Her research career spans over 18 years, attaining her PhD in 2008. Her current research team aims to develop new strategies to target cells of the bone environment to prevent tumour growth both within bone and spreading from bone to other sites. Using a specialised live animal imaging technique, Michelle visualises interactions between single tumour cells and bone cells, investigating how these interactions control the growth and behaviour of tumour cells in bone. Her work has recently attracted a number of competitive grants and spans collaborations with academic and industry partners internationally.Rachael Babin is the Editor-in-Chief of The Oncology Newsletter and Publisher of www.oncologynews.com.au. The Oncology Newsletter is a weekly publication for healthcare professionals with an interest in oncology, featuring the latest news, views and reviews. Click here to subscribe. We hope you enjoy this podcast - thank you for listening. 

All three nitric oxide synthase (NOS) isoforms are expressed in atherosclerotic plaques. NOS enzymes in general catalyse NO production. However, under conditions of substrate and cofactor deficiency, the enzyme directly catalyse superoxide formation. Considering this alternative chemistry, the effects of NOS on key events in spontaneous hyperlipidemia driven atherosclerosis have not been investigated yet. Here, we evaluate how endothelial nitric oxide synthase (eNOS) modulates leukocyte/endothelial- (L/E) and platelet/endothelial- (P/E) interactions in atherosclerosis and the production of nitric oxide (NO) and superoxide by the enzyme. Intravital microscopy (IVM) of carotid arteries revealed significantly increased L/E-interactions in apolipoproteinE/eNOS double knockout mice (apoE(-/-)/eNOS(-/-)), while P/E-interactions did not differ, compared to apoE(-/-). eNOS deficiency increased macrophage infiltration in carotid arteries and vascular cell adhesion molecule-1 (VCAM-1) expression, both in endothelial and smooth muscle cells. Despite the expression of other NOS isoforms (inducible NOS, iNOS and neuronal NOS, nNOS) in plaques, Electron Spin Resonance (ESR) measurements of NO showed significant contribution of eNOS to total circulating and vascular wall NO production. Pharmacological inhibition and genetic deletion of eNOS reduced vascular superoxide production, indicating uncoupling of the enzyme in apoE(-/-) vessels. Overt plaque formation, increased vascular inflammation and L/E- interactions are associated with significant reduction of superoxide production in apoE(-/-)/eNOS(-/-) vessels. Therefore, lack of eNOS does not cause an automatic increase in oxidative stress. Uncoupling of eNOS occurs in apoE(-/-) atherosclerosis but does not negate the enzyme's strong protective effects.

The BM is a key organ of hematopoiesis and also has an important role in the immune system. The BM microenvironment is a complex, highly vascularized 3D structure composed of different cell types and extracellular matrix. Intense cellular traffic takes place from the peripheral blood to the BM and vice versa. However, the precise arrangement and microscopic dimensions of this environment have only been inferred so far from static imaging of sectioned tissue. We developed a new model to characterize and analyze the 3D microanatomy of murine skull BM in its physiological state using intravital MPM. This technology offers deep tissue penetration, low phototoxicity, superior image contrast and 3D resolution compared to other microscopy techniques. This makes MPM a powerful tool to investigate the BM, overcoming its anatomic inaccessibility. To quantify the dimensions of the BM compartment, we used high molecular weight FITC-dextran and Rhodamine 6G, which delineated the intra- and extravascular space, respectively. Measurements were generated using the 3D visualization and measurement software VoxBlast 3.1 after using a thresholding technique carried out by Adobe Photoshop 6.0. Results were expressed as the ratio of intravascular to extravascular space for different microvascular segments. Moreover, we performed adoptive transfer experiments with isolated naïve B-cells and TCM and studied their location within the BM compartment. The new approach presented here will be a useful tool for further in vivo investigations of cell behavior, trafficking and interactions in the BM.

The global burden of chronic kidney diseases remains an ongoing medical challenge. Therapies that can halt or reverse advanced renal injury are not yet available. Increasing numbers of patients progress to the end-stage renal failure and require renal replacement therapy, the latter being associated with significant mortality, a lower quality of life, and high costs for national health systems. Thus, new treatment strategies that slow down, halt or even revert progressive renal damage are requested. Chemokines and their receptors are involved in the pathogenesis of renal diseases. They mediate leukocytes and macrophages recruitment and activation during initiation as well as progression of renal inflammation. Infiltrating leukocytes are the major source for proinflammatory and profibrotic cytokines and are therefore critical for mediating fibroblast proliferation, differentiation into myofibroblasts, matrix production, and tubular atrophy. Recent advances in the understanding of the molecular mechanisms that regulate renal leukocyte recruitment suggest chemokines and chemokine receptors as novel targets for specific pharmacological intervention. The aim of the present thesis was to investigate the role of chemokine receptor CCR1 for the progression of chronic kidney diseases, e.g. Alport disease and diabetic nephropathy. Two different animal models were used: Col4A3-deficient mice and type 2 diabetic db/db mice with advanced diabetic nephropathy. We blocked CCR1 in Col4A3-deficient mice with BX417, a small molecule CCR1 antagonist, and BL5923, a novel orally available antagonist with a high specificity for human and murine CCR1 in uninephrectomized type 2 diabetic db/db mice, respectively. Treatment with BX471 (25mg/kg) from weeks 6 to 10 of life improved survival of COL4A3- deficient mice, characterized by glomerulosclerosis and subsequent progressive tubulointerstitial injury, leading to fatal end-stage renal disease (ESRD). Improvement was associated with less interstitial macrophages, apoptotic tubular epithelial cells, tubular atrophy, interstitial fibrosis, and less globally sclerotic glomeruli. BX471 reduced total renal Ccl5 mRNA expression by reducing the number of interstitial CCL5-positive cells in inflammatory cell infiltrates. Intravital microscopy of the cremaster muscle in male mice identified that BX471 or lack of CCR1 impaired leukocyte adhesion to activated vascular endothelium and transendothelial leukocyte migration, whereas leukocyte rolling and interstitial migration were not affected. Furthermore, in activated murine macrophages, BX471 completely blocked CCL3-induced CCL5 production. When CCR1 was blocked with BL5923 (60mg/kg, b.i.d), the interstitial recruitment of ex vivo labeled macrophages was markedly decreased in uninephrectomized male db/db mice with type 2 diabetes. Similarly, BL5923 orally administered from month 5 to 6 of life reduced the numbers of interstitial macrophages in uninephrectomized db/db mice. This was associated with reduced numbers of Ki-67 proliferating tubular epithelial and interstitial cells, tubular atrophy, and interstitial fibrosis in uninephrectomized db/db mice. Glomerular pathology and proteinuria were not affected by the CCR1 antagonist. BL5923 reduced renal mRNA expression of Ccl2, Ccr1, Ccr2, Ccr5, Tgf-β1, and collagen I-α1 when compared to untreated uninephrectomized male db/db mice of the same age. Thus, we identified a previously unrecognized role for CCR1-dependent recruitment of interstitial macrophages for the progression of chronic kidney disease in Alport disease and diabetic nephropathy. These data identify CCR1 as a potential therapeutic target for Alport disease and late stage diabetic nephropathy or other progressive nephropathies associated with interstitial macrophage infiltrates.

Experimental studies in ischemia-reperfusion and sepsis indicate that activated protein C (APC) has direct anti-inflammatory effects at a cellular level. In vivo, however, the mechanisms of action have not been characterized thus far. Intravital multifluorescence microscopy represents an elegant way of studying the effect of APC on endotoxin-induced leukocyte-endothelial-cell interaction and nutritive capillary perfusion failure. These studies have clarified that APC effectively reduces leukocyte rolling and leukocyte firm adhesion in systemic endotoxemia. Protection from leukocytic inflammation is probably mediated by a modulation of adhesion molecule expression on the surface of leukocytes and endothelial cells. Of interest, the action of APC and antithrombin in endotoxin-induced leukocyte-endothelial-cell interaction differs in that APC inhibits both rolling and subsequent firm adhesion, whereas antithrombin exclusively reduces the firm adhesion step. The biological significance of this differential regulation of inflammation remains unclear, since both proteins are capable of reducing sepsis-induced capillary perfusion failure. To elucidate whether the action of APC and antithrombin is mediated by inhibition of thrombin, the specific thrombin inhibitor hirudin has been examined in a sepsis microcirculation model. Strikingly, hirudin was not capable of protecting from sepsis-induced microcirculatory dysfunction, but induced a further increase of leukocyte-endothelial-cell interactions and aggravated capillary perfusion failure when compared with nontreated controls. Thus, the action of APC on the microcirculatory level in systemic endotoxemia is unlikely to be caused by a thrombin inhibition-associated anticoagulatory action.

Bei Patienten, die mit dem humanen Immundefizienzvirus-1 (HIV-1) infiziert sind, kommt es häufig zu krankhaften Veränderungen des Endothels, die zu einer Fehlfunktion des Gefäßsystems führen. Klinischer Ausdruck dieser als acquired immune deficiency syndrome (AIDS)-assoziierten Vaskulopathie bezeichneten Veränderungen sind Schädigungen des Aortenendothels, die mit einer erhöhten Adhäsion mononukleärer Zellen an das Endothel einhergehen, Defekte der Blut-Hirn-Schranke, die zur Entstehung von Demenz beitragen, sowie das Kaposi-Sarkom (KS), das durch eine sehr starke Extravasation von T-Zellen und Monozyten gekennzeichnet ist. In dieser Arbeit wird gezeigt, dass das regulatorische HIV-1-Tat-Protein und das inflammatorische Zytokin TNF-a synergistisch die Adhäsion der promonozytären Zelllinie U937 und von PBMZ an humane mikrovaskuläre Endothelzellen (HMVEZ) erhöht. Die adhäsionsfördernde Wirkung wurde selektiv bei HIV-1-Tat beobachtet, andere virale Proteine des HIV-1, wie Negativfaktor (Nef) und das Glykoprotein gp41, hatten keinen Einfluss auf die Adhäsion. Anhand zellspezifischer Marker wurde gezeigt, dass HIV-1-Tat in periphere mononukleäre Blutzellen (PBMZ) spezifisch die Adhäsion von Monozyten und T-Zellen erhöhte, jedoch nicht von B-Zellen. Intravital-mikroskopische Untersuchungen an der Maus bestätigten in vivo, dass HIV-1-Tat und TNF-a synergistisch die Adhäsion von Leukozyten an das Endothel erhöhten. HIV-1-Tat reguliert die Expression einer großen Anzahl zellulärer Gene. Diese Fehlregulation durch HIV-1-Tat könnte an der Enstehung der AIDS-assoziierten Vaskulopathie beteiligt sein. Im zweiten Teil dieser Arbeit wird die parakrine Wirkung von HIV-1-Tat auf die Genexpression in Monozyten mittels der suppressed subtractive hybridization (SSH)-Methode untersucht. Hierbei wurde O-linked N-Acetylglucosamine-transferase (OGT) als Gen identifiziert, dessen Expression durch HIV-1-Tat unterdrückt wird. Bisher ist bekannt, dass OGT ein Repressor der basalen Transkription und der SP-1-regulierten Transkription ist. Die Expression von OGT wurde sowohl auf mRNA-Ebene als auch auf Protein-Ebene durch HIV-1-Tat und VEGF121 gehemmt, wobei die Regulierung über den VEGF-Rezeptor Flt-1 vermittelt wurde. Weitere Faktoren wie inflammatorische Zytokine (TNF-a, IL-1b, IFN-g und IL-2), angiogene Wachstumsfaktoren (bFGF und VEGF165) und Chemokine (IL-8, MIP-1a, IP-10, MCP-1 und SDF-1a) hatten keine hemmende Wirkung auf die OGT-Expression. Die schnelle Abnahme von intrazellulärem OGT-Protein wurde weder durch lysosomale Proteasen noch durch Proteasen des Proteasoms verursacht. Expressionsstudien an PBMZ von fünf verschiedenen Probanden zeigten, dass bei zwei Probanden die OGT-Konzentration durch HIV-1-Tat zunahm, bei zweien nahm sie ab und bei einer Person gab es keine Veränderung. Diese Ergebnisse belegen, dass HIV-1-Tat entscheidend an der Entstehung der AIDS-assoziierten Vaskulopathie, insbesondere von KS, beteiligt sein könnte. Die Repression von OGT durch HIV-1-Tat könnte die weitreichende Wirkung des HIV-1-Tat-Proteins auf zelluläre und virale Gene erklären.

Dynamic visualization of the intravascular events leading to the extravasation of leukocytes into tissues by intravital microscopy has significantly expanded our understanding of the underlying molecular processes. In contrast, the detailed observation of leukocyte transendothelial and interstitial migration in vivo has been hampered by the poor image contrast of cells within turbid media that is obtainable by conventional brightfield microscopy. Here we present a microscopic method, termed reflected light oblique transillumination microscopy, that makes use of the optical interference phenomena generated by oblique transillumination to visualize subtle gradients of refractive indices within tissues for enhanced image contrast. Using the mouse cremaster muscle, we demonstrate that this technique makes possible the reliable quantification of extravasated leukocytes as well as the characterization of morphological phenomena of leukocyte transendothelial and interstitial migration.

Inhibition of angiogenesis might be a therapeutic approach to prevent joint destruction caused by the overgrowing synovial tissue during chronic joint inflammation. The aim of this study was to investigate angiogenesis in the knee joint of mice with antigen-induced arthritis (AIA) by means of intravital microscopy. In 14 mice (C57BL6/129Sv) intravital microscopic assessment was performed on day 8 after AIA induction in two groups (controls, AIA). Synovial tissue was investigated by intravital fluorescence microscopy using FITC-dextran (150 kD). Quantitative assessment of vessel density was performed according to the following categories: functional capillary density (FCD, vessels 10 mum) and FVD of vessels with angiogenic criteria (convoluted vessels, abrupt changes of diameter, vessels which are generated by sprouting and progressively pruned and remodelled). Microvessel count was performed using immunohistochemistry. There was no significant difference in FCD between the control group (337 +/- 9 cm/cm(2); mean +/-SEM) and the AIA group (359 +/- 13 cm/cm(2)). The density of vessels larger than 10 gm diameter was significantly increased in animals with AIA (135 +/- 10 vs. 61 +/- 5 cm/cm(2) in control). The density of blood vessels with angiogenic criteria was enhanced in arthritic animals (79 +/- 17 vs. 12 +/- 2 cm/cm(2) in control). There was a significant increase in the microvessel count in arthritic animals (297 +/- 25 vs. 133 +/- 16 mm(-2) in control). These findings demonstrate that angiogenesis in murine AIA can be assessed quantitatively using intravital microscopy. Further studies will address antiangiogenic strategies in AIA.

Growing evidence supports the substantial pathophysiological impact of platelets on the development of acute lung injury. Methods for studying these cellular mechanisms in vivo are not present yet. The aim of this study was to develop a model enabling the quantitative analysis of platelet kinetics and platelet-endothelium interaction within consecutive segments of the pulmonary microcirculation in vivo. New Zealand White rabbits were anesthetized and ventilated. Autologous platelets were separated from blood and labeled ex vivo with rhodamine 6G. After implantation of a thoracic window, microhemodynamics and kinetics of platelets were investigated by intravital microscopy. Velocities of red blood cells (RBCs) and platelets were measured in arterioles, capillaries and venules, and the number of platelets adhering to the microvascular endothelium was counted. Kinetics of unstimulated platelets was compared with kinetics of thrombin-activated platelets. Velocity of unstimulated platelets was comparable to RBC velocity in all vessel segments. Unstimulated platelets passed the pulmonary microcirculation without substantial platelet-endothelial interaction. In contrast, velocity of activated platelets was decreased in all vascular segments indicating platelet margination and temporal platelet-endothelium interaction. Thrombin-activated platelets adhered to arteriolar endothelium; in capillaries and venules adherence of platelets was increased 8-fold and 13-fold, respectively. In conclusion, using intravital microscopy platelet kinetics were directly analyzed in the pulmonary microcirculation in vivo for the first time. In contrast to leukocytes, no substantial platelet-endothelium interaction occurs in the pulmonary microcirculation without any further stimulus. In response to platelet activation, molecular mechanisms enable adhesion of platelets in arterioles and venules as well as retention of platelets within capillaries. Copyright (C) 2002 S. Karger AG, Basel.

Background. The OPS imaging technique has been introduced for in vivo assessment of microcirculation in humans. The aim of this study was to validate the new technique against intravital fluorescence microscopy (IFM) for the visualization of colon microcirculation in a murine model of inflammatory bowel disease (IBD). Method. IBD was induced in Balb/c mice by dextran sulfate sodium, controls received normal water. In each animal, both the CYTOSCAN(TM) A/R and IFM were used to image the microcirculation (n = 7 in each group). The postcapillary venular diameter was analyzed on the colon muscularis and mucosa. Results: The venular diameter correlated significantly between both methods representing the good correspondence between both methods. Conclusion: Our study demonstrates that the new technique for visualization of microcirculation without use of fluorescent dyes, the OPS imaging, allows for quantitative measurement of a key microcirculatory parameters of the mouse colon. Copyright (C) 2002 S. Karger AG, Basel.

The Cytoscan(TM) Model E-II (Cytometrics Inc., Philadelphia, Pa., USA) is a newly developed instrument which functions as an intravital microscope and is small and easily portable. Through the use of orthogonal polarization spectral (OPS) imaging, the Cytoscan Model E-II delivers images of the microcirculation which are comparable to those achieved with intravital fluorescence videomicroscopy (IFM), but without the use of fluorescent dyes. The purpose of this study was to validate the Cytoscan Model E-II instrument against IFM. The experiments were carried out on striated muscle in the dorsal skinfold chamber of the awake Syrian hamster. The following parameters were measured in identical regions of interest in the same animal under baseline conditions and 0.5 and 2 h after a 4-hour period of pressure-induced ischemia: arteriolar diameter, venular diameter and venular red blood cell velocity. Bland-Altman plots showed good agreement between the two techniques for venular red blood cell velocity. As expected, arteriolar and venular diameters as measured by the Cytoscan were on average 5 mum smaller than the values from IFM, since the Cytoscan measures the red blood cell column width and IFM measures luminal diameter. Thus, OPS imaging can be used to make valid measurements of microvascular diameter and red blood cell velocity in tissues. Copyright (C) 2000 S. Karger AG, Basel.