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ABSTRACT: A computational model is potentially a powerful tool to apprehend complex phenomena like solid tumour growth and to predict the outcome of therapies. To that end, the confrontation of the model with experiments is essential to validate this tool. In this study, we develop a computational model specifically dedicated to the interpretation of tumour growth as observed in a mouse model with a dorsal skinfold chamber. Observation of the skin vasculature at the dorsal window scale shows a sparse network of a few main vessels of several hundreds micrometers in diameter. However observation at a smaller scale reveals the presence of a dense and regular interconnected network of capillaries about ten times smaller. We conveniently designate this structure as the submicrovascular network (SMVN).(1) The question that we wish to answer concerns the necessity of explicitly taking into account the SMVN in the computational model to describe the tumour evolution observed in the dorsal chamber. For that, simulations of tumour growth realised with and without the SMVN are compared and lead to two distinct scenarios. Parameters that are known to strongly influence the tumour evolution are then tested in the two cases to determine to which extent those parameters can be used to compensate the observed differences between these scenarios. Explicit modelling of the smallest vessels appears mandatory although not necessarily under the form of a regular grid. A compromise between the two investigated cases can thus be reached.
Microvascular Research 06/2012; 84(2):188-204. · 2.83 Impact Factor
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International Journal of Cancer 03/2011; 128(6):1502-3. · 5.44 Impact Factor
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ABSTRACT: Our purpose is to test if Pluronic® fluorescent nanomicelles can be used for in vivo two-photon imaging of both the normal and the tumor vasculature. The nanomicelles were obtained after encapsulating a hydrophobic two-photon dye: di-stryl benzene derivative, in Pluronic block copolymers. Their performance with respect to imaging depth, blood plasma staining, and diffusion across the tumor vascular endothelium is compared to a classic blood pool dye Rhodamin B dextran (70 kDa) using two-photon microscopy. Pluronic nanomicelles show, like Rhodamin B dextran, a homogeneous blood plasma staining for at least 1 h after intravenous injection. Their two-photon imaging depth is similar in normal mouse brain, using 10 times less injected mass. In contrast with Rhodamin B dextran, no extravasation is observed in leaky tumor vessels due to their large size: 20-100 nm. In conclusion, Pluronic nanomicelles can be used as a blood pool dye, even in leaky tumor vessels. The use of Pluronic block copolymers is a valuable approach for encapsulating two-photon fluorescent dyes that are hydrophobic and not suitable for intravenous injection.
Journal of Biomedical Optics 03/2011; 16(3):036001. · 3.16 Impact Factor
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ABSTRACT: Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such as serum or feeder cell layers by recombinant cytokines or growth factors. Another example is the control of the oxygen pressure. For many years cell cultures have been done under atmospheric oxygen pressure which is much higher than the one experienced by stem cells in vivo. A consequence of cell metabolism is that cell culture conditions are constantly changing. Therefore, the development of high sensitive monitoring processes and control algorithms is required for ensuring cell culture medium homeostasis. Stem cells also sense the physical constraints of their microenvironment. Rigidity, stiffness, and geometry of the culture substrate influence stem cell fate. Hence, nanotopography is probably as important as medium formulation in the optimization of stem cell culture conditions. Recent advances include the development of synthetic bioinformative substrates designed at the micro- and nanoscale level. On going research in many different fields including stem cell biology, nanotechnology, and bioengineering suggest that our current way to culture cells in Petri dish or flasks will soon be outdated as flying across the Atlantic Ocean in the Lindbergh's plane.
Journal of Cellular Biochemistry 11/2010; 111(4):801-7. · 2.87 Impact Factor
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ABSTRACT: The purpose is to evaluate effects of a new radiotherapy protocol, microbeam radiation therapy, on the artery wall. In previous studies on animal models, it was shown that capillaries recover well from hectogray doses of X-rays delivered in arrays of narrow (< or = 50 microm) beams with a minimum spacing of 200 microm. Here, short- and long-term effects of comparable microplanar beam configurations on the saphenous artery of the mouse hind leg were analyzed in situ by use of nonlinear optics and compared with histopathologic findings.
The left hind leg of normal mice including the saphenous artery was irradiated by an array of 26 microbeams of synchrotron X-rays (50 microm wide, spaced 400 microm on center) with peak entrance doses of 312 Gy and 2,000 Gy.
The artery remained patent, but narrow arterial smooth muscle cell layer segments that were in the microplanar beam paths became atrophic and fibrotic in a dose-dependent pattern. The wide tunica media segments between those paths hypertrophied, as observed in situ by two-photon microscopy and histopathologically.
Clinical risks of long-delayed disruption or occlusion of nontargeted arteries from microbeam radiation therapy will prove less than corresponding risks from broad-beam radiosurgery, especially if peak doses are kept below 3 hectograys.
International journal of radiation oncology, biology, physics 08/2010; 77(5):1545-52. · 4.59 Impact Factor
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ABSTRACT: Cell aggregates are a tool for in vitro studies of morphogenesis, cancer invasion, and tissue engineering. They respond to mechanical forces as a complex rather than simple liquid. To change an aggregate's shape, cells have to overcome energy barriers. If cell shape fluctuations are active enough, the aggregate spontaneously relaxes stresses ("fluctuation-induced flow"). If not, changing the aggregate's shape requires a sufficiently large applied stress ("stress-induced flow"). To capture this distinction, we develop a mechanical model of aggregates based on their cellular structure. At stress lower than a characteristic stress tau*, the aggregate as a whole flows with an apparent viscosity eta*, and at higher stress it is a shear-thinning fluid. An increasing cell-cell tension results in a higher eta* (and thus a slower stress relaxation time t(c)). Our constitutive equation fits experiments of aggregate shape relaxation after compression or decompression in which irreversibility can be measured; we find t(c) of the order of 5 h for F9 cell lines. Predictions also match numerical simulations of cell geometry and fluctuations. We discuss the deviations from liquid behavior, the possible overestimation of surface tension in parallel-plate compression measurements, and the role of measurement duration.
Proceedings of the National Academy of Sciences 09/2009; 106(41):17271-5. · 9.68 Impact Factor
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Clément Ricard,
Manuel Fernández,
Jérôme Gastaldo,
Lucie Dupin,
Laurette Somveille,
Régine Farion,
Herwig Requardt,
Jean-Claude Vial,
Hélène Elleaume,
Christoph Segebarth, Boudewijn van der Sanden
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ABSTRACT: The purpose of this study is to measure the effects of a tomographic synchrotron irradiation on healthy mouse brain. The cerebral cortexes of healthy nude mice were irradiated with a monochromatic synchrotron beam of 79 keV at a dose of 15 Gy in accordance with a protocol of photoactivation of cisplatin previously tested in our laboratory. Forty-eight hours, one week and one month after irradiation, the blood brain barrier (BBB) permeability was measured in the irradiated area with intravital multiphoton microscopy using fluorescent dyes with molecular weights of 4 and 70 kDa. Vascular parameters and gliosis were also assessed using quantitative immunohistochemistry. No extravasation of the fluorescent dyes was observed in the irradiated area at any measurement time (48 h, 1 week, 1 month). It appears that the BBB remains impermeable to molecules with a molecular weight of 4 kDa and above. The vascular density and vascular surface were unaffected by irradiation and no gliosis was induced. These findings suggest that a 15 Gy/79 keV synchrotron irradiation does not induce important damage on brain vasculature and tissue on the short term following irradiation.
Journal of Synchrotron Radiation 08/2009; 16(Pt 4):477-83. · 2.73 Impact Factor
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ABSTRACT: We report the synthesis of new nanosized fluorescent probes based on bio-compatible polyethylene-polypropylene glycol (Pluronic) materials. In aqueous solution, mini-emulsification of Pluronic with a high fluorescent di-stryl benzene-modified derivative, exhibiting a two-photon absorption cross section as high as 2500 Goeppert-Mayer units at 800 nm, leads to nanoparticles exhibiting a hydrodynamic radius below 100 nm. We have demonstrated that these new probes with luminescence located in the spectral region of interest for bio-imaging (the yellow part of the visible spectrum) allow deep (500 microm) bio-imaging of the mice brain vasculature. The dose injected during our experiments is ten times lower when compared to the classical commercial rhodamine-B isothicyanate-Dextran system but gives similar results to homogeneous blood plasma staining. The mean fluorescent signal intensity stayed constant during more than 1 h.
Nanotechnology 07/2009; 20(23):235102. · 3.98 Impact Factor
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Raphaël Serduc,
Thomas Christen,
Jean Laissue,
Régine Farion,
Audrey Bouchet, Boudewijn van der Sanden,
Christoph Segebarth,
Elke Bräuer-Krisch,
Géraldine Le Duc,
Alberto Bravin,
Chantal Rémy,
Emmanuel L Barbier
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ABSTRACT: The aim of this work focuses on the description of the short-term response of a 9L brain tumor model and its vasculature to microbeam radiation therapy (MRT) using magnetic resonance imaging (MRI). Rat 9L gliosarcomas implanted in nude mice brains were irradiated by MRT 13 days after tumor inoculation using two orthogonal arrays of equally spaced 28 planar microbeams (25 microm width, 211 microm spacing and dose 500 Gy). At 1, 7 and 14 days after MRT, apparent diffusion coefficient, blood volume and vessel size index were mapped by MRI. Mean survival time after tumor inoculation increased significantly between MRT-treated and untreated groups (23 and 28 days respectively, log-rank test, p < 0.0001). A significant increase of apparent diffusion coefficient was observed 24 h after MRT in irradiated tumors versus non-irradiated ones. In the untreated group, both tumor size and vessel size index increased significantly (from 7.6 +/- 2.2 to 19.2 +/- 4.0 mm(2) and +23%, respectively) between the 14th and the 21st day after tumor cell inoculation. During the same period, in the MRT-treated group, no difference in tumor size was observed. The vessel size index measured in the MRT-treated group increased significantly (+26%) between 14 and 28 days of tumor growth. We did not observe the significant difference in blood volume between the MRT-treated and untreated groups. MRT slows 9L tumor growth in a mouse brain but MRI results suggest that the increase in survival time after our MRT approach may be rather due to a cytoreduction than to early direct effects of ionizing radiation on tumor vessels. These results suggest that MRT parameters need to be optimized to further damage tumor vessels.
Physics in Medicine and Biology 07/2008; 53(13):3609-22. · 2.83 Impact Factor
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Raphaël Serduc,
Yohan van de Looij,
Gilles Francony,
Olivier Verdonck, Boudewijn van der Sanden,
Jean Laissue,
Régine Farion,
Elke Bräuer-Krisch,
Erik Albert Siegbahn,
Alberto Bravin,
Yolanda Prezado,
Christoph Segebarth,
Chantal Rémy,
Hana Lahrech
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ABSTRACT: Cerebral edema is one of the main acute complications arising after irradiation of brain tumors. Microbeam radiation therapy (MRT), an innovative experimental radiotherapy technique using spatially fractionated synchrotron x-rays, has been shown to spare radiosensitive tissues such as mammal brains. The aim of this study was to determine if cerebral edema occurs after MRT using diffusion-weighted MRI and microgravimetry. Prone Swiss nude mice's heads were positioned horizontally in the synchrotron x-ray beam and the upper part of the left hemisphere was irradiated in the antero-posterior direction by an array of 18 planar microbeams (25 mm wide, on-center spacing 211 mm, height 4 mm, entrance dose 312 Gy or 1000 Gy). An apparent diffusion coefficient (ADC) was measured at 7 T 1, 7, 14, 21 and 28 days after irradiation. Eventually, the cerebral water content (CWC) was determined by microgravimetry. The ADC and CWC in the irradiated (312 Gy or 1000 Gy) and in the contralateral non-irradiated hemispheres were not significantly different at all measurement times, with two exceptions: (1) a 9% ADC decrease (p < 0.05) was observed in the irradiated cortex 1 day after exposure to 312 Gy, (2) a 0.7% increase (p < 0.05) in the CWC was measured in the irradiated hemispheres 1 day after exposure to 1000 Gy. The results demonstrate the presence of a minor and transient cellular edema (ADC decrease) at 1 day after a 312 Gy exposure, without a significant CWC increase. One day after a 1000 Gy exposure, the CWC increased, while the ADC remained unchanged and may reflect the simultaneous presence of cellular and vasogenic edema. Both types of edema disappear within a week after microbeam exposure which may confirm the normal tissue sparing effect of MRT.
Physics in Medicine and Biology 03/2008; 53(5):1153-66. · 2.83 Impact Factor
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Ali Hayek,
Alexei Grichine,
Thomas Huault,
Clément Ricard,
Frédéric Bolze, Boudewijn Van Der Sanden,
Jean-Claude Vial,
Yves Mély,
Alain Duperray,
Patrice Lilian Baldeck,
Jean-François Nicoud
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ABSTRACT: Because of the spreading of nonlinear microscopies in biology, there is a strong demand for specifically engineered probes in these applications. Herein, we report on the imaging properties in living cells and nude mice brains of recently developed water soluble blue fluorophores that show efficient diffusion through cell membranes and blood-brain barriers. They are characterized by two-photon absorption cross-sections of 100-150 Goeppert-Mayer range in the near IR and fluorescence efficiencies of up to 72% in water. They were found to stain homogeneously the cytoplasm of cultured living cells within minutes. Moreover, their diffusion times and fluorescence characteristics in the cytoplasm suggest a hydrophobic association with intracellular membranes. Their intracellular fluorescent decays were found to be almost mono-exponential, a very favorable feature for fluorescence lifetime imaging. Two photon images of living cells were obtained with a good signal to noise ratio using laser powers in the sub-milliwatt range. This allows continuous imaging without significant photobleaching for tens of minutes. In addition, these fluorophores allowed in vivo three-dimensional two-photon imaging of mice cortex vasculatures and extra vasculature structures, with no sign of toxicity.
Microscopy Research and Technique 11/2007; 70(10):880-5. · 1.79 Impact Factor
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ABSTRACT: Knowledge of the blood volume per unit volume of brain tissue is important for understanding brain function in health and disease. We describe a direct method using two-photon laser scanning microscopy to obtain in vivo the local capillary blood volume in the cortex of anesthetized mouse. We infused fluorescent dyes in the circulating blood and imaged the blood vessels, including the capillaries, to a depth of 600 microm below the dura at the brain surface. Capillary cortical blood volume (CCBV) was calculated without any form recognition and segmentation, by normalizing the total fluorescence measured at each depth and integrating the collected intensities all over the stack. Theoretical justifications are presented and numerical simulations were performed to validate this method which was weakly sensitive to background noise. Then, CCBV had been estimated on seven healthy mice between 2%+/-0.3% and 2.4%+/-0.4%. We showed that this measure of CCBV is reproductible and that this method is highly sensitive to the explored zones in the cortex (vessel density and size). This method, which dispenses with form recognition, is rapid and would allow to study in vivo temporal and highly resolute spatial variations of CCBV under different conditions or stimulations.
Journal of Cerebral Blood Flow & Metabolism 06/2007; 27(5):1072-81. · 5.01 Impact Factor
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Raphaël Serduc,
Pascale Vérant,
Jean-Claude Vial,
Régine Farion,
Linda Rocas,
Chantal Rémy,
Taoufik Fadlallah,
Elke Brauer,
Alberto Bravin,
Jean Laissue,
Hans Blattmann, Boudewijn van der Sanden
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ABSTRACT: The purpose of this study was to assess the early effects of microbeam irradiation on the vascular permeability and volume in the parietal cortex of normal nude mice using two-photon microscopy and immunohistochemistry.
The upper part of the left hemisphere of 55 mice was irradiated anteroposteriorly using 18 vertically oriented beams (width 25 microm, interdistance 211 microm; peak entrance doses: 312 or 1000 Gy). At different times after microbeam exposure, the microvasculature in the cortex was analyzed using intravital two-photon microscopy after intravascular injection of fluorescein isothiocyanate (FITC)-dextrans and sulforhodamine B (SRB). Changes of the vascular volume were observed at the FITC wavelength over a maximum depth of 650 mum from the dura. The vascular permeability was detected as extravasations of SRB.
For all times (12 h to 1 month) after microbeam irradiation and for both doses, the FITC-dextran remained in the vessels. No significant change in vascular volume was observed between 12 h and 3 months after irradiation. Diffusion of SRB was observed in microbeam irradiated regions from 12 h until 12 days only after a 1000 Gy exposure.
No radiation damage to the microvasculature was detected in normal brain tissue after a 312 Gy microbeam irradiation. This dose would be more appropriate than 1000 Gy for the treatment of brain tumors using crossfired microbeams.
International Journal of Radiation OncologyBiologyPhysics 05/2006; 64(5):1519-27. · 4.11 Impact Factor
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ABSTRACT: Brain pathologies, including stroke and tumors, are associated with a variable degree of breakdown of the blood-brain barrier (BBB), which can usefully be studied in animal models. We describe a new optical technique for quantifying extravasation in the cortex of the living mouse and for imaging intraparenchymal tissue. Leakiness of the BBB was induced by microbeam x-irradiation. Two fluorescent dyes were simultaneously infused intravenously, one of high molecular weight (fluorescein-labeled dextran, 70 kDa, green fluorescence) and one of low molecular weight (sulforhodamine B, 559 Da, red fluorescence). A two-photon microscope, directed through a cranial window, obtained separate images of the two dyes in the cortex. The gains of the two channels were adjusted so that the signals coming from within the vessels were equal. Subtraction of the image of the fluorescein-dextran from that of the sulforhodamine B gave images in which the vasculature was invisible and the sulforhodamine B in the parenchyma could be imaged with high resolution. Algorithms are presented for rapidly quantifying the extravasation without the need for shape recognition and for calculating the permeability of the BBB. Sulforhodamine B labeled certain intraparenchymal cells; these cells, and other details, were best observed using the subtraction method.
Journal of Biomedical Optics 13(1):011002. · 3.16 Impact Factor
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ABSTRACT: Until now, the imaging of elastic fibers was restricted to tissue sections using the endofluorescence properties of elastin or histological dyes. Methods to study their morphology in vivo and in situ have been lacking. We present and characterize a new application of a fluorescent dye for two-photon microscopy: sulforhodamine B (SRB), which is shown to specifically stain elastic fibers in vivo. SRB staining of elastic fibers is demonstrated to be better than using elastin endofluorescence for two-photon microscopy. Our imaging method of elastic fibers is shown to be suitable for simultaneous imaging with both other fluorescent intravital dyes and second-harmonic generation (SHG). We illustrate these findings with intravital imaging of elastic and collagen fibers in muscle epimysium and endomysium and in blood vessel walls. We expect SRB staining to become a key method to study elastic fibers in vivo.
Journal of Biomedical Optics 12(6):064017. · 3.16 Impact Factor
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ABSTRACT: Staining and imaging glial cells in vivo while observing the microvasculature could help understand brain physiology, namely neuronal-glial-vascular communication. Two-photon excitation microscopy provides a means to monitor these interactions at the cellular level in living animals, but the cells of interest must be fluorescent. Injecting dyes intravenously is a rapid and quasi noninvasive method to stain cells in the brain. It necessitates that the dye is soluble in the blood plasma and crosses the blood brain barrier (BBB). We demonstrate here, using two-photon imaging, that sulforhodamine B (SRB) crosses the BBB and stains in vivo, specifically mouse astrocytes. This is confirmed by experiments on primary neurons and astrocytes cultures showing the preferential SRB staining of the latter. SRB is rapidly eliminated from the blood, which allows repeated injections in longitudinal studies.
Journal of Biomedical Optics 13(6):064028. · 3.16 Impact Factor
