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.

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.

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.

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.

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.

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.

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.

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.

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.

Cerebrovascular effects of sodium nitroprusside in the anaesthetized baboon: a positron emission tomographic study.

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.

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.

Transforming growth factor alpha-induced expression of type 1 plasminogen activator inhibitor in astrocytes rescues neurons from excitotoxicity.

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.

Sp1 and Smad transcription factors co-operate to mediate TGF-beta-dependent activation of amyloid-beta precursor protein gene transcription.

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.

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.

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.

Penumbral microcirculatory changes associated with peri-infarct depolarizations in the rat.

BACKGROUND AND PURPOSE: This study was designed to investigate the influence of peri-infarct depolarization elicited by occlusion of the middle cerebral artery on the dynamics of the microcirculation. METHODS: The microcirculation in the frontoparietal cortex of 9 rats was visualized in real time through a closed cranial window with the use of laser-scanning confocal fluorescence microscopy combined with intravenous fluorescein isothiocyanate (FITC)-dextran and FITC-labeled erythrocytes. The direct current potential/electrocorticogram was continuously monitored. Intraluminal focal ischemia was induced for 2 hours in 6 rats anesthetized with halothane and mechanically ventilated. Reperfusion was monitored for 1 hour. Three rats underwent sham operation. Brains were removed 24 hours after occlusion and processed for histology. RESULTS: In control conditions, the velocity of fluorescent erythrocytes through capillaries was 0.51+/-0.19 mm/s (mean+/-SD), and the diameter of the arterioles studied was 33+/-12 microm. Under ischemia, erythrocyte velocity through capillaries was significantly decreased to 0.33+/-0.14 mm/s, while arteriole diameter did not change significantly. During spontaneous peri-infarct depolarizations, arteriole diameter was significantly increased (119+/-23% of baseline), while capillary erythrocyte velocity was further decreased by 14+/-34%. The direction of arteriolar blood flow episodically and transiently reversed during approximately half of the peri-infarct depolarizations. The decrease in capillary erythrocyte velocity was more pronounced (23+/-37%) in these cases. After reperfusion, the microcirculatory variables rapidly returned to baseline. All rats in the ischemic group had infarcts 24 hours after occlusion. CONCLUSIONS: Peri-infarct depolarization has an adverse influence on penumbral microcirculation, reducing capillary perfusion by erythrocytes, despite dilatation of arterioles. These findings suggest that a steal phenomenon contributes to the deleterious effect of these depolarizations.

Selective blockade of endothelin-B receptors exacerbates ischemic brain damage in the rat.

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.

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.