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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 p t O 2 from [ 18 F]-FMISO images in glioblastoma and less aggressive glioma patients in order to quantitatively assess tumor hypoxia. An ancillary objective was to compare estimated p t O 2 values to other biomarkers: perfusion weighted imaging (PWI) and tumor metabolism obtained from 1 H-MR mono-voxel spectroscopy (MRS). Ten patients with glioblastoma (GBM) and three patients with less aggressive glioma (nGBM) were enrolled. All patients had [ 18 F]-FMISO and multiparametric MRI (anatomic, PWI, MRS) scans. A non-linear regression was performed to generate p t O 2 maps based on normal appearing gray (NAGM) and white matter (NAWM) for each patient. As expected, a marked [ 18 F]-FMISO uptake was observed in GBM patients. The p t O 2 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 p t O 2 maps, PWI-derived rCBV maps and MRS-derived lipid and lactate concentration strengthens the robustness of our approaches.

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 (S t O 2 -MRI) and the identification of hypoxia by a positron emission tomography (PET) biomarker with 3-[ 18 F]fluoro-1-(2-nitro-1-imidazolyl)-2-propanol ([ 18 F]-FMISO) as the leading radiopharmaceutical. Nonetheless, a formal validation of S t O 2 -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, S t O 2 -MRI and [ 18 F]-FMISO PET were performed sequentially. Under hypoxemia conditions, S t O 2 -MRI revealed a decrease in oxygen saturation in the brain. Nonetheless, [ 18 F]-FMISO PET, pimonidazole immunohistochemistry and molecular biology were insensitive to hypoxia. Within the context of tumours, S t O 2 -MRI was able to detect hypoxia in the hypoxic models, mimicking [ 18 F]-FMISO PET with high sensitivity/specificity. Altogether, our data clearly support that, in brain pathologies, S t O 2 -MRI could be a robust and specific imaging biomarker to assess hypoxia.

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.

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.

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.

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.

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 (O 2 ) availability could be a major regulator of this transition and that the intratumoral O 2 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.

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.

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., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Neurotrophins are a family of growth factors that attenuate several forms of pathological neuronal cell death and may represent a putative therapeutic approach to neurodegenerative diseases. In Alzheimer disease, amyloid-beta (Abeta) is thought to play a central role in the neuronal death occurring in brains of patients. In the present study, we evaluate the ability of neurotrophin-3 (NT-3) to protect neurons against the toxicity induced by aggregated Abeta. We showed that in primary cultures of cortical neurons, NT-3 reduces Abeta-induced apoptosis by limiting caspase-8, caspase-9, and caspase-3 cleavage. This neuroprotective effect of NT-3 was concomitant to an increased level of Akt phosphorylation and was abolished by an inhibitor of the phosphatidylinositol-3 kinase (PI-3K), LY294002. In parallel, NT-3 treatment reduced Abeta induced caspase-3 processing to control levels. In an attempt to link PI-3K/Akt to caspase inhibition, we evaluated the influence of the PI-3K/Akt axis on the expression of a member of the inhibitors of apoptosis proteins (IAPs), the neuronal apoptosis inhibitory protein-1. We demonstrated that NT-3 induces an up-regulation of neuronal apoptosis inhibitory protein-1 expression in neurons that promotes the inhibition of Abeta-induced neuronal apoptosis. Together, these findings demonstrate that NT-3 signaling counters Abeta-dependent neuronal cell death and may represent an innovative therapeutic intervention to limit neuronal death in Alzheimer disease.

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.

The effects of sodium nitroprusside (SNP), a potent hypotensive agent, on cerebral blood flow (CBF) have been extensively studied in clinical and experimental situations but the results remain controversial. Whereas its properties would predict a dilatation of cerebral blood vessels, most studies report either no change or a decrease in CBF. The aim of this study was to investigate the effects of SNP on CBF, cerebral blood volume (CBV), and cerebral oxygen metabolism (CMRO2), by means of positron emission tomography in the anaesthetized baboon. Measurements were performed during normotension (mean arterial pressure (MABP): 97+/-16 mm Hg) and repeated following SNP-induced hypotension (MABP: 44+/-9 mm Hg). Sodium nitroprusside led to an increase in CBF and CBV (+30% and +37%, respectively, P<0.05), whereas no change in CMRO2 was noted. Linear regression analysis of CBF values as a function of MABP confirmed that CBF increases when MABP is reduced by SNP. The comparison between these cerebrovascular changes and those found during trimetaphan-induced hypotension in our previously published studies further argues for a direct dilatatory effect of SNP on cerebral blood vessels.

Tissue-type plasminogen activator (tPA) has been involved in both physiological and pathological glutamatergic-dependent processes, such as synaptic plasticity, seizure, trauma, and stroke. In a previous study, we have shown that the proteolytic activity of tPA enhances the N-methyl-D-aspartate (NMDA) receptor-mediated signaling in neurons (Nicole, O., Docagne, F., Ali, C., Margaill, I., Carmeliet, P., MacKenzie, E. T., Vivien, D., and Buisson, A. (2001) Nat. Med. 7, 59-64). Here, we show that tPA forms a direct complex with the amino-terminal domain (ATD) of the NR1 subunit of the NMDA receptor and cleaves this subunit at the arginine 260. Furthermore, point mutation analyses show that arginine 260 is necessary for both tPA-induced cleavage of the ATD of NR1 and tPA-induced potentiation of NMDA receptor signaling. Thus, tPA is the first binding protein described so far to interact with the ATD of NR1 and to modulate the NMDA receptor function.

The present work aimed 1) to evaluate whether an increase in galanin or galanin receptors could be induced in the nucleus basalis magnocellularis (nbm) by degeneration of the basalocortical neurons from the cortex and 2) to analyze the consequences of such an increase on cortical activity. First, a mild ischemic insult to the frontoparietal cortex was performed to induce the degeneration of the basalocortical system; galanin immunoreactivity, galanin binding sites, and cholinergic muscarinic receptors were quantified through immunocytochemistry and autoradiography. Second, galanin infusions in the nbm were undertaken to mimic a local increase of the galaninergic innervation; cortical acetylcholine release, cerebral glucose use, and cerebral blood flow were then measured as indices of cortical activity. As a result of the cortical ischemic lesion, the postsynaptic M1 and presynaptic M2 muscarinic receptors were found to be reduced in the altered cortex. In contrast, galaninergic binding capacity and fiber density were found to be increased in the ipsilateral nbm in parallel with a local decrease in the cholinergic markers such as the muscarinic M1 receptor density. Galanin infusion into the nbm inhibited the cortical acetylcholine release and cerebral blood flow increases elicited by the activation of the cholinergic basalocortical system but failed to affect acetylcholine release, cerebral blood flow, and cerebral glucose use when injected alone in the nbm. These results demonstrate that degeneration of the basalocortical system from the cortex induces an increase in galaninergic markers in the nbm, a result that might suggest that the galaninergic overexpression described in the basal forebrain of patients with Alzheimer's disease can result from a degeneration of the cholinergic basalocortical system from the cortex. Because galanin was found to reduce the activity of the basalocortical cholinergic system only when this one is activated, galanin might exert its role rather during activation deficits than under resting conditions such as the resting cortical hypometabolism, which is characteristic of Alzheimer's disease.

Abnormal deposition of Abeta (amyloid-beta peptide) is one of the hallmarks of AD (Alzheimer's disease). This peptide results from the processing and cleavage of its precursor protein, APP (amyloid-beta precursor protein). We have demonstrated previously that TGF-beta (transforming growth factor-beta), which is overexpressed in AD patients, is capable of enhancing the synthesis of APP by astrocytes by a transcriptional mechanism leading to the accumulation of Abeta. In the present study, we aimed at further characterization of the molecular mechanisms sustaining this TGF-beta-dependent transcriptional activity. We report the following findings: first, TGF-beta is capable of inducing the transcriptional activity of a reporter gene construct corresponding to the +54/+74 region of the APP promoter, named APP(TRE) (APP TGF-beta-responsive element); secondly, although this effect is mediated by a transduction pathway involving Smad3 (signalling mother against decapentaplegic peptide 3) and Smad4, Smad2 or other Smads failed to induce the activity of APP(TRE). We also observed that the APP(TRE) sequence not only responds to the Smad3 transcription factor, but also the Sp1 (signal protein 1) transcription factor co-operates with Smads to potentiate the TGF-beta-dependent activation of APP. TGF-beta signalling induces the formation of nuclear complexes composed of Sp1, Smad3 and Smad4. Overall, the present study gives new insights for a better understanding of the fine molecular mechanisms occurring at the transcriptional level and regulating TGF-beta-dependent transcription. In the context of AD, our results provide additional evidence for a key role for TGF-beta in the regulation of Abeta production.

Tissue-type plasminogen activator (tPA) is available for the treatment of thromboembolic stroke in humans. However, adverse effects of tPA have been observed in animal models of ischemic brain injuries. In the present study, we have used a synthetic tPA inhibitor, named 2,7-bis-(4-amidino-benzylidene)-cycloheptan-1-one dihydrochloride (tPA stop), to investigate the role of endogenous tPA in the cerebral parenchyma. In mouse cortical cell cultures, we observed that although tPA stop reduced N-methyl-D-aspartic acid (NMDA)-mediated excitotoxic neuronal death, it failed to modulate alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole propanoic acid or kainate-mediated necrosis. In addition, we found that tPA stop could prevent the deleterious effects of both endogenous and exogenous tPA during NMDA exposure. At the functional level, tPA stop was found to prevent tPA-dependent potentiation of NMDA receptor-evoked calcium influx. The relevance of those findings was strengthened by the observation of a massive reduction of NMDA-induced excitotoxic lesion in rats when tPA stop was co-injected. Altogether, these data demonstrate that the blockade of the endogenous proteolytic activity of tPA in the cerebral parenchyma could be a powerful neuroprotective strategy raised against brain pathologies associated with excitotoxicity.

A number of cytokines including neurotrophins have been tested for their neuroprotective activity against different paradigms of neuronal death. However, as for neurotrophin-3 (NT-3), their mechanisms of action have not been fully identified. By using cultures of mouse cortical neurons, we have investigated the molecular mechanisms by which neurotrophin-3 could protect cortical neurons against apoptosis. In a model of caspase-dependent apoptosis leading to the recruitment of active initiators caspase-8 and -9 and of executioner caspase-3, we have evidenced that NT-3 displayed an anti-apoptotic effect in a dose-dependent manner. First, we showed that, in cultured cortical neurons, NT-3 could promote extracellular signal-regulated protein kinase/mitogen-activated protein kinase (ERK/MAPK) and phosphatidylinositol-3' (PI-3) kinase/Akt phosphorylation. Second, we showed that although the blockade of the Akt pathway prevented the anti-apoptotic effect of NT-3, blockade of the ERK pathway did not. Altogether, our data demonstrate that NT-3 displayed an anti-apoptotic effect on cultured cortical neurons through a mechanism involving the recruitment of the PI-3 kinase/Akt signaling pathway.

In the last few years, it has been evidenced that serine proteases play key roles in the mammalian brain, both in physiological and pathological conditions. It has been well established that among these serine proteases, the tissue-type plasminogen activator (t-PA) is critically involved in development, plasticity, and pathology of the nervous system. However, its mechanism of action remains to be further investigated. By using pharmacological and immunological approaches, we have evidenced in the present work that t-PA should be considered as a neuromodulator. Indeed, we have observed that: (i). neuronal depolarization induces a release of t-PA; (ii). this release of t-PA is sensitive to exocytosis inhibition and calcium chelation; (iii). released t-PA modulates NMDA receptor signaling and (iv). astrocytes are able to recapture extracellular t-PA through a low-density lipoprotein (LDL) receptor-related protein (LRP)-dependent mechanism.

1. Necrosis and apoptosis are the two fundamental hallmarks of neuronal death in stroke. Nevertheless, thrombolysis, by using the recombinant serine protease t-PA, remains until now the only approved treatment of stroke in man. 2. Over the last years, the cytokine termed Transforming Growth Factor-beta1 (TGF-beta1) has been found to be strongly up-regulated in the central nervous system following ischemia-induced brain damage. 3. Recent studies have shown a neuroprotective activity of TGF-beta1 against ischemia-induced neuronal death. In vitro, TGF-beta1 protects neurons against excitotoxicity by inhibiting the t-PA-potentiated NMDA-induced neuronal death through a mechanism involving the up-regulation of the type-1 plasminogen activator inhibitor (PAI-1) in astrocytes 4. In addition, TGF-beta1 has been recently characterized as an antiapoptotic factor in a model of staurosporine-induced neuronal death through a mechanism involving activation of the extracellular signal-regulated kinase 1/2 (Erk1/2) and a concomitant increase phosphorylation of the antiapoptotic protein Bad. 5. Altogether, these observations suggest that either TGF-beta signaling or TGF-beta1-modulated genes could be good targets for the development of new therapeutic strategies for stroke in man.

Neuronal death after ischemia-induced brain damage depends largely upon the activation of the N-methyl-D-aspartate (NMDA) excitatory glutamate receptor that is a target for many putative neuroprotective agents. Whereas the NMDA receptors mediate ischemic brain damage, blocking them is deleterious in humans. Here, the authors investigated whether nitrous oxide or xenon, which are gaseous anesthetics with a remarkably safe clinical profile that have been recently demonstrated as effective inhibitors of the NMDA receptor, may reduce the following: (1) ischemia-induced brain damage in vivo, when given after occlusion of the middle cerebral artery (MCAO), a condition needed to make these potentially neuroprotective agents therapeutically valuable; or (2) NMDA-induced Ca2+ influx in cortical cell cultures, a major critical event involved in excitotoxic neuronal death. The authors have shown that both nitrous oxide at 75 vol% and xenon at 50 vol% reduce ischemic neuronal death in the cortex by 70% and further decrease NMDA-induced Ca2+ influx by 30%. In addition, xenon at 50%, but not nitrous oxide at 75 vol%, further decreases ischemic brain damage in the striatum (a subcortical structure that is known to be resistant to neuroprotective interventions). However, at a higher concentration (75 vol%), xenon exhibits potentially neurotoxic effects. The mechanisms of the neuroprotective and potentially neurotoxic effects of nitrous oxide and xenon, as well as the possible therapeutic implications in humans, are discussed.

Background: We studied the potential neuroprotective effects of halothane and desflurane, compared with the awake state, on infarct size following 2 h of intraluminal middle cerebral artery occlusion (MCAo) and 22 h of reperfusion., Methods: Male Sprague-Dawley rats were anaesthetized with desflurane or halothane, intubated, and mechanically ventilated. Mean arterial pressure (MAP), blood gases, and pH were controlled. Body temperature was maintained at 37.5-38 degrees C. Animals were assigned to one of four groups according to the anaesthetic type (halothane or desflurane) and the duration of anaesthesia: "short-duration", during the preparation only; "long-duration", during both preparation and ischaemia. Twenty-four hours after MCAo, infarcts were visualized by staining with 2,3,5-triphenyltetrazolium chloride. Two additional groups of rats were subjected to the same protocol as that of long-duration halothane and long-duration desflurane with additional pericranial temperature measurements made., Results: Physiological parameters were comparable between the groups but MAP was higher (P<0.0001) in the short-duration groups. In the short-duration groups, cerebral infarct volumes were not significantly different between anaesthetics (short-duration halothane: 288 (61) mm(3), mean (SD); short-duration desflurane: 269 (71) mm(3), P>0.56). Compared with the awake state (short-duration groups), halothane and desflurane significantly reduced infarct volumes (long-duration halothane: 199 (54) mm(3), P<0.0047 vs short-duration halothane; long-duration desflurane: 121 (55) mm(3), P<0.0001 vs short-duration desflurane). The mean infarct volume in the long-duration desflurane group was significantly lower than that in the long-duration halothane group (P<0.0053). Pericranial temperatures were similar in the desflurane and halothane long-duration groups (P>0.17)., Conclusions: In rats, desflurane-induced neuroprotection against focal cerebral ischaemia was greater than that conferred by halothane.

We previously reported differential alterations of the mu, delta, and kappa opioid receptors following permanent middle cerebral artery occlusion. The present work studied the evolution of opioid receptor types following transient focal cerebral ischemia (tMCAO), as well as the putative predictive potential of early neurochemical alterations on the delayed ischemic damage. delta receptors were significantly decreased as early as 6 h post tMCAO (-22% approximately -57% vs. sham group), followed by a decrease in the mu binding site density at 24 h post tMCAO (-18% approximately -65%), in infarcted and penumbral cortices. Finally, early decreases in cortical opioid mu and delta receptor densities were found to significantly correlate (P < 0.001, r(2) = 0.48 and 0.75, respectively) with the occurrence of delayed histological damage. The high correlation between decreases in mu and delta receptor densities at 6 h post tMCAO and the histological damage that occurred at 24 h post tMCAO suggests that these early neurochemical alterations could be used as predictive markers of delayed ischemic damage.

Accumulation of the amyloid-beta peptide (Abeta) in the brain is crucial for development of Alzheimer's disease. Expression of transforming growth factor-beta1 (TGF-beta1), an immunosuppressive cytokine, has been correlated in vivo with Abeta accumulation in transgenic mice and recently with Abeta clearance by activated microglia. Here, we demonstrate that TGF-beta1 drives the production of Abeta40/42 by astrocytes leading to Abeta production in TGF-beta1 transgenic mice. First, TGF-beta1 induces the overexpression of the amyloid precursor protein (APP) in astrocytes but not in neurons, involving a highly conserved TGF-beta1-responsive element in the 5'-untranslated region (+54/+74) of the APP promoter. Second, we demonstrated an increased release of soluble APP-beta which led to TGF-beta1-induced Abeta generation in both murine and human astrocytes. These results demonstrate that TGF-beta1 potentiates Abeta production in human astrocytes and may enhance the formation of plaques burden in the brain of Alzheimer's disease patients.

The synthesis, pharmacological evaluation and modelisation of 7-methoxyindazole (7-MI) and related alkoxyindazoles as novel inhibitors of neuronal nitric oxide synthase are presented. 7-MI remains the most active compound of this series in an in vitro enzymatic assay of neuronal nitric oxide synthase activity. Modeling studies of the interaction of 7-substituted indazole derivatives complexed with nNOS and the relationship with their respective biological activities suggest that a bulky substitution on position-7 is responsible for a steric hindrance effect which does not allow these compounds to interact with nNOS in the same way as 7-NI and 7-MI.

Although angiopoietin-1 (Ang-1) is recognized as an endothelial growth factor, its presence in brain following an ischemic event suggests a role in the evolution of neuronal damage. Using primary neuronal cultures, we showed that neurons express Ang-1 and possess the functional angiopoietin-receptor Tie-2, which is phosphorylated in the presence of Ang-1. We further investigated in vitro whether Ang-1 could protect neurons against either excitotoxic necrosis or apoptosis induced by serum deprivation (SD). A neuroprotective effect for Ang-1 was detected exclusively in the apoptotic paradigm. Treatment of cells with the phosphatidyl-inositol 3-kinase (PI3-K) inhibitor, LY294002, inhibited Ang-1-induced phosphorylation of Akt, restored the cleavage of the effector caspase-3, and reduced the protective effect of Ang-1 against SD-induced toxicity. These findings suggest that Ang-1 has a neuroprotective effect against apoptotic stress and that this effect is dependent on the PI3-K/Akt pathway and inhibition of caspase-3 cleavage. This study provides evidence that Ang-1 is not just angiogenic but also neuroprotective. The understanding of neuroprotective mechanisms induced by Ang-1 may promote strategies based on the pleiotropic effects of angiogenic factors. Such approaches could be useful for the treatment of brain diseases in which both neuronal death and angiogenesis are involved.

Although transforming growth factor (TGF)-alpha, a member of the epidermal growth factor (EGF) family, has been shown to protect neurons against excitotoxic and ischemic brain injuries, its mechanism of action remains unknown. In the present study, we used in vitro models of apoptotic or necrotic paradigms demonstrating that TGF-alpha rescues neurons from N-methyl-D-aspartate (NMDA)-induced excitotoxic death, with the obligatory presence of astrocytes. Because neuronal tissue-type plasminogen activator (t-PA) release was shown to potentiate NMDA-induced excitotoxicity, we observed that TGF-alpha treatment reduced NMDA-induced increase of t-PA activity in mixed cultures of neurons and astrocytes. In addition, we showed that although TGF-alpha induces activation of the extracellular signal-regulated kinases (ERKs) in astrocytes, it failed to activate p42/p44 in neurons. Finally, we showed that TGF-alpha, by an ERK-dependent mechanism, stimulates the astrocytic expression of PAI-1, a t-PA inhibitor, which mediates the neuroprotective activity of TGF-alpha against NMDA-mediated excitotoxic neuronal death. Taken together, we indicate that TGF-alpha rescues neurons from NMDA-induced excitotoxicity in mixed cultures through inhibition of t-PA activity, involving PAI-1 overexpression by an ERK-dependent pathway in astrocytes.

Necrosis and apoptosis are the two fundamental hallmarks of neuronal death in stroke. Nevertheless, thrombolysis, by means of the recombinant serine protease t-PA, remains until now the only approved treatment of stroke in man. Over the last years, the cytokine termed Transforming Growth Factor-beta 1 (TGF-beta 1) has been found to be strongly up regulated in the central nervous system following ischemia-induced brain damage. Recent studies have shown a neuroprotective activity of TGF-beta 1 against ischemia-induced neuronal death. In vitro, TGF-beta 1 protects neurons against excitotoxicity by inhibiting the t-PA-potentiated NMDA-induced neuronal death through a mechanism involving the up-regulation of the type-1 plasminogen activator inhibitor (PAI-1) in astrocytes. Altogether, these observations suggest that either TGF-beta signaling or TGF-beta 1-modulated genes could be good targets for the development of new therapeutic strategies for stroke in man.

Background and Purpose: Endothelins act through 2 receptors, namely, ET(A) and ET(B). In the cerebral circulation, ET(A) mediates marked and prolonged vasoconstriction, and its blockade increases cerebral blood flow (CBF) and reduces ischemic brain damage. However, the role of ET(B) receptors remains unclear. In this study we examined, in rats, the kinetics of expression of ET(B) and the effects of ET(B) blockade on changes in CBF and brain damage after focal cerebral ischemia and N-methyl-D-aspartate (NMDA)-induced excitotoxic injury., Methods: Rats were subjected to transient (60 minutes) focal cerebral ischemia or cortical injection of NMDA. The selective ET(B) antagonist BQ-788 was injected intracerebroventricularly 30 minutes before and 30 minutes after the onset of ischemia. Cortical perfusion was monitored by laser-Doppler flowmetry. The volume of infarction or NMDA-induced cortical lesion was assessed at 24 hours after the insult. The reverse transcription-polymerase chain reaction technique was used to assess ET(B) expression., Results: Cerebral ischemia failed to alter the expression of ET(B) mRNA in both acute and chronic stages. Administration of BQ-788 resulted in an increase in infarction volume (178%; P<0.05) accompanied by a decrease in residual CBF (-26.7% versus control; P<0.01). In these animals we found a positive correlation between the volume of infarction and the severity of the decrease in CBF. NMDA-induced cortical lesions were not affected by the administration of BQ-788., Conclusions: Our results suggest that the ET(B) antagonist BQ-788 induces deleterious effects that are mediated by the reduction of residual blood flow after ischemia and argue that the optimal therapeutic strategy in stroke would be to target the use of selective ET(A) antagonists and not mixed ET(A)/ET(B) antagonists.