Simon Roussel

A Potential Role for Erythropoietin in Focal Permanent Cerebral Ischemia in Mice

The present study describes, for the first time, a temporal and spatial cellular expression of erythropoietin (Epo) and Epo receptor (Epo-R) with the evolution of a cerebral infarct after focal permanent ischemia in mice. In addition to a basal expression of Epo in neurons and astrocytes, a postischemic Epo expression has been localized specifically to endothelial cells (1 day), microglia/macrophage-like cells (3 days), and reactive astrocytes (7 days after occlusion). Under these conditions, the Epo-R expression always precedes that of Epo for each cell type. These results support the hypothesis that there is a continuous formation of Epo, with its corresponding receptor, during the active evolution of a focal cerebral infarct and that the Epo/Epo-R system might be implicated in the processes of neuroprotection and restructuring (such as angiogenesis and gliosis) after ischemia. To support this hypothesis, a significant reduction in infarct volume (47%; P < 0.0002) was found in mice treated with recombinant Epo 24 hours before induction of cerebral ischemia. Based on the above, we propose that the Epo/Epo-R system is an endogenous mechanism that protects the brain against damages consequent to a reduction in blood flow, a mechanism that can be amplified by the intracerebroventricular application of exogenous recombinant Epo.

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.

A Transforming Growth Factor-β Antagonist Unmasks the Neuroprotective Role of This Endogenous Cytokine in Excitotoxic and Ischemic Brain Injury

Various studies describe increased concentrations of transforming growth factor-β (TGF-β) in brain tissue after acute brain injury. However, the role of endogenously produced TGF-β after brain damage to the CNS remains to be clearly established. Here, the authors examine the influence of TGF-β produced after an episode of cerebral ischemia by injecting a soluble TGF-β type II receptor fused with the Fc region of a human immunoglobulin (TβRIIs-Fc). First, this molecular construct was characterized as a selective antagonist of TGF-β. Then, the authors tested its ability to reverse the effect of TGF-β 1 on excitotoxic cell death in murine cortical cell cultures. The addition of 1 μg/mL of TβRIIs-Fc to the exposure medium antagonized the neuroprotective activity of TGF-β 1 in N-methyl-D-aspartate (NMDA)-induced excitotoxic cell death. These results are consistent with the hypothesis that TGF-β 1 exerts a negative modulatory action on NMDA receptor-mediated excitotoxicity. To determine the role of TGF-β 1 produced in response to brain damage, the authors used a model of an excitotoxic lesion induced by the intrastriatal injection of 75 nmol of NMDA in the presence of 1.5 μg of TβRIIs-Fc. The intrastriatal injection of NMDA was demonstrated to induce an early upregulation of the expression of TGF-β 1 mRNA. Furthermore, when added to the excitotoxin, TβRIIs-Fc increased (by 2.2-fold, P < 0.05) the lesion size. These observations were strengthened by the fact that an intracortical injection of TβRIIs-Fc in rats subjected to a 30-minute reversible cerebral focal ischemia aggravated the volume of infarction. In the group injected with the TGF-β 1 antagonist, a 3.5-fold increase was measured in the infarction size (43.3 ± 9.5 versus 152.8 ± 46.3 mm3; P < 0.05). In conclusion, by antagonizing the influence of TGF-β in brain tissue subjected to excitotoxic or ischemic lesion, the authors markedly exacerbated the resulting extent of necrosis. These results suggest that, in response to such insults, brain tissue responds by the synthesis of a neuroprotective cytokine, TGF-β1, which is involved in the limitation of the extent of the injury. The pharmacologic potentiation of this endogenous defensive mechanism might represent an alternative and novel strategy for the therapy of hypoxic-ischemic cerebral injury.

In vivo MRI tracking of exogenous monocytes/macrophages targeting brain tumors in a rat model of glioma

This study has shown that murine monocytes/macrophages (Mo/Ma) can be labeled simply and efficiently with large, green-fluorescent, micrometer-sized particles of iron-oxide (MPIO). Neither size nor proliferation rate of the Mo/Ma is significantly affected by this labeling. The labeled Mo/Ma have been administered intravenously to rats that had developed a glioma following stereotactic injection of C6 cells. The labeled Mo/Ma were shown to target the brain tumors, a process that could be monitored non-invasively using T2*-weighted MRI. MRI observations were confirmed by Prussian blue staining, lectin staining and fluorescence histology. Overall, the results of this study suggest that the use of Mo/Ma may be envisaged in the clinic for vectorizing therapeutic agents toward gliomas.

Delayed Hypoxic Postconditioning Protects Against Cerebral Ischemia in the Mouse

Background and Purpose— Inspired from preconditioning studies, ischemic postconditioning, consisting of the application of intermittent interruptions of blood flow shortly after reperfusion, has been described in cardiac ischemia and recently in stroke. It is well known that ischemic tolerance can be achieved in the brain not only by ischemic preconditioning, but also by hypoxic preconditioning. However, the existence of hypoxic postconditioning has never been reported in cerebral ischemia. Methods— Adult mice subjected to transient middle cerebral artery occlusion underwent chronic intermittent hypoxia starting either 1 or 5 days after ischemia and brain damage was assessed by T2-weighted MRI at 43 days. In addition, we investigated the potential neuroprotective effect of hypoxia applied after oxygen glucose deprivation in primary neuronal cultures. Results— The present study shows for the first time that a late application of hypoxia (5 days) after ischemia reduced delayed thalamic atrophy. Furthermore, hypoxia performed 14 hours after oxygen glucose deprivation induced neuroprotection in primary neuronal cultures. We found that hypoxia-inducible factor-1α expression as well as those of its target genes erythropoietin and adrenomedullin is increased by hypoxic postconditioning. Further studies with pharmacological inhibitors or recombinant proteins for erythropoietin and adrenomedullin revealed that these molecules participate in this hypoxia postconditioning-induced neuroprotection. Conclusions— Altogether, this study demonstrates for the first time the existence of a delayed hypoxic postconditioning in cerebral ischemia and in vitro studies highlight hypoxia-inducible factor-1α and its target genes, erythropoietin and adrenomedullin, as potential effectors of postconditioning.

The ischaemic penumbra.

The concept of an ischaemic penumbra, surrounding a focal cerebral lesion, is now widely accepted, although no universal definition of the ‘penumbra’ exists. In the present review, we consider the penumbra as that volume of brain tissue at the periphery of a focal, irreversibly damaged area that is threatened by recruitment into necrosis. Implicit to such a definition are several secondary concepts. First, the penumbra is both spatial, in that it surrounds the densely ischaemic core, but it is also temporal, in that its evolution toward infarction is a relatively progressive phenomenon. The pertinent literature is summarized. Second, penumbral tissue is potentially salvageable; the most recent animal studies are reviewed. Third, because electrically silent and pathologically damaged tissues have identical functional characteristics, it is evident that most clinical rating scales, be they neurological, behavioural, or psychological, are poorly adapted to address the problem of the penumbra. Finally, the penumbral tissue is remarkably and intensively ‘active’: multiple processes of cell death and repair occur and involve molecular mechanisms, electrophysiology and the vasculature.

Selective Blockade of Endothelin-B Receptors Exacerbates Ischemic Brain Damage in the Rat

Background and Purpose— Endothelins act through 2 receptors, namely, ETA and ETB. In the cerebral circulation, ETA mediates marked and prolonged vasoconstriction, and its blockade increases cerebral blood flow (CBF) and reduces ischemic brain damage. However, the role of ETB receptors remains unclear. In this study we examined, in rats, the kinetics of expression of ETB and the effects of ETB 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 ETB 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 ETB expression. Results— Cerebral ischemia failed to alter the expression of ETB 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 ETB 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 ETA antagonists and not mixed ETA/ETB antagonists.

The nature of penumbral depolarizations following focal cerebral ischemia in the rat

It has been previously suggested that the transient ischemic depolarizations (IDs), thought involved in the gradual expansion of ischemic injury in the first hours following middle cerebral artery occlusion (MCAo), are akin to spreading depression (SD). However, previous studies indicate that the characteristics of these events are heterogeneous (unlike those of SDs). We therefore sought to determine whether different types of IDs exist or not. Using four cortical microelectrodes, we compared the spatial and the temporal characteristics of IDs that occur following intraluminal MCAo in halothane-anesthetized rats to those of electrically induced SDs. An average 4.6+/-3.2 series of events, sequentially affecting the four electrodes, were recorded in 5 h following the induction of ischemia. The distribution of ID duration disclosed two types: short IDs (<7 min, 53% of all events) and long IDs (>7 min; 9% of all events). Most long IDs occurred within the first 30 min and as the initial electrophysiological event. Later on and often restricted to a single or reduced number of recording sites, intermittent IDs were of reduced amplitude or even replaced entirely by suppressed electrocorticographic activity (38% of all events). While the amplitude, duration and spreading characteristics were similar between short IDs and SDs provoked in the cortex of non-ischemic rats, those of long IDs were markedly different. Our results indicate that two types of IDs exist and confirm that most IDs (short ones) are similar in nature to SDs. Long IDs may represent a penumbral anoxic depolarization (AD), reversed by an improvement of perfusion, in the early stages of ischemia. Furthermore, we show that intermittent blockade of depolarization waves occurs and that its incidence increases with time. This blockade may reflect adaptive mechanisms which take place to prevent further depolarizations, the nature of which remains to be determined. The present description of electrophysiological abnormalities might have implications for anti-depolarization therapy in focal cerebral ischemia and to interpret the results of non-invasive techniques which enable the imaging of depolarized areas following stroke.

Crosstalk between HIF-1 and ROCK pathways in neuronal differentiation of mesenchymal stem cells, neurospheres and in PC12 neurite outgrowth.

This study demonstrates that the Rho-kinase (ROCK) inhibitor, Y-27632, potentiates not only the effect of cobalt chloride (CoCl(2)) but also that of deferoxamine, another HIF-1 inducer, on mesenchymal stem cell (MSC) neuronal differentiation. HIF-1 is essential for CoCl(2)+/-Y-27632-induced MSC neuronal differentiation, since agents inhibiting HIF-1 abolish the changes of morphology and cell cycle arrest-related gene or protein expressions (p21, cyclin D1) and the increase of neuronal marker expressions (Tuj1, NSE). Y-27632 potentiates the CoCl(2)-induced decrease of cyclin D1 and nestin expressions, the increase of HIF-1 activation and EPO expression, and decreases pVHL expression. Interestingly, CoCl(2) decreases RhoA expression, an effect potentiated by Y-27632, revealing crosstalk between HIF-1 and RhoA/ROCK pathways. Moreover, we demonstrate a synergistic effect of CoCl(2) and Y-27632 on neurosphere differentiation into neurons and PC12 neurite outgrowth underlining that a co-treatment targeting both HIF-1 and ROCK pathways might be relevant to differentiate stem cells into neurons.

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.