FMISO-PET-derived brain oxygen tension maps: application to glioblastoma and less aggressive gliomas.

Quantitative imaging modalities for the analysis of hypoxia in brain tumors are lacking. The objective of this study was to generate absolute maps of tissue ptO2 from [18F]-FMISO images in glioblastoma and less aggressive glioma patients in order to quantitatively assess tumor hypoxia. An ancillary objective was to compare estimated ptO2 values to other biomarkers: perfusion weighted imaging (PWI) and tumor metabolism obtained from 1H-MR mono-voxel spectroscopy (MRS). Ten patients with glioblastoma (GBM) and three patients with less aggressive glioma (nGBM) were enrolled. All patients had [18F]-FMISO and multiparametric MRI (anatomic, PWI, MRS) scans. A non-linear regression was performed to generate ptO2 maps based on normal appearing gray (NAGM) and white matter (NAWM) for each patient. As expected, a marked [18F]-FMISO uptake was observed in GBM patients. The ptO2 based on patient specific calculations was notably low in this group (4.8 ± 1.9 mmHg, p < 0.001) compared to all other groups (nGBM, NAGM and NAWM). The rCBV was increased in GBM (1.4 ± 0.2 when compared to nGBM tumors 0.8 ± 0.4). Lactate (and lipid) concentration increased in GBM (27.8 ± 13.8%) relative to nGBM (p < 0.01). Linear, nonlinear and ROC curve analyses between ptO2 maps, PWI-derived rCBV maps and MRS-derived lipid and lactate concentration strengthens the robustness of our approaches.

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.

Hypoxia induces macrophage polarization and re-education toward an M2 phenotype in U87 and U251 glioblastoma models.

Hypoxia is a common feature of solid tumors, particularly in glioblastoma (GBM), and known to be a poor prognosis factor in GBM patients. The growth of GBM is also associated with a marked inflammation partially characterized by an accumulation of macrophage (MΦ) of the M2 phenotype. However, the transition between M1 MΦ (antitumoral) and M2 MΦ (protumoral) phenotypes is a dynamic process. We made the assumption that oxygen (O2) availability could be a major regulator of this transition and that the intratumoral O2 gradient is of importance. We evaluated, in vivo, the impact of hypoxia on MΦ tropism and polarization in two models of human GBM, well differentiated by their degree of hypoxia. MΦ migration in the tumor was more pronounced in the more hypoxic tumor of the two GBM models. In the more hypoxic of the models, we have shown that MΦ migrated at the tumor site only when hypoxia takes place. We also demonstrated that the acquisition of the M2 phenotype was clearly an evolving phenomenon with hypoxia as the major trigger for this transition. In support of these in vivo finding, M0 but also M1 MΦ cultured in moderate or severe hypoxia displayed a phenotype close to that of M2 MΦ whose phenotype was further reinforced by severe hypoxia. These results highlight the role of hypoxia in the aggressiveness of GBM, in part, by transforming MΦ such that a protumoral activity is expressed.

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.

Imaging Modalities to Assess Oxygen Status in Glioblastoma.

Hypoxia, the result of an inadequacy between a disorganized and functionally impaired vasculature and the metabolic demand of tumor cells, is a feature of glioblastoma. Hypoxia promotes the aggressiveness of these tumors and, equally, negatively correlates with a decrease in outcome. Tools to characterize oxygen status are essential for the therapeutic management of patients with glioblastoma (i) to refine prognosis, (ii) to adapt the treatment regimen, and (iii) to assess the therapeutic efficacy. While methods that are focal and invasive in nature are of limited use, non-invasive imaging technologies have been developed. Each of these technologies is characterized by its singular advantages and limitations in terms of oxygenation status in glioblastoma. The aim of this short review is, first, to focus on the interest to characterize hypoxia for a better therapeutic management of patients and, second, to discuss recent and pertinent approaches for the assessment of oxygenation/hypoxia and their direct implication for patient care.

Chronic arterial hypertension impedes glioma growth: a multiparametric MRI study in the rat.

Glioblastoma is the most aggressive brain tumor and is almost always fatal. These tumors are highly vascularized and angiogenesis is one of the pre-eminent mechanisms underlying their growth. Chronic arterial hypertension (CAH) is a common and worldwide pathology that markedlly alters the structure and function of the vasculature. Yet, essential hypertension is associated in the brain with potential locally impaired vasoreactivity, disturbed perfusion supply and hypoxia phenomena. Even though CAH is a global burden and has an important impact on brain function, nothing is known about the way this frequent pathology would interact with the evolution of glioma. We sought to determine if arterial hypertension influences gliobastoma growth. In the present study, rat glioma C6 tumor cells were implanted in the caudate-putamen of spontaneously hypertensive rats (SHR) or their normotensive controls, the Wistar-Kyoto (WKY) rats. The evolution of the tumor was sequentially analyzed by multiparametric magnetic resonance imaging and the inflammatory response was examined by histochemistry. We found that CAH significantly attenuates the growth of the tumor as, at 21 days, the volume of the tumor was 85.4±34.7 and 126.1±28.8 mm(3), respectively, in hypertensive and normotensive rats (P<0.02). Moreover, cerebral blood volume and cerebral blood flow were greater in the tumors of hypertensive rats (P<0.05). The lesser growth of the tumor observed in normotensive animals was not due to an enhanced rejection of the tumor cells in WKY rats, the inflammatory response being similar in both groups. For the first time, these results show that CAH impedes the growth of glioblastoma and illustrate the need to further study the impact of hypertension on the evolution of brain tumors.

2-[18F]fludarabine, a novel positron emission tomography (PET) tracer for imaging lymphoma: a micro-PET study in murine models.

PURPOSE: Fludarabine has proven to be of considerable efficacy in the treatment of low-grade lymphomas. We have developed the labeling of this drug with fluorine-18 and evaluated 2-[(18)F]fludarabine as a novel positron emission tomography (PET) probe for in vivo imaging. PROCEDURES: Preclinical studies were conducted with 2-[(18)F]fludarabine, in parallel with 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG), in Swiss CD-1 and CB17 severely combined immunodeficient (SCID) mice, both as tumor-free control groups, and SCID mice bearing RL lymphomas. RESULTS: In Swiss mice, micro-PET studies with 2-[(18)F]fludarabine showed a distribution restricted to the organs of excretion and the spleen, the latter being less evident in SCID animals. In lymphoma-bearing SCID mice, 2-[(18)F]fludarabine demonstrated a rapid tumor uptake over the first 20 min which subsequently plateaued and provided an improved contrast than that of [(18)F]FDG. CONCLUSION: This radiotracer merits further evaluation to establish its clinical usefulness to image low-grade lymphoma in humans in future clinical investigations.

Synthesis and in vitro characterisation of ifenprodil-based fluorescein conjugates as GluN1/GluN2B N-Methyl-D-aspartate receptor antagonists.

GluN2B-containing NMDA receptors are involved in many important physiological functions and play a pivotal role in mediating pain as well as in several neurodegenerative disorders. We aimed to develop fluorescent probes to target the GluN2B subunit selectively in order to allow better understanding of the relationships between receptor localisation and physiological importance. Ifenprodil, known as the GluNR2B antagonist of reference, was chosen as the template for the elaboration of probes. We had previously reported a fluorescein conjugate that was shown (by confocal microscopy imaging of DS-red-labelled cortical neurons) to bind specifically to GluN2B. To elaborate this probe, we explored the influence of both the nature and the attachment point of the spacer between the fluorophore and the parent compound, ifenprodil. We performed chemical modifications of ifenprodil at the benzylic position and on the phenol ring by introducing secondary amine or amide functions and evaluated alkyl chains from two to 20 bonds either including or not including secondary amide functions as spacers. The previously developed probe was found to display the greatest activity in the inhibition of NMDA-induced Ca(2+) influx by calcium imaging experiments on HEK293 cells transfected with the cDNA encoding for GluN1-1A and GluN2B. Further investigations revealed that this probe had a neuroprotective effect equivalent to that of ifenprodil in a standard test for neurotoxicity. Despite effects of lesser amplitude with these probes relative to ifenprodil, we demonstrated that they displaced [(3) H]ifenprodil in mouse brain slices in a similar manner.

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.

Detection of glioblastoma response to temozolomide combined with bevacizumab based on µMRI and µPET imaging reveals [18F]-fluoro-L-thymidine as an early and robust predictive marker for treatment efficacy.

The individualized care of glioma patients ought to benefit from imaging biomarkers as precocious predictors of therapeutic efficacy. Contrast enhanced MRI and [(18)F]-fluorodeoxyglucose (FDG)-PET are routinely used in clinical settings; their ability to forecast the therapeutic response is controversial. The objectives of our preclinical study were to analyze sensitive µMRI and/or µPET imaging biomarkers to predict the efficacy of anti-angiogenic and/or chemotherapeutic regimens. Human U87 and U251 orthotopic glioma models were implanted in nude rats. Temozolomide and/or bevacizumab were administered. µMRI (anatomical, diffusion, and microrheological parameters) and µPET ([(18)F]-FDG and [(18)F]-fluoro-l-thymidine [FLT]-PET) studies were undertaken soon (t(1)) after treatment initiation compared with late anatomical µMRI evaluation of tumor volume (t(2)) and overall survival. In both models, FDG and FLT uptakes were attenuated at t(1) in response to temozolomide alone or with bevacizumab. The distribution of FLT, reflecting intratumoral heterogeneity, was also modified. FDG was less predictive for treatment efficacy than was FLT (also highly correlated with outcome, P < .001 for both models). Cerebral blood volume was significantly decreased by temozolomide + bevacizumab and was correlated with survival for rats with U87 implants. While FLT was highly predictive of treatment efficacy, a combination of imaging biomarkers was superior to any one alone (P < .0001 in both tumors with outcome). Our results indicate that FLT is a sensitive predictor of treatment efficacy and that predictability is enhanced by a combination of imaging biomarkers. These findings may translate clinically in that individualized glioma treatments could be decided in given patients after PET/MRI examinations.

Angiopoietin-2 is vasoprotective in the acute phase of cerebral ischemia.

Most forms of cerebral ischemia are characterized by damage to the entire neurovascular unit, which leads to an increase in the permeability of the blood-brain barrier (BBB). In response to permanent focal cerebral ischemia in mice, we detected an early concomitant increase in the expression of the vascular endothelial growth factor (VEGF), a key inducer of vascular leakage and pathological blood vessel growth, and of angiopoietin-2 (Ang2), which is closely associated with VEGF in vascular remodeling. Thus, the aim of this study was to evaluate the role of Ang2 alone, or in combination with VEGF, in the acute phase of cerebral ischemia. The effect of these angiogenic factors on the ischemic lesion volume was evaluated by magnetic resonance imaging. We observed that timely administration of VEGF exacerbates ischemic damage. In contrast, Ang2 decreases the ischemic volume and this beneficial effect is maintained in the presence of VEGF. This investigation reports, for the first time, a protective role of Ang2 following cerebral ischemia, an action associated with a reduced BBB permeability. We propose that Ang2 represents a pertinent molecular target for the treatment of cerebral ischemia since acute brain damage may be limited by a pharmacological protection of the vascular compartment.

Effects of urotensin-II on cerebral blood flow and ischemia in anesthetized rats.

Urotensin-II (U-II) is a cyclic peptide identified recently in many mammalian species including man. U-II and its receptor are expressed in the central nervous system, in the cardiovascular system and in other peripheral tissues. Although this peptide has been reported initially to be a potent vasoconstrictor, increasing evidence shows that its vascular actions strongly depend on species and vascular beds. Here we analyzed the effects of U-II administration on cerebral blood flow (CBF) under physiological conditions and following cerebral ischemia in rats. Although intravenous injection of U-II had minimal effects on CBF as measured by the technique of laser Doppler flowmetry, its administration (10 nmol) into the lateral cerebral ventricle induced gradual and long lasting increase in CBF (+61% at 1 h post-injection, p<0.05). These U-II-mediated CBF increases were not related to the transient systemic pressor actions of the peptide and were reduced by nitric oxide synthase inhibition (61 vs 17%, p<0.05). Intracerebroventricular administration of U-II following the induction of cerebral ischemia, failed to alter residual CBF in the affected cerebral hemisphere. Nonetheless, following reperfusion (90 min after ischemia), U-II-treated animals displayed a remarkable hyperperfusion compared to vehicle-treated rats (+168%, p<0.05). The volume of infarction was significantly increased in U-II-treated rats (+40%, p<0.05). These results provide the first evidence that U-II increases cerebral blood flow when administered into the cerebral ventricle and exacerbates brain damage following an ischemic insult.

Intraluminal thread model of focal stroke in the non-human primate.

The common marmoset (Callithrix jacchus), a New World monkey, has recently been used as a model of focal cerebral ischaemia. Here, we sought to develop a stroke model in this species using an intraluminal approach to occlude the middle cerebral artery (MCA). This technically simple procedure allows both transient and permanent ischaemia with minimal morbidity. Ten common marmosets underwent either transient (3 h) or permanent ischaemia by the insertion of a nylon filament through the external carotid artery up to the origin of the MCA. Cerebral blood flow (CBF) was monitored by the laser-Doppler flowmetry technique. Sensorimotor functions were regularly evaluated, and histologic, immunohistochemical, and magnetic resonance imaging analyses were performed 8 days after the occlusion. The surgical procedure was achieved straightforwardly without postoperative mortality or cerebral haemorrhage. All animals displayed a consistent decrease in CBF that remained stable over 3 h. Infarction affected both cortical and subcortical structures. Although not statistically significant, the volume of infarction was smaller in marmosets subjected to transient ischaemia compared to those permanently occluded (237+/-139 and 358+/-118 mm3, respectively). In all the behavioural tests used, reperfused marmosets exhibited fewer neurologic and functional impairments compared to permanently occluded ones. We show the feasibility of the induction of permanent or transient focal cerebral ischaemia in the marmoset using an intraluminal approach with minimal invasion. This model could be suitable as an advanced screening for potential stroke therapies in which behavioural, imaging, and histologic analyses can be compared.

Spontaneously hypertensive rats are highly vulnerable to AMPA-induced brain lesions.

BACKGROUND AND PURPOSE: Whereas the effects of chronic arterial hypertension on the cerebral vasculature have been widely studied, its effects on brain tissue have been studied less so. Here we examined if spontaneously hypertensive rats (SHRs) or the normotensive control Wistar Kyoto rats (WKYs) made hypertensive by renal artery stenosis (R-WKYs) are vulnerable to an excitotoxic brain lesion provoked by an overactivation of glutamate receptors. METHODS: Lesion volumes were quantified by histology in WKYs and SHRs subjected to striatal administration of N-methyl-d-aspartate (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA). The expression of AMPA receptors subunits and calcium/calmodulin kinase-II alpha was analyzed by real-time polymerase chain reaction and Western blot. RESULTS: NMDA (50 and 75 nmol) induced similar lesions in both SHRs (10+/-2 mm(3) and 16+/-4 mm(3), respectively) and WKYs (11+/-2 mm(3) and 19+/-7 mm(3), respectively). However, AMPA-induced (2.5 and 5 nmol) lesions were significantly greater in 14-week-old SHRs (14+/-3 mm(3) and 20+/-5 mm(3), respectively) than WKYs (4+/-2 mm(3), P<0.05 and 7+/-4 mm(3), P<0.001, respectively). Furthermore, normotensive 7-week-old SHRs also displayed an aggravated AMPA-induced lesion compared with age-matched WKYs (10+/-3 mm(3) vs 6+/-3 mm(3); P<0.05). Neither NMDA nor AMPA produced increased lesion volumes in R-WKYs (12+/-3 mm(3) and 5+/-4 mm(3), respectively) compared with WKYs. Striatal levels of AMPA receptors subunits, GluR1 and GluR2, were not different between SHRs and WKYs. However, SHRs displayed an increase in phosphorylated form of GluR1 at Ser-831 (P<0.05), as well as in calcium/calmodulin kinase-II alpha (P<0.002). Selective inhibition of this kinase by KN-93 reduced AMPA-induced damage in SHRs (P<0.01 vs vehicle). CONCLUSIONS: These findings show that an increase in phosphorylated GluR1, which increases AMPA receptor conductance, may be involved in the vulnerability of SHRs to AMPA.

Long-term alterations in mu, delta and kappa opioidergic receptors following middle cerebral artery occlusion in mice.

Alterations in the opioidergic system may play a role in the molecular mechanisms underlying neurochemical responses to cerebral ischaemia. The present study aimed to determine the delayed expression of mu, delta and kappa opioid receptors, following 1, 2, 7, and 30 days of middle cerebral artery occlusion (MCAO) in mice. Using quantitative autoradiography, we highlighted significant decreases in mu, delta and kappa opioid receptor expression in ipsilateral cortices from day 1 post-MCAO. Moreover, in contralateral nucleus lateralis thalami pars posterior, ipsi- and contralateral nucleus medialis dorsalis thalami, and ipsilateral substantia nigra, pars reticulata (SNr), kappa receptors were increased; mu receptor densities were decreased in nucleus ventralis thalami, pars posterior (VThP), and SNr. delta-Binding sites were increased in the striatum on day 30 post-MCAO. The alterations in opioid receptors in cortical infarcts were correlated with strong histological damage. Further reductions in opioid receptor densities in cortical infarcts were observed at later time points. In subcortical brain regions, opioid receptor densities were also altered but no histological damage was seen, except in the VThP, in which cell density was increased on day 30. Delayed reductions in opioid receptor densities in the infarct appeared as the continuation of the early processes previously demonstrated. However, changes in subcortical opioid receptor expression may correlate with neuronal alterations in remote brain regions. Changes in opioidergic receptor expression in these regions may be involved in the long-term consequences of stroke and could be used as biomarker of neuronal alteration through the use of imaging techniques in the clinic.

Delayed administration of deferoxamine reduces brain damage and promotes functional recovery after transient focal cerebral ischemia in the rat.

The mechanisms underlying functional recovery after stroke are poorly understood. Brain-adaptive responses to the hypoxic stress elicited by ischemia could contribute to these mechanisms. Indeed, hypoxia-inducible factor-1 (HIF-1), one of the main transcriptional factors regulated by oxygen level, increases the expression of several beneficial genes such as erythropoietin, glucose transporter-1 and vascular endothelial growth factor. In order to strengthen the expression of these hypoxia-inducible factors, we administered deferoxamine, an iron chelator known to stabilize HIF-1alpha protein expression, and examined its effects on the functional deficits induced by ischemia. Anesthetized Sprague-Dawley rats were subjected to 60 min of intraluminal occlusion of the middle cerebral artery. Chronic deferoxamine treatment (300 mg/kg, s.c.), or its vehicle, started 24 h after ischemia and was continued bi-weekly until the animals were killed. Sensorimotor deficits were periodically assessed over 2 months, and at this end point, the lesion volume was determined by histology. Treatment with deferoxamine significantly decreased the size of brain damage (-28%) after ischemia and improved behavioral recovery. Indeed, neurological score and sensorimotor performances in the adhesive removal test recovered earlier in the deferoxamine-treated animals. Moreover, the long-lasting skilled forepaw reaching deficits were attenuated by deferoxamine. Although an antioxidant effect of deferoxamine cannot be excluded, the hypothesis that its beneficial effects could be mediated by an increase in HIF-1 target genes merits further investigations. Our data suggest that delayed administration of deferoxamine could represent an interesting therapeutical approach to treat focal cerebral ischemia.

NMDA receptor activation inhibits alpha-secretase and promotes neuronal amyloid-beta production.

Acute brain injuries have been identified as a risk factor for developing Alzheimer's disease (AD). Because glutamate plays a pivotal role in these pathologies, we studied the influence of glutamate receptor activation on amyloid-beta (Abeta) production in primary cultures of cortical neurons. We found that sublethal NMDA receptor activation increased the production and secretion of Abeta. This effect was preceded by an increased expression of neuronal Kunitz protease inhibitory domain (KPI) containing amyloid-beta precursor protein (KPI-APP) followed by a shift from alpha-secretase to beta-secretase-mediated APP processing. This shift is a result of the inhibition of the alpha-secretase candidate tumor necrosis factor-alpha converting enzyme (TACE) when associated with neuronal KPI-APPs. This KPI-APP/TACE interaction was also present in AD brains. Thus, our findings reveal a cellular mechanism linking NMDA receptor activation to neuronal Abeta secretion. These results suggest that even mild deregulation of the glutamatergic neurotransmission may increase Abeta production and represent a causal risk factor for developing AD.

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.

Tissue-type plasminogen activator crosses the intact blood-brain barrier by low-density lipoprotein receptor-related protein-mediated transcytosis.

BACKGROUND: Accumulating evidence demonstrates a critical involvement of tissue-type plasminogen activator (tPA) in pathological and physiological brain conditions. Determining whether and how vascular tPA can cross the blood-brain barrier (BBB) to enter the brain is thus important, not only during stroke but also in physiological conditions. METHODS AND RESULTS: In the present work, we provide evidence in vivo that intravenous injection of tPA increases NMDA-induced striatal lesion in the absence of BBB leakage. Accordingly, we show that tPA crosses the BBB both after excitotoxic lesion and in control conditions. Indeed, vascular injected tPA can be detected within the brain parenchyma and in the cerebrospinal fluid. By using an in vitro model of BBB, we have confirmed that tPA can cross the intact BBB. Its passage was blocked at 4 degrees C, was saturable, and was independent of its proteolytic activity. We have shown that tPA crosses the BBB by transcytosis, mediated by a member of the LDL receptor-related protein family. CONCLUSIONS: We demonstrate that blood-derived tPA can reach the brain parenchyma without alteration of the BBB. The molecular mechanism of the passage of tPA from blood to brain described here could represent an interesting target to improve thrombolysis in stroke.