Jesús Pascual-Brazo

Bioluminescence imaging of stroke-induced endogenous neural stem cell response

Brain injury following stroke affects neurogenesis in the adult mammalian brain. However, a complete understanding of the origin and fate of the endogenous neural stem cells (eNSCs) in vivo is missing. Tools and technology that allow non-invasive imaging and tracking of eNSCs in living animals will help to overcome this hurdle. In this study, we aimed to monitor eNSCs in a photothrombotic (PT) stroke model using in vivo bioluminescence imaging (BLI). In a first strategy, inducible transgenic mice expressing firefly luciferase (Fluc) in the eNSCs were generated. In animals that received stroke, an increased BLI signal originating from the infarct region was observed. However, due to histological limitations, the identity and exact origin of cells contributing to the increased BLI signal could not be revealed. To overcome this limitation, we developed an alternative strategy employing stereotactic injection of conditional lentiviral vectors (Cre-Flex LVs) encoding Fluc and eGFP in the subventricular zone (SVZ) of Nestin-Cre transgenic mice, thereby specifically labeling the eNSCs. Upon induction of stroke, increased eNSC proliferation resulted in a significant increase in BLI signal between 2days and 2weeks after stroke, decreasing after 3months. Additionally, the BLI signal relocalized from the SVZ towards the infarct region during the 2weeks following stroke. Histological analysis at 90days post stroke showed that in the peri-infarct area, 36% of labeled eNSC progeny differentiated into astrocytes, while 21% differentiated into mature neurons. In conclusion, we developed and validated a novel imaging technique that unequivocally demonstrates that nestin(+) eNSCs originating from the SVZ respond to stroke injury by increased proliferation, migration towards the infarct region and differentiation into both astrocytes and neurons. In addition, this new approach allows non-invasive and specific monitoring of eNSCs over time, opening perspectives for preclinical evaluation of candidate stroke therapeutics.

Serotonin 5-HT 4 Receptors: A New Strategy for Developing Fast Acting Antidepressants?

The regulation of the activity of brain monoaminergic systems has been the focus of attention of many studies since the first antidepressant drug emerged 50 years ago. The search for novel antidepressants is deeply linked to the search for fast-acting strategies, taking into account that 2-4 weeks of treatment with classical antidepressant are required before clinical remission of the symptoms becomes evident. In the recent years several hypotheses have been proposed on the basis of the existence of alterations in brain synaptic plasticity in major depression. Recent evidences support a role for 5-HT4 receptors in the pathogenesis of depression as well as in the mechanism of action of antidepressant drugs. In fact, chronic treatment with antidepressant drugs appears to modulate, at different levels, the signaling pathway associated to 5-HT4 receptors, as well as their levels of expression in the brain. Moreover, several experimental studies have identified this receptor subtype as a promising new target for fast-acting antidepressant strategy: the administration of partial agonists of this receptor induces a number of responses similar to those observed after chronic treatment with classical antidepressants, but with a rapid onset of action. They include efficacy in behavioral models of depression, rapid desensitization of 5-HT1A autoreceptors, and modifications in the expression of several molecular markers of brain neuroplasticity. Although much work remains to be done in order to clarify the real therapeutic potential of these drugs, the evidences reviewed below support the hypothesis that 5-HT4 receptor partial agonists could behave as rapid and effective antidepressants.

Neurogenesis as a new target for the development of antidepressant drugs.

Thirteen years have passed since the neurogenic hypothesis of depression was postulated. One of its aspects, that decreased neurogenesis could be causative of the onset of depression has been difficult to prove. Another aspect, the prediction that increasing neurogenesis would not only be supportive but also required to produce clinical results by antidepressants has gathered experimental validation. Thus a question arises: should new antidepressant strategies based solely on increasing neurogenesis be pursued? At the risk of disappointing the audience, we will not provide a straight answer to this question in this review, but we do hope to enlighten the reader regarding what is known about adult hippocampal neurogenesis, the indications and evidence of its involvement in the onset and treatment of depression, and the advances that have been made in the field in recent years. As we will recount here, the main body of support in favor of the neurogenic hypothesis of depression is based more on intimation than actual proof. However the rare examples that provide support are sufficiently robust to justify investment of resources and effort to clarify the issue, even if the involvement of neurogenesis, both in the etiology and the treatment of depression, is only partial and comprises only subtle components of this complex mental disorder.

Long-Term Fate Mapping Using Conditional Lentiviral Vectors Reveals a Continuous Contribution of Radial Glia-Like Cells to Adult Hippocampal Neurogenesis in Mice

Newborn neurons are generated throughout life in two neurogenic regions, the subventricular zone and the hippocampal dentate gyrus. Stimulation of adult neurogenesis is considered as an attractive endogenous repair mechanism to treat different neurological disorders. Although tremendous progress has been made in our understanding of adult hippocampal neurogenesis, important questions remain unanswered, regarding the identity and the behavior of neural stem cells in the dentate gyrus. We previously showed that conditional Cre-Flex lentiviral vectors can be used to label neural stem cells in the subventricular zone and to track the migration of their progeny with non-invasive bioluminescence imaging. Here, we applied these Cre-Flex lentiviral vectors to study neurogenesis in the dentate gyrus with bioluminescence imaging and histological techniques. Stereotactic injection of the Cre-Flex vectors into the dentate gyrus of transgenic Nestin-Cre mice resulted in specific labeling of the nestin-positive neural stem cells. The labeled cell population could be detected with bioluminescence imaging until 9 months post injection, but no significant increase in the number of labeled cells over time was observed with this imaging technique. Nevertheless, the specific labeling of the nestin-positive neural stem cells, combined with histological analysis at different time points, allowed detailed analysis of their neurogenic potential. This long-term fate mapping revealed that a stable pool of labeled nestin-positive neural stem cells continuously contributes to the generation of newborn neurons in the mouse brain until 9 months post injection. In conclusion, the Cre-Flex technology is a valuable tool to address remaining questions regarding neural stem cell identity and behavior in the dentate gyrus.

Chronic fluoxetine regulates both 5-HT1A receptor functionality and hippocampal b-catenin pathway in an animal model of depression

P105 Administration of late endothelial progenitor cells enhances cerebral infarction outcome after transient middle cerebral artery occlusion in rats C Moubarik, JL Codaccioni, L Velly, F Sabatier, F Dignat-George, MD Piercecchi, B Guillet, P Pisano Faculté de Pharmacie, unité INSERM U 608, Marseille, France; Département d’anesthésie réanimation, Marseille, France Endothelial progenitor cells (EPCs) are a novel promising option for treatment of patients with ischemic diseases. We characterized the effect of late outgrowth EPCs transplantation in a model of transient middle cerebral artery occlusion (MCAO) in rat. EPCs were obtained from human umbilical cord blood. Adult male SpragueDawley rats were subjected to 1 h of MCAO and allocated 24 h after MCAO to transplanted (4–5 · 10 late EPCs IV, n = 12) and control (PBS IV, n = 13). Body weight, adhesive-removal dot test and the mNNS test were performed over the 14 days following MCAO. Seven or 14 days after MCAO, brain infarct volumes, human transplanted cells localization, apoptosis and capillary density staining were done. Animal body weights were significantly higher in the transplanted group 7 days after MCAO (P < 0.05). Significantly higher neurological performance was found in transplanted rats compared to control rats (P < 0.05). Immunochemical staining showed that EPCs survived and were preferentially localized in the ischemic boundary zone. No difference has been observed in term of infarct volume (P = 0.98). At day 3, apoptotic cell number was significantly lower in transplanted rats (48.0 ± 18/mm) compared to control rats (130.0 ± 50/mm) (P < 0.05). Capillary densities showed were significantly increased (P= <0.05) at the boundary of the ischemic lesion in transplanted rats (n = 4; 2.80 ± 0.3) compared to control rats (n = 4; 1.72 ± 0.7) 14 days after MCAO. Late EPCs administered intravenously 24 h after MCAO enter the brain, survive, migrate, and improve functional recovery. This may be involved ischemia-induced apoptosis together with angiogenic response modulation.