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International audience; An optical-resolution photoacoustic microscope with modulated CW laser diodes allowing multi-channel imaging is presented that can be used for both imaging biological tissues and for targeted photo-dynamic therapy (PDT) varying the optical power and exposure time. The effects of this therapy are immediately monitored in order to optimize the time of irradiation. After the description of the experimental setup, in vitro and in vivo applications are presented on a synthetic sample and on the mouse ear using hemoglobin as endogenous and methylene blue as exogenous dye for imaging and PDT, respectively.

We present how a laser optical feedback imaging (LOFI) setup can be used for the optical detection of ultrasound in photo-acoustic tomography (PAT). A PAT image is reconstructed by an inversion algorithm using surface displacement measurements made at several locations with our LOFI setup and following the optical irradiation with a pulsed Nd:YAG laser of a sample with absorbing inclusions. The width of the reconstructed inclusions and the signal-to-noise ratio (SNR) of the reconstructed images are first studied on the numerical model of a sample with three absorbing inclusions (i.e., with three acoustic punctual sources). Finally, an experimental PAT image of a phantom composed of two polyamide tubes with an internal diameter of 800 μm filled with red ink and submerged at -3.5 mm depth in a tank filled with water is reconstructed. Experimentally, the water surface displacement measurements have been made with our LOFI vibrometer, which provides an amplitude sensitivity of 1 nm (for a single-shot measurement) in a detection bandwidth of roughly 1 MHz adapted to the detection of the polyamide tubes. Under our experimental conditions, the surface energy densities of the LOFI focalized beam for the detection and of the pulsed Nd:YAG laser used for the irradiation, are compatible with the maximum permissive exposure for future biomedical measurements. The SNR and the resolution of the reconstructed PAT images are in good agreement with the theoretical predictions.

Purpose This study demonstrates how to quantify the tumor blood volume fraction (BVf) using the dynamic Rapid-Steady-State-T1 (RSST1)-MRI method despite contrast agent (CA) leakage and without arterial input function (AIF) determination. Methods For vasculature impermeable to CAs, the BVf is directly quantified from the RSST1 signal amplitude. In case of CA extravasation, we propose a two-compartment model to describe the dynamic RSST1 signal increase. We applied the mathematical model in a pilot-study on a RG2-glioma model to compare extravasation of two Gd-based CAs. The BVf quantification using the mathematical model in a C6-glioma model (n = 8) with the clinical CA Gd-DOTA was validated using a ΔR2*-steady-state MRI method with an USPIO and by immunohistochemical staining of perfused vessels labeled with Hoechst-33342 dye in the same rats. Results BVf in tumor and in healthy brain tissues (0.034 ± 0.005 and 0.026 ± 0.004, respectively) derived from the dynamic RSST1 signal were confirmed by ΔR2*-steady-state MRI (0.036 ± 0.003 and 0.027 ± 0.002, respectively, correlation coefficient rS = 0.74) and by histology (0.036 ± 0.003 and 0.025 ± 0.004 respectively, rS = 0.87). Conclusion Straightforward tumor BVf quantification without AIF determination is demonstrated in presence of CA leakage. The method will facilitate angiogenesis assessment in longitudinal neuro-oncologic studies in particular when monitoring the response to antiangiogenic therapies. Magn Reson Med 73:1005–1014, 2015. © 2014 Wiley Periodicals, Inc.

International audience; Surgery is the first line therapy for glioma. However, glioma recurs in 90 % of the patients in the resection margin. The impact of surgical brain injury (SBI) on glioma recurrence is largely overlooked. Herein, we review some of the mechanisms involved in tissue repair that may impact glioma recurrence at the resection margin. Many processes or molecules involved in tissue repair after brain injury are also critical for glioma growth. They include a wide array of secreted growth factors, cytokines and transcription factors including NFКB and STAT3 which in turn activate proliferative and anti-apoptotic genes and processes such as angiogenesis and inflammation. Because some residual glioma cells always remain in the tumor resection margin, there are now compelling arguments to suggest that some aspects of the brain tissue response to SBI can also participate to glioma recurrence at the resection margin. Brain tissue response to SBI recruits angiogenesis and inflammation that precede and then follow tumor recurrence at the resection margin. The healingresponse to SBI is double edged, as inflammation is involved in regeneration and healing, and has both pro- and anti-tumorigenic functions. A promising therapeutic approach is to normalize and re-educate the molecular and cellular responses at the resection margin to promote anti- tumorigenic processes involved in healing while inhibiting pro-tumorigenic activities. Manipulation of the inflamma- tory response to SBI to prevent local recurrence could also enhance the efficacy of other therapies such as immunotherapy. However, our current knowledge is far from sufficient to achieve this goal. Acknowledging, understanding and manipulating the double-edged role played by SBI in glioma recurrence is surely challenging, but it cannot be longer delayed.

The design of a patient-specific virtual tumour is an important step towards Personalized Medicine. However this requires to capture the description of many key events of tumour development, including angiogenesis, matrix remodelling, hypoxia, and cell state heterogeneity that will all influence the tumour growth kinetics and degree of tumour invasiveness. To that end, an integrated hybrid and multiscale approach has been developed based on data acquired on a preclinical mouse model as a proof of concept. Fluorescence imaging is exploited to build case-specific virtual tumours. Numerical simulations show that the virtual tumour matches the characteristics and spatiotemporal evolution of its real counterpart. We achieved this by combining image analysis and physiological modelling to accurately described the evolution of different tumour cases over a month. The development of such models is essential since a dedicated virtual tumour would be the perfect tool to identify the optimum therapeutic strategies that would make Personalized Medicine truly reachable and achievable.

International audience; No abstract

For pulsed arterial spin labeling techniques (e.g., FAIR), mismatches between the imaging and inversion slice profile result in a nonperfusion-related offset. Several methods have been proposed to reduce subtraction errors in FAIR imaging. Here an acquisition method for FAIR experiments based on adiabatic principles is proposed. It is shown that with adiabatic pulses the same pulse can be used for labeling and echo refocusing, thereby reducing the mismatch between imaging and labeling slice. A twofold reduction in subtraction errors compared to 5-lobe sinc excitation was shown both experimentally and by simulation.

Multipotent mesenchymal stromal cells (MSC), have the potential to differentiate into cells of the mesenchymal lineage and have non-progenitor functions including immunomodulation. The demonstration that MSCs are perivascular cells found in almost all adult tissues raises fascinating perspectives on their role in tissue maintenance and repair. However, some controversies about the physiological role of the perivascular MSCs residing outside the bone marrow and on their therapeutic potential in regenerative medicine exist. In brain, perivascular MSCs like pericytes and adventitial cells, could constitute another stem cell population distinct to the neural stem cell pool. The demonstration of the neuronal potential of MSCs requires stringent criteria including morphological changes, the demonstration of neural biomarkers expression, electrophysiological recordings, and the absence of cell fusion. The recent finding that brain cancer stem cells can transdifferentiate into pericytes is another facet of the plasticity of these cells. It suggests that the perversion of the stem cell potential of pericytes might play an even unsuspected role in cancer formation and tumor progression.

Among the noninvasive imaging modalities, contrast enhanced magnetic resonance (MR) imaging is the most powerful tool with which to visualize vascularity. Common pathology only shows microvessel density, whereas dynamic MR imaging is sensitive to the total endothelial surface area of perfused vessels. Therefore, dynamic MR imaging may be of additional value in tumor staging and in evaluating therapies that affect the perfused microvessel density or surface area, such as chemo-, radiation, or anti-angiogenic therapy. In urinary bladder cancer, this technique results in improved local and nodal staging, in improved separation of transurethral granulation tissue and edema from malignant tumor, and in improved evaluation of the effect of chemotherapy. In prostate cancer, dynamic MR imaging may be of help in problematic cases. This technique can assist in determining seminal vesicle infiltration, in depicting of minimal capsular penetration, and in recognizing tumors within the transitional zone. Also, based on very rapid enhancement, very poorly differentiated tumors can be recognized. Evaluation of the effects of therapy is another promising area, however a lot of research remain to be done. This article reviews some basics of fast enhancement techniques, provides practical information, and shows recent developments, in using these fast techniques for staging and grading of bladder and prostate cancer, and for evaluating the effect of therapy.

To assess angiogenesis noninvasively in a C6 rat brain tumor model, the rapid-steady-state- T1 (RSST1) magnetic resonance imaging (MRI) method was used for microvascular blood volume fraction (BVf) quantification with a novel contrast agent gadolinium per (3,6 anhydro) α-cyclodextrin (Gd-ACX). In brain tissue contralateral to the tumor, equal BVfs were obtained with Gd-ACX and the clinically approved gadoterate meglumine (Gd-DOTA). Contrary to Gd-DOTA, which leaks out of the tumor vasculature, Gd-ACX was shown to remain vascular in the tumor tissue allowing quantification of the tumor BVf. We sought to confirm the obtained tumor BVf using an independent method: instead of using a ‘standard’ two-dimensional histologic method, we study here how vascular morphometry combined with a stereological technique can be used for three-dimensional assessment of the vascular volume fraction ( VV). The VV is calculated from the vascular diameter and length density. First, the technique is evaluated on simulated data and the healthy rat brain vasculature and is then applied to the same C6 tumor vasculature previously quantified by RSST1-MRI with Gd-ACX. The mean perfused VV and the BVf obtained by MRI in tumor regions are practically equal and the technique confirms the spatial heterogeneity revealed by MRI.

International audience; PURPOSE: 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. METHODS AND MATERIALS: 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. RESULTS: 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. CONCLUSIONS: 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 audience; 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.

International audience; 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.

Since the early nineties, multiphoton microscopy has become a powerful tool to investigate morphological and physiological parameters in vivo or on thick ex vivo sections. To stain structures of interest many dyes have been developed and two-photon properties (cross section, excitation and emission spectra) of existing ones have been characterized. Recently, our team has shown a new property of sulforhodamine B (SRB). This dye has the ability to bind specifically elastic fibers. The observation of elastin using its endofluorescence properties was already widely described but required long exposition delays up to 10s and the imaging depth was limited to approximately 50 μm. With a multiphoton microscope and SRB, it is possible to observe elastic fibers directly in the living animal or on thick tissue sections with a micrometric spatial resolution in less than one second per image with an imaging depth of ~ 200 μm. Moreover, with an appropriate set of filters, we can acquire simultaneously the SRB and the second harmonic generation (SHG) signals of collagen fibers. Here, we report various applications of this new staining method on different arterial rings. The layers of the arterial wall, as well as, the elastic lamellae are observed and are numbered. With the addition of a nuclear stain such as the Hoechst 33342, a more accurate morphological study of the arterial walls can be accomplished. Finally, an intravital observation of the saphenous artery morphology is presented.

International audience; PURPOSE: 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. METHODS AND MATERIALS: 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. RESULTS: 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. CONCLUSION: 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.

The control of the efficiency of therapy on brain tissues requires an imaging at the microscopic scale and preferably in vivo. Here, we show that the intravital two-photon excited fluorescence microscopy already known for its capability in imaging the microvasculature can also be used to get three dimensional images of other cortical tissues in the brain of living mouse by taking profit of the dense capillary network to irrigate them. The technique which uses the differential imaging of two dyes: One being diffusing through the blood brain barrier, while the other don’t, gives access to tissues which are usually difficult to image in vivo.

Flow-sensitive alternating inversion recovery (FAIR) is a noninvasive method for perfusion imaging. It has been shown that the FAIR signal may depend on hemodynamic parameters other than perfusion, the most important one being transit delays of labeled spins to the observed tissue. These parameters are expected to change with ischemia. The goal of this study was to assess the effect of these changes on the interpretation of FAIR results in the case of altered perfusion. This was investigated in a rat model of transient cerebral ischemia. It was shown that the ratio of FAIR signal in the infarct compared to the contralateral side was lower at short inflow times, which suggests that transit times affected the effective FAIR signal. The FAIR results were compared with those from functional histology and dynamic susceptibility contrast MRI, and the findings indicated that the altered kinetics of the FAIR signal were related to reduced and delayed inflow in the infarct region—not to a decrease in the number of functional vessels. Magn Reson Med 51:312–320, 2004. © 2004 Wiley-Liss, Inc.

Purpose To investigate the responses of two experimental rat tumors to single and fractionated X-ray doses whether or not combined with Motexafin gadolinium (MGd), and the distribution of MGd in R3327-MATLyLu (MLL) tumors using MRI. Methods L44 lung tumor in BN rats and MLL prostate tumor in Copenhagen rats were grown subcutaneously. MGd at concentrations of 8.7 to 25.1 μmol/kg was administered 2 h before or just before treatments with single and fractionated X-ray doses. Tumor volume growth delay was the endpoint used. The two-dimensional distribution of the MGd concentration in time was analyzed simultaneously in slices through the center of MLL tumors using MRI. Directly after the MRI experiments, tumor sections were stained for cytoplasm, nuclei, and microvessel endothelium. Results MGd at different concentrations administered a few minutes or 2 h before X-ray doses produced no radiation enhancement in the two tumor models. The MGd concentration as determined by MRI was maximal 5 min after injection and decreased slowly thereafter. In a representative section at the center of the MLL tumor, the microvessel density is nearly homogeneous and correlates with a nearly homogeneous MGd distribution. Hardly any MGd is taken up in underlying muscle tissue. Conclusions No radiosensitization was observed for the different irradiation regimens. The distribution of MGd is nearly homogeneous in the MLL tumor and hardly any MGd is taken up in underlying muscles. Our negative results on radiosensitivity in our two tumor models raise questions about the efficacy of MGd as a general radiosensitizing agent.

Item does not contain fulltext PURPOSE: To investigate the physiological origins responsible for the varying blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) responses to carbogen (95% O(2)/5% CO(2)) breathing observed with different tumor types. MATERIALS AND METHODS: Susceptibility contrast-enhanced MRI using the exogenous blood pool contrast agent NC100150 to determine blood volume and vessel size, and immunohistochemical-derived morphometric parameters, were determined in GH3 prolactinomas and RIF-1 fibrosarcomas, both grown in mice, which exhibited very different BOLD responses to carbogen. RESULTS: Administration of NC100150 increased the R(2)* and R(2) rates of both tumor types, and indicated a significant four-fold larger blood volume in the GH3 tumor. The ratio deltaR(2)*/deltaR(2) showed that the capillaries in the GH3 were two-fold larger than those in the RIF-1, in agreement with morphometric analysis. Carbogen breathing induced a significant 25% decrease in R(2)* in the GH3 prolactinoma, whereas the response in the RIF-1 fibrosarcoma was negligible. CONCLUSION: Low blood volume and small vessel size (and hence reduced hematocrit) are two reasons for the lack of R(2)* change in the RIF-1 with carbogen breathing. BOLD MRI is sensitive to erythrocyte-perfused vessels, whereas exogenous contrast agents interrogate the total perfused vascular volume. BOLD MRI, coupled with a carbogen challenge, provides information on functional, hemodynamic tumor vasculature.

A solid tumor presents a unique challenge as a system in which the dynamics of the relationship between vascularization, the physiological environment and metabolism are continually changing with growth and following treatment. Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) studies have demonstrated quantifiable linkages between the physiological environment, angiogenesis, vascularization and metabolism of tumors. The dynamics between these parameters continually change with tumor aggressiveness, tumor growth and during therapy and each of these can be monitored longitudinally, quantitatively and non-invasively with MRI and MRS. An important aspect of MRI and MRS studies is that techniques and findings are easily translated between systems. Hence, pre-clinical studies using cultured cells or experimental animals have a high connectivity to potential clinical utility. In the following review, leaders in the field of MR studies of basic tumor physiology using pre-clinical models have contributed individual sections according to their expertise and outlook. The following review is a cogent and timely overview of the current capabilities and state-of-the-art of MRI and MRS as applied to experimental cancers. A companion review deals with the application of MR methods to anticancer therapy.