Magnetic resonance imaging of hypoxia in acute stroke compared with fluorine-18 fluoromisonidazole-positron emission tomography: A cross-validation study?

Acute ischemic stroke results in an ischemic core surrounded by a tissue at risk, named the penumbra, which is potentially salvageable. One way to differentiate the tissues is to measure the hypoxia status. The purpose of the current study is to correlate the abnormal brain tissue volume derived from magnetic resonance-based imaging of brain oxygen saturation (St O2 -MRI) to the fluorine-18 fluoromisonidazole ([18 F]FMISO) positron emission tomography (PET) volume for hypoxia imaging validation, and to analyze the ability of St O2 -MRI to depict the different hypoxic tissue types in the acute phase of stroke. In a pertinent model of stroke in the rat, the volume of tissue with decreased St O2 -MRI signal and that with increased uptake of [18 F]FMISO were equivalent and correlated (r = 0.706; p = 0.015). The values of St O2 in the tissue at risk were significantly greater than those quantified in the core of the lesion, and were less than those for healthy tissue (52.3% ± 2.0%; 43.3% ± 1.9%, and 67.9 ± 1.4%, respectively). A threshold value for St O2 of ≈60% as the cut-off for the identification of the tissue at risk was calculated. Tissue volumes with reduced St O2 -MRI correlated with the final lesion (r = 0.964, p < 0.0001). The findings show that the St O2 -MRI approach is sensitive for the detection of hypoxia and for the prediction of the final lesion after stroke. Once validated in acute clinical settings, this approach might be used to enhance the stratification of patients for potential therapeutic interventions.

Multimodal imaging based on MRI and PET reveals [(18)F]FLT PET as a specific and early indicator of treatment efficacy in a preclinical model of recurrent glioblastoma.

PURPOSE: The primary objective of this study was to compare the ability of PET and MRI biomarkers to predict treatment efficacy in a preclinical model of recurrent glioblastoma multiforme. METHODS: MRI (anatomical, diffusion, vasculature and oxygenation) and PET ([(18)F]FDG and [(18)F]FLT) parameters were obtained 3 days after the end of treatment and compared with late tumour growth and survival. RESULTS: Early after tumour recurrence, no effect of treatment with temozolomide combined with bevacizumab was observed on tumour volume as assessed by T2-W MRI. At later times, the treatment decreased tumour volume and increased survival. Interestingly, at the earlier time, temozolomide + bevacizumab decreased [(18)F]FLT uptake, cerebral blood volume and oedema. [(18)F]FLT uptake, oedema and cerebral blood volume were correlated with overall survival but [(18)F]FLT uptake had the highest specificity and sensitivity for the early prediction of treatment efficacy. CONCLUSION: The present investigation in a preclinical model of glioblastoma recurrence underscores the importance of multimodal imaging in the assessment of oedema, tumour vascular status and cell proliferation. Finally, [(18)F]FLT holds the greatest promise for the early assessment of treatment efficacy. These findings may translate clinically in that individualized treatment for recurrent glioma could be prescribed for patients selected after PET/MRI examinations.

Molecular mechanisms of skeletal muscle atrophy in a mouse model of cerebral ischemia.

BACKGROUND AND PURPOSE: Loss of muscle mass and function is a severe complication in patients with stroke that contributes to promoting physical inactivity and disability. The deleterious consequences of skeletal muscle mass loss underline the necessity to identity the molecular mechanisms involved in skeletal muscle atrophy after cerebral ischemia. METHODS: Transient focal cerebral ischemia (60 minutes) was induced by occlusion of the right middle cerebral artery in C57BL/6J male mice. Skeletal muscles were removed 3 days later and analyzed for the regulation of critical determinants of muscle mass homeostasis (Akt/mammalian target of rapamycin pathway, myostatin-Smad2/3 and bone morphogenetic protein-Smad1/5/8 signaling pathways, ubiquitin-proteasome and autophagy-lysosome proteolytic pathways). RESULTS: Cerebral ischemia induced severe sensorimotor deficits associated with muscle mass loss of the paretic limbs. Mechanistically, cerebral ischemia repressed Akt/mammalian target of rapamycin pathway and increased expression of key players of ubiquitin-proteasome pathway (MuRF1 [muscle RING finger-1], MAFbx [muscle atrophy F-box], Musa1 [muscle ubiquitin ligase of SCF complex in atrophy-1]), together with a marked increase in myostatin expression, in both paretic and nonparetic skeletal muscles. The Smad1/5/8 pathway was also activated. CONCLUSIONS: Our data fit with a model in which a repression of Akt/mammalian target of rapamycin pathway and an increase in the expression of key players of ubiquitin-proteasome pathway are critically involved in skeletal muscle atrophy after cerebral ischemia. Cerebral ischemia also caused an activation of bone morphogenetic protein-Smad1/5/8 signaling pathway, suggesting that compensatory mechanisms are also concomitantly activated to limit the extent of skeletal muscle atrophy.

Noninvasive assessment of hypoxia with 3-[18F]-fluoro-1-(2-nitro-1-imidazolyl)-2-propanol ([18F]-FMISO): a PET study in two experimental models of human glioma.

Despite multiple advances in cancer therapies, patients with glioblastoma (GBM) still have a poor prognosis. Numerous glioma models are used not only for the development of innovative therapies but also to optimize conventional ones. Given the significance of hypoxia in drug and radiation resistance and that hypoxia is widely observed among GBM, the establishment of a reliable method to map hypoxia in preclinical human models may contribute to the discovery and translation of future and more targeted therapies. The aim of this study was to compare the hypoxic status of two commonly used human orthotopic glioma models (U87 and U251) developed in rats and studied by noninvasive hypoxia imaging with 3-[18F]fluoro-1-(2-nitro-1-imidazolyl)-2-propanol-micro-positron emission tomography ([18F]-FMISO-µPET). In parallel, because of the relationships between angiogenesis and hypoxia, we used magnetic resonance imaging (MRI), histology, and immunohistochemistry to characterize the tumoral vasculature. Although all tumors were detectable in T2-weighted MRI and 2-deoxy-2-[18F]fluoro-d-glucose-µPET, only the U251 model exhibited [18F]-FMISO uptake. Additionally, the U251 tumors were less densely vascularized than U87 tumors. Our study demonstrates the benefits of noninvasive imaging of hypoxia in preclinical models to define the most reliable one for translation of future therapies to clinic based on the importance of intratumoral oxygen tension for the efficacy of chemotherapy and radiotherapy.

TNF receptor I sensitizes neurons to erythropoietin- and VEGF-mediated neuroprotection after ischemic and excitotoxic injury.

CNS neurons use robust cytoprotective mechanisms to ensure survival and functioning under conditions of injury. These involve pathways induced by endogenous neuroprotective cytokines such as erythropoietin (EPO). Recently, in contrast to its well known deleterious roles, TNF has also been shown to exhibit neuroprotective properties. In the present study, we investigated the molecular mechanisms by which TNF receptor (TNFR)I mediates neuroprotection by comparing the gene expression profiles of lesioned cortex from WT and TNFRI KO mice after permanent middle cerebral artery occlusion. Several known neuroprotective molecules were identified as TNFRI targets, notably members of the Bcl-2 family, DNA repair machinery and cell cycle, developmental, and differentiation factors, neurotransmitters and growth factors, as well as their receptors, including EPO receptor (EPOR), VEGF, colony-stimulating factor receptor 1, insulin-like growth factor (IGF), and nerve growth factor (NGF). Further analysis showed that induction of EPOR and VEGF expression in primary cortical neurons after glucose deprivation (GD) largely depended on TNFRI and was further up-regulated by TNF. Also, EPO- and VEGF-induced neuroprotection against GD, oxygen-glucose deprivation, and NMDA excitotoxicity depended significantly on TNFRI presence. Finally, EPO prevented neuronal damage induced by kainic acid in WT but not TNFRI KO mice. Our results identify cross-talk between tissue protective cytokines, specifically that TNFRI is necessary for constitutive and GD-induced expression of EPOR and VEGF and for EPO-mediated neuroprotection.

Multimodal evaluation of hypoxia in brain metastases of lung cancer and interest of hypoxia image-guided radiotherapy.

Lung cancer patients frequently develop brain metastases (BM). Despite aggressive treatment including neurosurgery and external-radiotherapy, overall survival remains poor. There is a pressing need to further characterize factors in the microenvironment of BM that may confer resistance to radiotherapy (RT), such as hypoxia. Here, hypoxia was first evaluated in 28 biopsies from patients with non­small cell lung cancer (NSCLC) BM, using CA-IX immunostaining. Hypoxia characterization (pimonidazole, CA-IX and HIF-1α) was also performed in different preclinical NSCLC BM models induced either by intracerebral injection of tumor cells (H2030-Br3M, H1915) into the cortex and striatum, or intracardial injection of tumor cells (H2030-Br3M). Additionally, [18F]-FMISO-PET and oxygen-saturation-mapping-MRI (SatO2-MRI) were carried out in the intracerebral BM models to further characterize tumor hypoxia and evaluate the potential of Hypoxia-image-guided-RT (HIGRT). The effect of RT on proliferation of BM ([18F]-FLT-PET), tumor volume and overall survival was determined. We showed that hypoxia is a major yet heterogeneous feature of BM from lung cancer both preclinically and clinically. HIGRT, based on hypoxia heterogeneity observed between cortical and striatal metastases in the intracerebrally induced models, showed significant potential for tumor control and animal survival. These results collectively highlight hypoxia as a hallmark of BM from lung cancer and the value of HIGRT in better controlling tumor growth.

Detection and quantification of remote microglial activation in rodent models of focal ischaemia using the TSPO radioligand CLINDE.

PURPOSE: Neuroinflammation is involved in stroke pathophysiology and might be imaged using radioligands targeting the 18 kDa translocator protein (TSPO). METHODS: We studied microglial reaction in brain areas remote from the primary lesion site in two rodent models of focal cerebral ischaemia (permanent or transient) using [125I]-CLINDE, a promising TSPO single photon emission computed tomography radioligand. RESULTS: In a mouse model of permanent middle cerebral artery occlusion (MCAO), ex vivo autoradiographic studies demonstrated, besides in the ischaemic territory, accumulation of [125I]-CLINDE in the ipsilateral thalamus with a binding that progressed up to 3 weeks after MCAO. [125I]-CLINDE binding markedly decreased in animals pre-injected with either unlabelled CLINDE or PK11195, while no change was observed with flumazenil pre-treatment, demonstrating TSPO specificity. In rats subjected to transient MCAO, [125I]-CLINDE binding in the ipsilateral thalamus and substantia nigra pars reticulata (SNr) was significantly higher than that in contralateral tissue. Moreover, [125I]-CLINDE binding in the thalamus and SNr was quantitatively correlated to the ischaemic volume assessed by MRI in the cortex and striatum, respectively. CONCLUSION: Clinical consequences of secondary neuronal degeneration in stroke might be better treated thanks to the discrimination of neuronal processes using in vivo molecular imaging and potent TSPO radioligands like CLINDE to guide therapeutic interventions.

VCAM-1 targeted alpha-particle therapy for early brain metastases.

BACKGROUND: Brain metastases (BM) develop frequently in patients with breast cancer. Despite the use of external beam radiotherapy (EBRT), the average overall survival is short (6 months from diagnosis). The therapeutic challenge is to deliver molecularly targeted therapy at an early stage when relatively few metastatic tumor cells have invaded the brain. Vascular cell adhesion molecule 1 (VCAM-1), overexpressed by nearby endothelial cells during the early stages of BM development, is a promising target. The aim of this study was to investigate the therapeutic value of targeted alpha-particle radiotherapy, combining lead-212 (212Pb) with an anti-VCAM-1 antibody (212Pb-αVCAM-1). METHODS: Human breast carcinoma cells that metastasize to the brain, MDA-231-Br-GFP, were injected into the left cardiac ventricle of nude mice. Twenty-one days after injection, 212Pb-αVCAM-1 uptake in early BM was determined in a biodistribution study and systemic/brain toxicity was evaluated. Therapeutic efficacy was assessed using MR imaging and histology. Overall survival after 212Pb-αVCAM-1 treatment was compared with that observed after standard EBRT. RESULTS: 212Pb-αVCAM-1 was taken up into early BM with a tumor/healthy brain dose deposition ratio of 6 (5.52e108 and 0.92e108) disintegrations per gram of BM and healthy tissue, respectively. MRI analyses showed a statistically significant reduction in metastatic burden after 212Pb-αVCAM-1 treatment compared with EBRT (P < 0.001), translating to an increase in overall survival of 29% at 40 days post prescription (P < 0.01). No major toxicity was observed. CONCLUSIONS: The present investigation demonstrates that 212Pb-αVCAM-1 specifically accumulates at sites of early BM causing tumor growth inhibition.

Behavioral deficits after distal focal cerebral ischemia in mice: Usefulness of adhesive removal test.

Distal occlusion of the middle cerebral artery (dMCAo), which closely mimics human stroke, is one of the most used animal models. However, although assessment of histological and functional outcome is increasingly recommended for preclinical studies, the latter is often excluded because of the high difficulties to estimate, especially in mice, behavioral impairments. The aim of our study was to deeply screen functional consequences of distal permanent MCAo in mice to target relevant behaviors for future studies. A set of sensorimotor and cognitive tests were performed during 3 weeks postsurgery in 2 groups of mice. Afterward, brain infarctions were estimated by histological staining or magnetic resonance imaging. Overall, while no long-term functional impairments could be detected, the adhesive removal was the only test showing a deficit. Interestingly, this sensorimotor impairment was correlated to cortical damage 3 weeks after surgery. In conclusion, despite the fact that dMCAo-induced deficits could not be evidenced by most of our behavioral tests, the authors showed that the adhesive removal test was the only one, sensitive enough, to highlight a long-term deficit. This result suggests therefore that this mouse model of ischemia is relevant to efficiently assess therapeutic strategies with histological but also behavioral analysis, provided that relevant tests are used.

64Cu-ATSM/64Cu-Cl2 and their relationship to hypoxia in glioblastoma: a preclinical study.

BACKGROUND: Diacetyl-bis(N4-methylthiosemicarbazone), labeled with 64Cu (64Cu-ATSM) has been suggested as a promising tracer for imaging hypoxia. However, various controversial studies highlighted potential pitfalls that may disable its use as a selective hypoxic marker. They also highlighted that the results may be tumor location dependent. Here, we first analyzed uptake of Cu-ATSM and its less lipophilic counterpart Cu-Cl2 in the tumor over time in an orthotopic glioblastoma model. An in vitro study was also conducted to investigate the hypoxia-dependent copper uptake in tumor cells. We then further performed a comprehensive ex vivo study to compare 64Cu uptake to hypoxic markers, specific cellular reactions, and also transporter expression. METHODS: µPET was performed 14 days (18F-FMISO), 15 days (64Cu-ATSM and 64Cu-Cl2), and 16 days (64Cu-ATSM and 64Cu-Cl2) after C6 cell inoculation. Thereafter, the brains were withdrawn for further autoradiography and immunohistochemistry. C6 cells were also grown in hypoxic workstation to analyze cellular uptake of Cu complexes in different oxygen levels. RESULTS: In vivo results showed that Cu-ASTM and Cu-Cl2 accumulated in hypoxic areas of the tumors. Cu-ATSM also stained, to a lesser extent, non-hypoxic regions, such as regions of astrogliosis, with high expression of copper transporters and in particular DMT-1 and CTR1, and also characterized by the expression of elevated astrogliosis. In vitro results show that 64Cu-ATSM showed an increase in the uptake only in severe hypoxia at 0.5 and 0.2% of oxygen while for 64Cu-Cl2, the cell retention was significantly increased at 5% and 1% of oxygen with no significant rise at lower oxygen percentages. CONCLUSION: In the present study, we show that Cu-complexes undoubtedly accumulate in hypoxic areas of the tumors. This uptake may be the reflection of a direct dependency to a redox metabolism and also a reflection of hypoxic-induced overexpression of transporters. We also show that Cu-ATSM also stained non-hypoxic regions such as astrogliosis.

FLIP(L) protects neurons against in vivo ischemia and in vitro glucose deprivation-induced cell death.

Knowledge of the molecular mechanisms that underlie neuron death after stroke is important to allow the development of effective neuroprotective strategies. In this study, we investigated the contribution of death receptor signaling pathways to neuronal death after ischemia using in vitro and in vivo models of ischemic injury and transgenic mice that are deficient in tumor necrosis factor receptor I (TNFRI KO) or show neuron-specific overexpression of the long isoform of cellular Fas-associated death domain-like interleukin-1-beta-converting enzyme-inhibitory protein (FLIP(L)). Caspase 8 was activated in brain lesions after permanent middle cerebral artery occlusion (pMCAO) and in cortical neurons subjected to glucose deprivation (GD) and was necessary for GD-induced neuron death. Thus, neurons treated with zIETD-FMK peptide or overexpressing a dominant-negative caspase 8 mutant were fully protected against GD-induced death. The presence of the neuroprotective TNFRI was necessary for selectively sustaining p50/p65NF-kappaB activity and the expression of the p43 cleavage form of FLIP(L), FLIP(p43), an endogenous inhibitor of caspase 8, in pMCAO lesions and GD-treated neurons. Moreover, TNF pretreatment further upregulated p50/p65NF-kappaB activity and FLIP(p43) expression in neurons after GD. The knock-down of FLIP in wild-type (WT) neurons using a short hairpin RNA revealed that FLIP(L) is essential for TNF/TNFRI-mediated neuroprotection after GD. Furthermore, the overexpression of FLIP(L) was sufficient to rescue TNFRI KO neurons from GD-induced death and to enhance TNF neuroprotection in WT neurons, and neuron-specific expression of FLIP(L) in transgenic mice significantly reduced lesion volume after pMCAO. Our results identify a novel role for the TNFRI-NF-kappaB-FLIP(L) pathway in neuroprotection after ischemia and identify potential new targets for stroke therapy.

A heparan sulfate-based matrix therapy reduces brain damage and enhances functional recovery following stroke.

Alteration of the extracellular matrix (ECM) is one of the major events in the pathogenesis of brain lesions following ischemic stroke. Heparan sulfate mimetics (HSm) are synthetic pharmacologically active polysaccharides that promote ECM remodeling and tissue regeneration in various types of lesions. HSm bind to growth factors, protect them from enzymatic degradation and increase their bioavailability, which promotes tissue repair. As the ECM is altered during stroke and HSm have been shown to restore the ECM, we investigated the potential of HSm4131 (also named RGTA-4131®) to protect brain tissue and promote regeneration and plasticity after a stroke. Methods: Ischemic stroke was induced in rats using transient (1 h) intraluminal middle cerebral artery occlusion (MCAo). Animals were assigned to the treatment (HSm4131; 0.1, 0.5, 1.5, or 5 mg/kg) or vehicle control (saline) groups at different times (1, 2.5 or 6 h) after MCAo. Brain damage was assessed by MRI for the acute (2 days) and chronic (14 days) phases post-occlusion. Functional deficits were evaluated with a battery of sensorimotor behavioral tests. HSm4131-99mTc biodistribution in the ischemic brain was analyzed between 5 min and 3 h following middle cerebral artery reperfusion. Heparan sulfate distribution and cellular reactions, including angiogenesis and neurogenesis, were evaluated by immunohistochemistry, and growth factor gene expression (VEGF-A, Ang-2) was quantified by RT-PCR. Results: HSm4131, administered intravenously after stroke induction, located and remained in the ischemic hemisphere. HSm4131 conferred long-lasting neuroprotection, and significantly reduced functional deficits with no alteration of physiological parameters. It also restored the ECM, and increased brain plasticity processes, i.e., angiogenesis and neurogenesis, in the affected brain hemisphere. Conclusion: HSm represent a promising ECM-based therapeutic strategy to protect and repair the brain after a stroke and favor functional recovery.

M2 macrophages are more resistant than M1 macrophages following radiation therapy in the context of glioblastoma.

In some highly inflammatory tumors, such as glioblastoma (GB), macrophages (MΦ) represent the most abundant population of reactive cells. MΦ, initially denoted as M0 MΦ, can be polarized into two further phenotypes: the antitumor M1 MΦ, and the protumor M2 MΦ. The three phenotypes can reside simultaneously in the tumor mass and various external factors may influence MΦ polarization. Radiotherapy is a common modality of cancer treatment aiming to target tumor cells. However, the specific effects of X-ray radiation on the inflammatory cells are, so far, controversial and not fully understood. In the present investigation, we have first analyzed, in vivo, the effect of X-ray radiation on MΦ present in GB tumors. We have observed a decrease in MΦ number paralleled by an increase in the proportion of M2 MΦ. To understand this phenomenon, we then evaluated, in vitro, the effects of X-rays on the MΦ phenotypes and survival. We have found that X-ray radiation failed to modify the phenotype of the different MΦ. However, M1 MΦ were more sensitive to ionizing radiation than M2 MΦ, both in normoxia and in hypoxia, which could explain the in vivo observations. To conclude, M2 MΦ are more radioresistant than M0 and M1 MΦ and the present study allows us to propose that X-ray radiotherapy could contribute, along with other phenomena, to the increased density in the protumor M2 MΦ in GB.

Pharmacological inhibition of myostatin improves skeletal muscle mass and function in a mouse model of stroke.

In stroke patients, loss of skeletal muscle mass leads to prolonged weakness and less efficient rehabilitation. We previously showed that expression of myostatin, a master negative regulator of skeletal muscle mass, was strongly increased in skeletal muscle in a mouse model of stroke. We therefore tested the hypothesis that myostatin inhibition would improve recovery of skeletal muscle mass and function after cerebral ischemia. Cerebral ischemia (45 minutes) was induced by intraluminal right middle cerebral artery occlusion (MCAO). Swiss male mice were randomly assigned to Sham-operated mice (n = 10), MCAO mice receiving the vehicle (n = 15) and MCAO mice receiving an anti-myostatin PINTA745 (n = 12; subcutaneous injection of 7.5 mg.kg-1 PINTA745 immediately after surgery, 3, 7 and 10 days after MCAO). PINTA745 reduced body weight loss and improved body weight recovery after cerebral ischemia, as well as muscle strength and motor function. PINTA745 also increased muscle weight recovery 15 days after cerebral ischemia. Mechanistically, the better recovery of skeletal muscle mass in PINTA745-MCAO mice involved an increased expression of genes encoding myofibrillar proteins. Therefore, an anti-myostatin strategy can improve skeletal muscle recovery after cerebral ischemia and may thus represent an interesting strategy to combat skeletal muscle loss and weakness in stroke patients.

Imaging of brain oxygenation with magnetic resonance imaging: A validation with positron emission tomography in the healthy and tumoural brain.

The partial pressure in oxygen remains challenging to map in the brain. Two main strategies exist to obtain surrogate measures of tissue oxygenation: the tissue saturation studied by magnetic resonance imaging (StO2-MRI) and the identification of hypoxia by a positron emission tomography (PET) biomarker with 3-[18F]fluoro-1-(2-nitro-1-imidazolyl)-2-propanol ([18F]-FMISO) as the leading radiopharmaceutical. Nonetheless, a formal validation of StO2-MRI against FMISO-PET has not been performed. The objective of our studies was to compare the two approaches in (a) the normal rat brain when the rats were submitted to hypoxemia; (b) animals implanted with four tumour types differentiated by their oxygenation. Rats were submitted to normoxic and hypoxemic conditions. For the brain tumour experiments, U87-MG, U251-MG, 9L and C6 glioma cells were orthotopically inoculated in rats. For both experiments, StO2-MRI and [18F]-FMISO PET were performed sequentially. Under hypoxemia conditions, StO2-MRI revealed a decrease in oxygen saturation in the brain. Nonetheless, [18F]-FMISO PET, pimonidazole immunohistochemistry and molecular biology were insensitive to hypoxia. Within the context of tumours, StO2-MRI was able to detect hypoxia in the hypoxic models, mimicking [18F]-FMISO PET with high sensitivity/specificity. Altogether, our data clearly support that, in brain pathologies, StO2-MRI could be a robust and specific imaging biomarker to assess hypoxia.

Carbogen-induced increases in tumor oxygenation depend on the vascular status of the tumor: A multiparametric MRI study in two rat glioblastoma models.

The alleviation of hypoxia in glioblastoma with carbogen to improve treatment has met with limited success. Our hypothesis is that the eventual benefits of carbogen depend on the capacity for vasodilation. We examined, with MRI, changes in fractional cerebral blood volume, blood oxygen saturation, and blood oxygenation level dependent signals in response to carbogen. The analyses were performed in two xenograft models of glioma (U87 and U251) recognized to have different vascular patterns. Carbogen increased fractional cerebral blood volume, blood oxygen saturation, and blood oxygenation level dependent signals in contralateral tissues. In the tumor core and peritumoral regions, changes were dependent on the capacity to vasodilate rather than on resting fractional cerebral blood volume. In the highly vascularised U87 tumor, carbogen induced a greater increase in fractional cerebral blood volume and blood oxygen saturation in comparison to the less vascularized U251 tumor. The blood oxygenation level dependent signal revealed a delayed response in U251 tumors relative to the contralateral tissue. Additionally, we highlight the considerable heterogeneity of fractional cerebral blood volume, blood oxygen saturation, and blood oxygenation level dependent within U251 tumor in which multiple compartments co-exist (tumor core, rim and peritumoral regions). Finally, our study underlines the complexity of the flow/metabolism interactions in different models of glioblastoma. These irregularities should be taken into account in order to palliate intratumoral hypoxia in clinical trials.

VEGF-induced BBB permeability is associated with an MMP-9 activity increase in cerebral ischemia: both effects decreased by Ang-1.

After cerebral ischemia, angiogenesis, by supplying for the deficient perfusion, may be a beneficial process for limiting neuronal death and promoting tissue repair. In this study, we showed that the combination of Ang-1 and vascular endothelial growth factor (VEGF) provides a more adapted therapeutic strategy than the use of VEGF alone. Indeed, we showed on a focal ischemia model that an early administration of VEGF exacerbates ischemic damage, because of its effects on blood-brain barrier (BBB) permeability. In contrast, a coapplication of Ang-1 and VEGF leads to a significant reduction of the ischemic and edema volumes by 50% and 42%, respectively, in comparison with VEGF-treated mice. We proposed that Ang-1 blocks the BBB permeability effect of VEGF in association with a modulation of matrix metalloproteinase (MMP) activity. Indeed, we showed on both ischemic in vivo and BBB in vitro models that VEGF enhances BBB damage and MMP-9 activity and that Ang-1 counteracts both effects. However, we also showed a synergic angiogenic effect of Ang-1 and VEGF in the brain. Taken together, these results allow to propose that, in cerebral ischemia, the combination of Ang-1 and VEGF could be used early to promote the formation of mature neovessels without inducing side effects on BBB permeability.

Silencing erythropoietin receptor on glioma cells reinforces efficacy of temozolomide and X-rays through senescence and mitotic catastrophe.

Hypoxia-inducible genes may contribute to therapy resistance in glioblastoma (GBM), the most aggressive and hypoxic brain tumours. It has been recently reported that erythropoietin (EPO) and its receptor (EPOR) are involved in glioma growth. We now investigated whether EPOR signalling may modulate the efficacy of the GBM current treatment based on chemotherapy (temozolomide, TMZ) and radiotherapy (X-rays). Using RNA interference, we showed on glioma cell lines (U87 and U251) that EPOR silencing induces a G2/M cell cycle arrest, consistent with the slowdown of glioma growth induced by EPOR knock-down. In vivo, we also reported that EPOR silencing combined with TMZ treatment is more efficient to delay tumour recurrence and to prolong animal survival compared to TMZ alone. In vitro, we showed that EPOR silencing not only increases the sensitivity of glioma cells to TMZ as well as X-rays but also counteracts the hypoxia-induced chemo- and radioresistance. Silencing EPOR on glioma cells exposed to conventional treatments enhances senescence and induces a robust genomic instability that leads to caspase-dependent mitotic death by increasing the number of polyploid cells and cyclin B1 expression. Overall these data suggest that EPOR could be an attractive target to overcome therapeutic resistance toward ionising radiation or temozolomide.

Effects of mesenchymal stem cell therapy, in association with pharmacologically active microcarriers releasing VEGF, in an ischaemic stroke model in the rat.

Few effective therapeutic interventions are available to limit brain damage and functional deficits after ischaemic stroke. Within this context, mesenchymal stem cell (MSC) therapy carries minimal risks while remaining efficacious through the secretion of trophic, protective, neurogenic and angiogenic factors. The limited survival rate of MSCs restricts their beneficial effects. The usefulness of a three-dimensional support, such as a pharmacologically active microcarrier (PAM), on the survival of MSCs during hypoxia has been shown in vitro, especially when the PAMs were loaded with vascular endothelial growth factor (VEGF). In the present study, the effect of MSCs attached to laminin-PAMs (LM-PAMs), releasing VEGF or not, was evaluated in vivo in a model of transient stroke. The parameters assessed were infarct volume, functional recovery and endogenous cellular reactions. LM-PAMs induced the expression of neuronal markers by MSCs both in vitro and in vivo. Moreover, the prolonged release of VEGF increased angiogenesis around the site of implantation of the LM-PAMs and facilitated the migration of immature neurons towards the ischaemic tissue. Nonetheless, MSCs/LM-PAMs-VEGF failed to improve sensorimotor functions. The use of LM-PAMs to convey MSCs and to deliver growth factors could be an effective strategy to repair the brain damage caused by a stroke.

Normobaric hypoxia induces tolerance to focal permanent cerebral ischemia in association with an increased expression of hypoxia-inducible factor-1 and its target genes, erythropoietin and VEGF, in the adult mouse brain.

Tolerance to cerebral ischemia is achieved by preconditioning sublethal stresses, such as ischemia or hypoxia, paradigms in which the decrease of O2 availability may constitute an early signal inducing tolerance. In accordance with this concept, this study shows that hypoxia induces tolerance against focal permanent ischemia in adult mice. Normobaric hypoxia (8% O2 of 1-hour, 3-hour, or 6-hour duration), performed 24 hours before ischemia, reduces infarct volume by approximately 30% when compared with controls. To elucidate the mechanisms underlying this neuroprotection, the authors investigated the effects of preconditioning on cerebral expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and its target genes, erythropoietin and vascular endothelial growth factor (VEGF). Hypoxia, whatever its duration (1 hour, 3 hours, 6 hours), rapidly increases the nuclear content of HIF-1alpha as well as the mRNA levels of erythropoietin and VEGF. Furthermore, erythropoietin and VEGF are upregulated at the protein level 24 hours after 6 hours of hypoxia. The authors' findings show that (1) hypoxia elicits a delayed, short-lasting (<72 hours) tolerance to focal permanent ischemia in the adult mouse brain; (2) HIF-1 target genes could contribute to the establishment of tolerance; and (3) this model might be a useful paradigm to further study the mechanisms of ischemic tolerance, to identify new therapeutic targets for stroke.