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International audience; In patients with metastatic breast cancer (MBC), brain metastases (BM) are associated with high morbidity and mortality. However, there is no validated serum biomarker that accurately predicts BM occurrence in these patients, and the role of serum biomarkers for prognosis remains unclear. Here, we evaluated the association of neurofilament light chain (NfL), ubiquitin C-terminal hydrolase L1 (UCHL1), glial fibrillary acidic protein (GFAP) and tau serum levels with BM presence and prognosis in patients with MBC. In serum samples from patients with MBC with (n = 100) and without BM (n = 47), we measured the biomarker serum levels using single molecule array (Simoa) technology (Neurology-4-Plex assay). To evaluate their accuracy to identify patients with BM, we determined the receiver operating characteristic curve and the area under the curve (AUC) for each biomarker and calculated their sensitivity and specificity. The median serum levels of NfL, UCHL1, tau and GFAP were significantly higher in patients with BM. The AUC for GFAP (0.82, 95% confidence interval [CI] 0.75-0.88) was significantly higher than those of the other biomarkers considered independently. Using the medians as cutoff values, elevated serum levels of NfL, UCHL1, tau and GFAP were associated with BM in univariate analysis, but only high GFAP levels in multivariate analysis (odd ratio 23.4, 95% CI 6.8-80.5, P

International audience; PurposeThe exact cellular types that form the human fovea remain a subject of debate, and few studies have been conducted on human macula to solve this question. The purpose of this study was to perform immunohistochemistry on fresh human samples to characterize the glial cells that form the human fovea.MethodsImmunohistochemistry was performed using antibodies against proteins expressed in astrocytes or in retinal Müller glial cells or both types of cells on six human macula obtained from eyes enucleated for peripheral intraocular tumors and on two postmortem eyes from healthy donors. The posterior poles of the enucleated eyes were cryosectioned and stained with antibodies against the glial proteins GFAP, vimentin, CRALBP, glutamine synthetase, and connexin 43.ResultsA population of cells positive for GFAP and negative for glutamine synthetase and CRALBP that express connexin 43 were identified at the roof of the foveal pit. These cells are distinct from the Müller cone cells described by Yamada and Gass, suggesting that another type of foveal glial cells, most likely astrocytes, are present in the human fovea.ConclusionsThis study showed that in humans, astrocytic glial cells cover the foveal pit. Their roles in macula homeostasis and mechanisms of macular disease remain to be determined.

International audience; Purpose: To analyze the influence of age on retinochoroidal wound healing processes and on glial growth factor and cytokine mRNA expression profiles observed after argon laser photocoagulation.Methods: A cellular and morphometric study was performed that used 44 C57Bl/6J mice: 4-week-old mice (group I, n=8), 6-week-old mice (group II, n=8), 10-12-week-old mice (group III, n=14), and 1-year-old mice (group IV, n=14). All mice in these groups underwent a standard argon laser photocoagulation (50 microm, 400 mW, 0.05 s). Two separated lesions were created in each retina using a slit lamp delivery system. At 1, 3, 7, 14, 60 days, and 4 months after photocoagulation, mice from each of the four groups were sacrificed by carbon dioxide inhalation. Groups III and IV were also studied at 6, 7, and 8 months after photocoagulation. At each time point the enucleated eyes were either mounted in Tissue Tek (OCT), snap frozen and processed for immunohistochemistry or either flat mounted (left eyes of groups III and IV). To determine, by RT-PCR, the time course of glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and monocyte chemotactic protein-1 (MCP-1) gene expression, we delivered ten laser burns (50 microm, 400 mW, 0.05 s) to each retina in 10-12-week-old mice (group III', n=10) and 1-year-old mice (group IV', n=10). Animals from Groups III' and IV' had the same age than those from Groups III and IV, but they received ten laser impacts in each eye and served for the molecular analysis. Mice from Groups III and IV received only two laser impacts per eye and served for the cellular and morphologic study. Retinal and choroidal tissues from these treated mice were collected at 16 h, and 1, 2, 3, and 7 days after photocoagulation. Two mice of each group did not receive photocoagulation and were used as controls.Results: In the cellular and morphologic study, the resultant retinal pigment epithelium interruption expanse was significantly different between the four groups. It was more concise and smaller in the oldest group IV (112.1 microm+/-11.4 versus 219.1 microm+/-12.2 in group III) p

Dexamphetamine (AMPH) is a psychostimulant drug that is used both recreationally and as medication for attention deficit hyperactivity disorder. Preclinical studies have demonstrated that repeated exposure to AMPH can induce damage to nerve terminals of dopamine (DA) neurons. We here assessed the underlying neurobiological changes in the DA system following repeated AMPH exposure and pre-treated rats with AMPH or saline (4 times 5 mg/kg s.c., 2 hours apart), followed by a 1-week washout period. We then used pharmacological MRI (phMRI) with a methylphenidate (MPH) challenge, as a sensitive and non-invasive in-vivo measure of DAergic function. We subsequently validated the DA-ergic changes post-mortem, using a.o. high-performance liquid chromatography (HPLC) and autoradiography. In the AMPH pre-treated group, we observed a significantly larger BOLD response to the MPH challenge, particularly in DA-ergic brain areas and their downstream projections. Subsequent autoradiography studies showed that AMPH pre-treatment significantly reduced DA transporter (DAT) density in the caudate-putamen (CPu) and nucleus accumbens, whereas HPLC analysis revealed increases in the DA metabolite homovanillic acid in the CPu. Our results suggest that AMPH pre-treatment alters DAergic responsivity, a change that can be detected with phMRI in rats. These phMRI changes likely reflect increased DA release together with reduced DAT binding. The ability to assess subtle synaptic changes using phMRI is promising for both preclinical studies of drug discovery, and for clinical studies where phMRI can be a useful tool to non-invasively investigate DA abnormalities, e.g. in neuropsychiatric disorders. ispartof: PLoS One vol:12 issue:2 pages:1-16 ispartof: location:United States status: published

Intermediate filaments (IFs) participate in directed cell migration, but how the IF network becomes polarized in motile cells is unclear. Leduc and Etienne-Manneville show that the turnover of IF mainly relies on actin-driven retrograde flow and microtubule-driven anterograde and retrograde transport. During cell migration, Cdc42-mediated polarity signaling inhibits dynein-dependent transport to promote the polarization of the IF network., Intermediate filaments (IFs) are key players in the control of cell morphology and structure as well as in active processes such as cell polarization, migration, and mechanoresponses. However, the regulatory mechanisms controlling IF dynamics and organization in motile cells are still poorly understood. In this study, we investigate the mechanisms leading to the polarized rearrangement of the IF network along the polarity axis. Using photobleaching and photoconversion experiments in glial cells expressing vimentin, glial fibrillary acidic protein, and nestin, we show that the distribution of cytoplasmic IFs results from a continuous turnover based on the cooperation of an actin-dependent retrograde flow and anterograde and retrograde microtubule-dependent transports. During wound-induced astrocyte polarization, IF transport becomes directionally biased from the cell center toward the cell front. Such asymmetry in the transport is mainly caused by a Cdc42- and atypical PKC–dependent inhibition of dynein-dependent retrograde transport. Our results show how polarity signaling can affect the dynamic turnover of the IF network to promote the polarization of the network itself.

International audience; The zebrafish adult brain contains numerous neural progenitors and is a good model to approach the general mechanisms of adult neural stem cell maintenance and neurogenesis. Here we use this model to test for a correlation between Fgf signaling and cell proliferation in adult progenitor zones. We report expression of Fgf signals (fgf3,4,8a,8b,17b), receptors (fgfr1-4), and targets (erm, pea3, dusp6, spry1,2,4, and P-ERK) and document that genes of the embryonic fgf8 synexpression group acquire strikingly divergent patterns in the adult brain. We further document the specific expression of fgf3, fgfr1-3, dusp6, and P-ERK in ventricular zones, which contain neural progenitors. In these locations, however, a comparison at the single-cell level of fgfr/P-ERK expression with bromo-deoxy-uridine (BrdU) incorporation and the proliferation marker MCM5 indicates that Fgf signaling is not specifically associated with proliferating progenitors. Rather, it correlates with the ventricular radial glia state, some of which only are progenitors. Together these results stress the importance of Fgf signaling in the adult brain and establish the basis to study its function in zebrafish, in particular in relation to adult neurogenesis.

International audience; Congenital deafness, affecting 1 in 1000 neonates, can lead to major problems in speech, cognitive and psychosocial development. Congenital deafness is mainly caused by mutations in connexins, hemi-channel proteins forming gap-junctions between supporting cells in the sensory epithelia. We describe a high tropism of AAV5 serotype for the supporting cells of the cochlea, both in vitro in postnatal day 4 mouse explants, and in vivo in the adult guinea-pig inner ear, through scala media perfusion. AAV5 transduction correlates with PDGFRalpha expression, previously reported as AAV5 receptor. This vector could be of major interest in addressing gene therapy approaches to deafness as well as for studying basic aspects of inner-ear development and hearing mechanisms.

International audience; In lower vertebrates, like fishes and amphibians, retina is able to self-regenerate whereas Mammalian retina has lost this property. Nevertheless, recently, it has turned out that retinal glial Müller cells were playing a role in neuronal regeneration of the adult rodent retina, in case of acute damages, by dedifferentiating and redifferentiating in glial and neural cells. The purpose of this study was to analyse the ability of mammalian Müller cells for forming neurospheres. First of all, rats Müller cells were isolated in a primary culture. Second, these cells were resuspended in two different culture media: the cells cultures in the Neurobasal-A medium kept a typical Müller cells morphology even after 15 days of EGF treatment, and the cells plated in the DMEM-F12 medium formed neurospheres from the third day in culture. The neurosphere cells expressed nestin, cellular retinaldehyde binding protein (CRALBP) and glial fibrillary acidic protein (GFAP). These results showed our capacity to isolate and propagate Müller cells-derived progenitor cells. Moreover, it allows us to control the number of progenitor cells and, in the future, to study their differentiation capacity.

International audience; Quantitative RT-PCR (qRT-PCR) and Western blotting studies on transporters at the blood-brain barrier (BBB) of isolated brain microvessels have produced conflicting data on their cellular distribution. A major problem is identifying cells expressing the genes of interest, since isolated brain microvessels are composed of several cell types and may be contaminated with mRNA or proteins from astrocytes and neurons. We isolated rat brain microvessels and examined microscopically samples at each step of isolation to evaluate microvessel purity. The expression of specific markers of endothelial cells (Glut-1, Flk-1), pericytes (Ng2), neurons (synaptophysin, Syn) and astrocytes (Gfap) was measured by qRT-PCR in order to select the protocol giving the least astrocyte and neuron mRNAs and the most endothelial mRNAs. We also evaluated the gene expression of drug transporters (Mdr1a, Mdr1b, Mrp1-5, Bcrp and Oatp-2) at each step to optimize their location in cells at the BBB. The Mdr1a, Mrp4, Bcrp and Oatp-2 gene profiles were similar to those of endothelium markers. The profiles of Mrp2 and Mrp3 closely resembled that of Ng2. Mrp5 and Mrp1 expression was not increased in the microvessel-enriched fraction, suggesting that they are ubiquitously expressed throughout the cortex parenchyma. We also evaluated by Western blotting the expression of P-gp, Mrp2, Gfap and Syn in the cortex and in the purest obtained microvessel fraction. Our results showed that P-gp expression strongly increased in microvessels whereas Mrp2 was not detected in any of the fraction. Surprisingly, Gfap expression increased in isolated microvessels whereas Syn was not detected. Our results showed that the strategy consisting of identifying gene expression at different steps of the protocol is useful to identify cells containing mRNA at the BBB and give overall similar results with protein expression.

International audience; Circadian rhythms in mammals are synchronized to the light (L)/dark (D) cycle through messages relaying in the master clock, the suprachiasmatic nucleus of the hypothalamus (SCN). Here, we provide evidence that the SCN undergoes rhythmic ultrastructural rearrangements over the 24-h cycle characterized by day/night changes of the glial, axon terminal, and/or somato-dendritic coverage of neurons expressing arginine vasopressin (AVP) or vasoactive intestinal peptide (VIP), the two main sources of SCN efferents. At nighttime, we noted an increase in the glial coverage of the dendrites of the VIP neurons (+29%) that was concomitant with a decrease in the mean coverage of the somata (-36%) and dendrites (-43%) of these neurons by axon terminals. Conversely, glial coverage of the dendrites of AVP neurons decreased (-19%) with parallel increase in the extent of somatal (+96%) and dendritic (+52%) membrane appositions involving these neurons. These plastic events were concomitant with daily fluctuations in quantitative expression of glial fibrillary acidic protein (GFAP), which were then used as an index of structural plasticity. The GFAP rhythm appeared to be strictly dependent on light entrainment, indicating that structural reorganization of the SCN may subserve synchronization of the clock to the L/D cycle. Other results presented reinforced this view while showing that circulating glucocorticoid hormones, which are known to modulate photic entrainment, were required to maintain amplitude of the GFAP rhythm to normal values.

Combination of ex vivo gene transfer and cell transplantation is now considered as a potentially useful strategy for the treatment of spinal cord injury. In a perspective of clinical application, autologous transplantation could be an option of choice. We analyzed the fate of adult rat cortical astrocytes genetically engineered with a lentiviral vector transplanted into a lesioned rat spinal cord. Cultures of adult rat cortical astrocytes were infected with an HIV-1-derived vector (TRIP-CMV-GFP) and labeled with the fluorescent dye Hoechst. Transfected and labeled astrocyte suspension was injected at T11 in rats in which spinal cord transection at T7-T8 levels had been carried out 1 week earlier. Six weeks after grafting, the animals were sacrificed and transplants were retrieved either by Hoechst fluorescence or by immunohistochemistry for detection of glial fibrillary acidic protein (GFAP) and vimentin. Grafted astrocytes expressing green fluorescent protein (GFP) were found both at the injection and transection sites. Genetically modified astrocytes thus survived, integrated, and migrated within the host parenchyma when grafted into the completely transected rat spinal cord. In addition, they retained some ability to express the GFP transgene for at least 6 weeks after transplantation. Adult astrocytes infected with lentiviral vectors can therefore be a valuable tool for the delivery of therapeutic factors into the lesioned spinal cord., Contract grant sponsor: Institut de Recherche sur la Moelle Epiniere;Contract grant sponsor: Association Francaise des Myopathies.

International audience; Urotensin II (UII) and Urotensin II-related peptide (URP) are structurally related paralog peptides that exert peripheral and central effects. UII binding sites have been partly described in brain, and those of URP have never been reported. We exhaustively compared [(125)I]-UII and -URP binding site distributions in the adult rat brain, and found that they fully overlapped at the regional level. We observed UII/URP binding sites in structures lining ventricles, comprising the sphenoid nucleus and cell rafts scattered on a line joining the fourth ventricle and its lateral recess. After injection of UII and URP in the lateral ventricle, we observed c-Fos-positive cell nuclei in areas close to the fourth ventricle, indicating that these receptors are functional. Different c-Fos-containing cell populations were activated. They were all positive for vimentin and glial fibrillary acidic protein (GFAP), excluding the possibility of an ependymal nature. In conclusion, this study demonstrated that UII and URP binding sites are totally overlapping and that these sites were functional in regions bordering the fourth ventricle. These data support a role for UII/URP at the interface between brain parenchyma and cerebrospinal fluid.; Octylglyceryl dextran-graft-poly(lactic acid) nanoparticles formulated by emulsification demonstrate potential for peptide delivery.

β-Amyloid (Aβ) oligomers initiate synaptotoxicity following their interaction with the plasma membrane. Several proteins including metabotropic glutamate type 5 receptors (mGluR5s) contribute to this process. We observed an overexpression of mGluR5s in reactive astrocytes surrounding Aβ plaques in brain sections from an Alzheimer's disease mouse model. In a simplified cell culture system, using immunocytochemistry and single molecule imaging, we demonstrated a rapid binding of Aβ oligomers on the plasma membrane of astrocytes. The resulting aggregates of Aβ oligomers led to the diffusional trapping and clustering of mGluR5s. Further, Aβ oligomers induced an increase in ATP release following activation of astroglial mGluR5s by its agonist. ATP slowed mGluR5s diffusion in astrocytes as well as in neurons co-cultured with astrocytes. This effect, which is purinergic receptor-dependent, was not observed in pure neuronal cultures. Thus, Aβ oligomer- and mGluR5-dependent ATP release by astrocytes may contribute to the overall deleterious effect of mGluR5s in Alzheimer's disease. GLIA 2013;61:1673-1686.

International audience; Antibodies normally do not cross the blood-brain barrier (BBB) and cannot bind an intracellular cerebral antigen. We demonstrate here for the first time that a new class of antibodies can cross the BBB without treatment. Camelids produce native homodimeric heavy-chain antibodies, the paratope being composed of a single-variable domain called VHH. Here, we used recombinant VHH directed against human glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Only basic VHHs (e.g., pI=9.4) were able to cross the BBB in vitro (7.8 vs. 0% for VHH with pI=7.7). By intracarotid and intravenous injections into live mice, we showed that these basic VHHs are able to cross the BBB in vivo, diffuse into the brain tissue, penetrate into astrocytes, and specifically label GFAP. To analyze their ability to be used as a specific transporter, we then expressed a recombinant fusion protein VHH-green fluorescent protein (GFP). These "fluobodies" specifically labeled GFAP on murine brain sections, and a basic variant (pI=9.3) of the fusion protein VHH-GFP was able to cross the BBB and to label astrocytes in vivo. The potential of VHHs as diagnostic or therapeutic agents in the central nervous system now deserves attention.

Glioblastomas are the most common CNS tumors in adults, and they remain resistant to current treatments. ErbB1 signaling is frequently altered in these tumors, which indicates that the erbB receptor family is a promising target for molecular therapy. However, data on erbB signaling in glioblastomas are still sparse. Therefore, we undertook a comprehensive analysis of erbB receptor and ligand expression profiles in a panel of nine glioblastomas that were compared to non-neoplastic cerebral tissue containing neocortex and corresponding portions of subcortical convolutional white matter and we determined the distribution patterns of erbB receptors among the main neural cell types that are present in these tumors, particularly the putative tumoral stem cell population. Using quantitative RT-PCR and western blot analysis, we showed that erbB1 signaling and erbB2 receptors exhibited highly variable deregulation profiles among tumors, ranging from under- to overexpression, while erbB3 and erbB4 were down-regulated. Immunohistochemistry revealed an important inter- and intra-tumoral heterogeneity in all four erbB expression profiles. However, each receptor exhibited a distinct repartition pattern among the GFAP-, Olig2-, NeuN- and CD133-positive populations. Interestingly, while erbB1 immunoreactivity was only detected in small subsets of CD133-positive putative tumoral stem cells, erbB3 immunoreactivity was prominent in this cell population thus suggesting that erbB3 may represent a new potential target for molecular therapy.

A 32-year-old immunocompetent female patient, 8 months pregnant, underwent three complex partial seizures beginning with visual hallucinations and secondarily generalized. She had been in good health until then. The pregnancy was normal and no high blood pressure was noted. Clinical examination revealed a left nystagmus, a right sensory hemibody deficiency and a right pyramidal syndrome. MRI showed an isolated intraparenchymal lesion in the left parieto-occipital region with an enhancement of the left lateral ventricle floor (Fig. 1a,b). The biological assessments, including hemogram, ionogram, CRP, liver enzymes and b2 microglobulin, were all normal. An elevated erythrocyte sedimentation rate was noted. CMV, HBV, HCV, and Lyme serologies were negative. Previous HSV, EBV, and varicella zoster virus infection markers were found. CSF examination revealed hyperproteinorachia (0.95 g/L) without cellular reaction. No tumoral cells were found. Thoracic and abdominal CT was normal. After the brain biopsy, a course of corticosteroids was started. The patient did not present with any other seizures. Five years after the first epileptic manifestations, follow-up MRI demonstrated resolution of the enhancing lesions (Fig. 2) and the patient was in good health with 10 mg of corticosteroids daily.

International audience; Accumulating evidence indicates that cerebral ischemia enhances neurogenesis in the adult brain. The mechanisms responsible for stem-cell development are poorly understood. Recent in vitro studies indicate the involvement of metalloproteinase (MMPs) in the regulation of proliferation and differentiation of neural progenitor cells. To elucidate if MMPs participate in neurogenesis-associated processes after ischemic insult, we aimed to establish spatial and temporal relationships between neural stem-cell development and the activity of MMPs in the adult brain hippocampus. Our results show that post ischemic acceleration in the proliferation of progenitors in the dentate gyrus is accompanied by increased activity of MMPs. On the contrary, in the damaged CA1 pyramidal layer the neurogenesis seems to be rather elusive. Simultaneously, the activity of MMPs fell below the control level. In conclusion, our results show that the activation of MMPs may, at least in part, contribute to ischemia-induced neurogenesis in the dentate gyrus of the adult brain.

International audience; In rodents, there is compelling evidence indicating that dynamic cell-to-cell communications involving cross talk between astroglial cells (such as astrocytes and specialised ependymoglial cells known as tanycytes) and neurones are important in regulating the secretion of gonadotrophin-releasing hormone (GnRH), the neurohormone that controls both sexual maturation and adult reproductive function. However, whether such astroglial cell-GnRH neurone interactions occur in the human brain is not known. In the present study, we used immunofluorescence to examine the anatomical relationship between GnRH neurones and glial cells within the hypothalamus of five women. Double-staining experiments demonstrated the ensheathment of GnRH neurone perikarya by glial fibrillary acidic protein (GFAP)-immunoreactive astrocyte processes in the periventricular zone of the tuberal region of the hypothalamus. GFAP immunoreactivity did not overlap that of GnRH at the GnRH neurone's projection site (i.e. the median eminence of the hypothalamus). Rather, human GnRH neuroendocrine fibres were found to be closely associated with vimentin or nestin-immunopositive radial glial processes likely belonging to tanycytes. In line with these light microscopy data, ultrastructural examination of GnRH-immunoreactive neurones showed numerous glial cells in direct apposition to pre-embedding-labelled GnRH cell bodies and/or dendrites in the infundibular nucleus, whereas postembedding immunogold-labelled GnRH nerve terminals were often seen to be enwrapped by glial cell processes in the median eminence. GnRH nerve button were sometimes visualised in close proximity to fenestrated pituitary portal blood capillaries and/or evaginations of the basal lamina that delineate the pericapillary space. In summary, these data demonstrate that GnRH neurones morphologically interact with astrocytes and tanycytes in the human brain and provide evidence that glial cells may contribute physiologically to the process by which the neuroendocrine brain controls the function of GnRH neurones in humans.

International audience; During the postnatal development, astrocytic cells in the neocortex progressively lose their neural stem cell (NSC) potential, whereas this peculiar attribute is preserved in the adult subventricular zone (SVZ). To understand this fundamental difference, many reports suggest that adult subventricular GFAP-expressing cells might be maintained in immature developmental stage. Here, we show that S100B, a marker of glial cells, is absent from GFAP-expressing cells of the SVZ and that its onset of expression characterizes a terminal maturation stage of cortical astrocytic cells. Nevertheless, when cultured in vitro, SVZ astrocytic cells developed as S100B expressing cells, as do cortical astrocytic cells, suggesting that SVZ microenvironment represses S100B expression. Using transgenic s100b-EGFP cells, we then demonstrated that S100B expression coincides with the loss of neurosphere forming abilities of GFAP expressing cells. By doing grafting experiments with cells derived from beta-actin-GFP mice, we next found that S100B expression in astrocytic cells is repressed in the SVZ, but not in the striatal parenchyma. Furthermore, we showed that treatment with epidermal growth factor represses S100B expression in GFAP-expressing cells in vitro as well as in vivo. Altogether, our results indicate that the S100B expression defines a late developmental stage after which GFAP-expressing cells lose their NSC potential and suggest that S100B expression is repressed by adult SVZ microenvironment.

International audience; During the postnatal development, astrocytic cells in the neocortex progressively lose their neural stem cell (NSC) potential, whereas this peculiar attribute is preserved in the adult subventricular zone (SVZ). To understand this fundamental difference, many reports suggest that adult subventricular GFAP-expressing cells might be maintained in immature developmental stage. Here, we show that S100B, a marker of glial cells, is absent from GFAP-expressing cells of the SVZ and that its onset of expression characterizes a terminal maturation stage of cortical astrocytic cells. Nevertheless, when cultured in vitro, SVZ astrocytic cells developed as S100B expressing cells, as do cortical astrocytic cells, suggesting that SVZ microenvironment represses S100B expression. Using transgenic s100b-EGFP cells, we then demonstrated that S100B expression coincides with the loss of neurosphere forming abilities of GFAP expressing cells. By doing grafting experiments with cells derived from beta-actin-GFP mice, we next found that S100B expression in astrocytic cells is repressed in the SVZ, but not in the striatal parenchyma. Furthermore, we showed that treatment with epidermal growth factor represses S100B expression in GFAP-expressing cells in vitro as well as in vivo. Altogether, our results indicate that the S100B expression defines a late developmental stage after which GFAP-expressing cells lose their NSC potential and suggest that S100B expression is repressed by adult SVZ microenvironment.

International audience; In this study, we used a neuronal cell line generated by transfection of rat olfactory epithelium with immortalizing recombinant oncogene E1A of adenovirus-2. The resulting 13.S.1.24 line of transformed cells expressed an antigenic phenotype of olfactory neuronal progenitors. Time-dependency assessments over 1 week of treatment indicated that apoptosis and differentiation induced by retinoic acid (RA) were concomitant. Indeed, RA altered the cell proliferation rate, but it also stimulated differentiation of surviving 13.S.1.24 cells into bipolar olfactory marker protein-immunoreactive neurons. To characterize the nature of the cells we used immunocytochemistry, optical imaging, scanning electron microscopy and atomic force microscopy.

International audience; In rodents, there is compelling evidence indicating that dynamic cell-to-cell communications involving cross talk between astroglial cells (such as astrocytes and specialised ependymoglial cells known as tanycytes) and neurones are important in regulating the secretion of gonadotrophin-releasing hormone (GnRH), the neurohormone that controls both sexual maturation and adult reproductive function. However, whether such astroglial cell-GnRH neurone interactions occur in the human brain is not known. In the present study, we used immunofluorescence to examine the anatomical relationship between GnRH neurones and glial cells within the hypothalamus of five women. Double-staining experiments demonstrated the ensheathment of GnRH neurone perikarya by glial fibrillary acidic protein (GFAP)-immunoreactive astrocyte processes in the periventricular zone of the tuberal region of the hypothalamus. GFAP immunoreactivity did not overlap that of GnRH at the GnRH neurone's projection site (i.e. the median eminence of the hypothalamus). Rather, human GnRH neuroendocrine fibres were found to be closely associated with vimentin or nestin-immunopositive radial glial processes likely belonging to tanycytes. In line with these light microscopy data, ultrastructural examination of GnRH-immunoreactive neurones showed numerous glial cells in direct apposition to pre-embedding-labelled GnRH cell bodies and/or dendrites in the infundibular nucleus, whereas postembedding immunogold-labelled GnRH nerve terminals were often seen to be enwrapped by glial cell processes in the median eminence. GnRH nerve button were sometimes visualised in close proximity to fenestrated pituitary portal blood capillaries and/or evaginations of the basal lamina that delineate the pericapillary space. In summary, these data demonstrate that GnRH neurones morphologically interact with astrocytes and tanycytes in the human brain and provide evidence that glial cells may contribute physiologically to the process by which the neuroendocrine brain controls the function of GnRH neurones in humans.

Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin-proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.

International audience; In contrast to mouse embryonic stem cells and in spite of overlapping gene expression profiles, neural stem cells (NSCs) isolated from the embryonic spinal cord do not respond to physiological morphogenetic stimuli provided by Sonic hedgehog and retinoic acid and do not generate motor neurons upon differentiation. Transcription factors expressed in motor neuron progenitors during embryogenesis include Pax6, Ngn2, Nkx6.1 and Olig2, whose expression precedes that of factors specifying motor neuron fate, including HB9, Islet1 and LIM3. We showed that all these factors were present in neural progenitors derived from mouse ES cells, whereas NSCs derived from the rat embryonic spinal cord expressed neither HB9 nor Islet1 and contained low levels of Nkx6.1 and LIM3. We constructed a lentivirus vector to express HB9 and GFP in NSCs and examined the consequences of HB9 expression on other transcription factors and cell differentiation. Compared to cell expressing GFP alone, NSCs expressing GFP and HB9 cycled less rapidly, downregulated Pax6 and Ngn2 mRNA levels, produced higher proportions of neurons in vitro and lower numbers of neurons after transplantation in the spinal cord of recipient rats. Oligodendrocytic and astrocytic differentiations were not affected. HB9 expressing NSCs did not express Islet1 or upregulate LIM3. They neither responded to Sonic hedgehog and retinoic acid nor produced cholinergic neurons. We concluded that forced HB9 expression affected neurogenesis but was not sufficient to confer motor neuron fate to NSCs.

International audience; We have shown previously that the tissue nonspecific alkaline phosphatase (TNAP) is selectively expressed in the synaptic cleft of sensory cortical areas in adult mammals and, by using sensory deprivation, that TNAP activity depends on thalamocortical activity. We further analyzed this structural functional relationship by comparing the developmental pattern of TNAP activity to the maturation of the thalamocortical afferents in the primate brain (Callithrix jacchus). Cortical expression of alkaline phosphatase (AP) activity reflects the sequential maturation of the modality-specific sensory areas. Within the visual cortex, the regional and laminar distribution of AP correlates with the differential maturation of the magno- and parvocellular streams. AP activity, which is transiently expressed in the white matter, exhibits a complementary distributional pattern with myelin staining. Ultrastructural analysis revealed that AP activity is localized exclusively to the myelin-free axonal segments, including the node of Ranvier. It was also found that AP activity is gradually expressed in parallel with the maturation of synaptic contacts in the neuropile. These data suggest the involvement of AP, in addition to neurotransmitter synthesis previously suggested in the adult, in synaptic stabilization and in myelin pattern formation and put forward a role of AP in cortical plasticity and brain disorders.

International audience; Unlike mammals, some fish, including carp and trout, have a continuously growing brain. The glial architecture of teleost brain has been intensively studied in the carp and few data exist on trout brain. In this study, using immunoblotting we characterized the topographic distribution of glial fibrillary acidic protein (GFAP) in larval and adult rainbow trout brain and studied by immunohistochemistry the distribution and morphology of GFAP-immunoreactive cell systems in the rainbow trout hindbrain and spinal cord. Immunoblotting yielded a double band with an apparent molecular weight of 50-52 kDa in the spinal cord homogenate in the trout larval and adult stages. In the adult hindbrain and forebrain, our antibody cross reacted also with a second band at a higher molecular weight (90 kDa). Because the forebrain contained this band alone the two brain regions might contain two distinct isoforms. Conversely, the larval total brain homogenate contained the heavy 90 kDa band alone. Hence the heavy band might be a GFAP protein dimer or vimentin/GFAP copolymer reflecting nerve fiber growth and elongation, or the two isoforms might indicate two distinct astroglial cell types as recently proposed in the zebrafish. In sections from trout hindbrain and spinal cord the antibody detected a GFAP-immunoreactive glial fiber system observed in the raphe and in the glial septa separating the nerve tracts. These radial glia fibers thickened toward the pial surface, where they formed glial end feet. The antibody also labeled perivascular glia around blood vessels in the white matter, and the ependymoglial plexus surrounding the ventricular surface in the grey matter. Last, it labeled round astrocytes. The GFAP-immunoreactive glial systems had similar distribution patterns in the adult and larval spinal cord suggesting early differentiation.

Basic fibroblast growth factor (bFGF) exerts a mitogenic effect on cortical neuroblasts, whereas neurotrophin 3 (NT3) promotes differentiation in these cells. Here we provide evidence that both the mitogenic effect of bFGF and the differentiation-promoting effect of NT3 are linked with modifications of cell cycle kinetics in mouse cortical precursor cells. We adapted an in vitro assay, which makes it possible to evaluate (1) the speed of progression of the cortical precursors through the cell cycle, (2) the duration of individual phases of the cell cycle, (3) the proportion of proliferative versus differentiative divisions, and (4) the influence on neuroglial differentiation. Contrary to what has been claimed previously, bFGF promotes proliferation via a change in cell cycle kinetics by simultaneously decreasing G1 duration and increasing the proportion of proliferative divisions. In contrast, NT3 lengthens G1 and promotes differentiative divisions. We investigated the molecular foundations of these effects and show that bFGF downregulates p27(kip1) and upregulates cyclin D2 expression. This contrasts with NT3, which upregulates p27(kip1) and downregulates cyclin D2 expression. Neither bFGF nor NT3 influences the proportion of glia or neurons in short to medium term cultures. The data point to links between the length of the G1 phase and the type of division of cortical precursors: differentiative divisions are correlated with long G1 durations, whereas proliferative divisions correlate with short G1 durations. The present results suggest that concerted mechanisms control the progressive increase in the cell cycle duration and proportion of differentiative divisions that is observed as corticogenesis proceeds.

Basic fibroblast growth factor (bFGF) exerts a mitogenic effect on cortical neuroblasts, whereas neurotrophin 3 (NT3) promotes differentiation in these cells. Here we provide evidence that both the mitogenic effect of bFGF and the differentiation-promoting effect of NT3 are linked with modifications of cell cycle kinetics in mouse cortical precursor cells. We adapted an in vitro assay, which makes it possible to evaluate (1) the speed of progression of the cortical precursors through the cell cycle, (2) the duration of individual phases of the cell cycle, (3) the proportion of proliferative versus differentiative divisions, and (4) the influence on neuroglial differentiation. Contrary to what has been claimed previously, bFGF promotes proliferation via a change in cell cycle kinetics by simultaneously decreasing G1 duration and increasing the proportion of proliferative divisions. In contrast, NT3 lengthens G1 and promotes differentiative divisions. We investigated the molecular foundations of these effects and show that bFGF downregulates p27(kip1) and upregulates cyclin D2 expression. This contrasts with NT3, which upregulates p27(kip1) and downregulates cyclin D2 expression. Neither bFGF nor NT3 influences the proportion of glia or neurons in short to medium term cultures. The data point to links between the length of the G1 phase and the type of division of cortical precursors: differentiative divisions are correlated with long G1 durations, whereas proliferative divisions correlate with short G1 durations. The present results suggest that concerted mechanisms control the progressive increase in the cell cycle duration and proportion of differentiative divisions that is observed as corticogenesis proceeds.

International audience; Chemical preconditioning using the mitochondrial toxin, 3-nitropropionic acid (3-NP) has been reported to induce neuroprotection against subsequent global ischemia. To investigate the underlying mechanisms, Mongolian gerbils were pretreated with either vehicle or 3-NP at the dose of 3 or 10 mg/kg, intraperitoneal, 3 days prior to a 5-min bilateral carotid artery occlusion followed by either 48 h or 7 days of blood recirculation. Neuronal damage was assessed by a cresyl violet/fuchsin acid staining. Induction of heat shock protein 72 (HSP72) and manganese superoxide dismutase (MnSOD) expression was evaluated by Western blotting. Astroglial and microglial activation was detected by immunohistochemistry (glial fibrillary acid protein) and by histochemistry (isolectin B4 staining), respectively. Present data show that the hippocampal neuronal damage induced by ischemia were of similar extent between the vehicle- and 3-NP-treated gerbils, whatever the dose tested, indicating that 3-NP did not induce tolerance to transient forebrain ischemia under our experimental conditions. The lack of difference in the post-ischemic level of HSP72 and MnSOD protein expression and in the intensity of astroglial and microglial activation represents further indirect indications of the absence of 3-NP preconditioning effect. In conclusion, although chemical preconditioning with 3-NP is a well-established phenomenon at least in vitro and in models of focal ischemia, the relevance of 3-NP as a preconditioning molecule towards global brain ischemia remains an open question.

Basic fibroblast growth factor (bFGF) exerts a mitogenic effect on cortical neuroblasts, whereas neurotrophin 3 (NT3) promotes differentiation in these cells. Here we provide evidence that both the mitogenic effect of bFGF and the differentiation-promoting effect of NT3 are linked with modifications of cell cycle kinetics in mouse cortical precursor cells. We adapted an in vitro assay, which makes it possible to evaluate (1) the speed of progression of the cortical precursors through the cell cycle, (2) the duration of individual phases of the cell cycle, (3) the proportion of proliferative versus differentiative divisions, and (4) the influence on neuroglial differentiation. Contrary to what has been claimed previously, bFGF promotes proliferation via a change in cell cycle kinetics by simultaneously decreasing G1 duration and increasing the proportion of proliferative divisions. In contrast, NT3 lengthens G1 and promotes differentiative divisions. We investigated the molecular foundations of these effects and show that bFGF downregulates p27(kip1) and upregulates cyclin D2 expression. This contrasts with NT3, which upregulates p27(kip1) and downregulates cyclin D2 expression. Neither bFGF nor NT3 influences the proportion of glia or neurons in short to medium term cultures. The data point to links between the length of the G1 phase and the type of division of cortical precursors: differentiative divisions are correlated with long G1 durations, whereas proliferative divisions correlate with short G1 durations. The present results suggest that concerted mechanisms control the progressive increase in the cell cycle duration and proportion of differentiative divisions that is observed as corticogenesis proceeds.

The morphogen sonic hedgehog (Shh) is implicated in neural tissue patterning and the growth of brain structures during embryogenesis and postnatal development and is also present in the adult brain. Shh signals through interaction with the tumour suppressor Patched (Ptc). This receptor for Shh is associated with Smoothened (Smo), a protein with high homology to the G-protein coupled receptors. However, little is known about the transduction mechanisms implicated in Shh signalling in the adult brain. The study described here shows that injection of aminoterminal myristoylated Shh (myrShhN) into the adult rat striatum robustly increases the levels of Ptc transcripts in selective brain areas including the subventricular zone (SVZ). The adult SVZ contains cell progenitors, which can proliferate and differentiate into new neurons and glia. In the myrShhN injected animals, proliferation and differentiation of these SVZ precursor cells were not affected as demonstrated by BrdU incorporation and immunohistochemistry performed with specific antibodies for nestin (uncommitted neural progenitors), PSA-NCAM (migrating neuroblasts) or GFAP (astrocytes). Together with the presence of Smo expressing cells and amino-terminal Shh (ShhN) protein in SVZ area of untreated animals, the data presented here supports the hypothesis that the Shh pathway may be activated in the adult brain, and that a niche for Shh signalling exists within the adult SVZ.

The present study demonstrates that bipotential neural precursors isolated from an early developmental stage of the sheep embryo nervous system can be maintained in vitro in an undifferentiated state for a long period. These precursors multiplied under the action of epidermal growth factor and basic fibroblast growth factor and formed free-floating aggregates of nestin-immunoreactive cells, called neurospheres. These precursors can undergo predominantly neural or glial differentiation according to the culture conditions. Medium supplemented with foetal calf serum mainly favoured cell differentiation predominantly into astrocytes, whereas the defined SATO medium favoured neuronal differentiation. Using various immunomarkers of neurones and astroglial cells, we described the course of differentiation of neuronal and astroglial cells in different culture conditions. The ability to grow neural precursors from common laboratory animals has been useful for studying the cellular and molecular mechanisms underlying the development of the central nervous system. Furthermore, neural progenitors are already being used for in vivo cell therapy in various neurodegenerative disorders. The ovine species is a well-known model for prion diseases, since scrapie is endemic in most countries and has been studied for a long time. In this respect, the availability of ovine neural precursors will add a new perspective to the study of the pathogenicity of prion diseases.

International audience; Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by selective degeneration of motoneurones. Familial ALS is an age-dependent autosomal dominant disorder in which mutations in the homodimeric enzyme Cu/Zn superoxide dismutase 1 (SOD1) is linked to the disease. An animal model for this disease is a transgenic mouse expressing the mutated human SOD1(G93A) gene. Recent electrophysiological data emphasised that the striking selective vulnerability of motoneurones might be due to their differential calcium buffering capacities. Therefore we have investigated, using immunohistochemistry, the expression of different calcium binding proteins in brainstem and spinal cord from normal and SOD1 mutated mice. Among the 13 calcium-binding proteins screened, only one, S100A6, a homodimeric calcium-binding protein able to bind four Zn(2+), appeared to be highly expressed in the SOD1 mutated mice. In brainstem, reactive astrocytes, but not motoneurones, from several regions, including nerve 12 root, were highly S100A6-positive. Hypoglossal nucleus was negative for S100A6. In dorsal root, reactive astrocytes from both white matter and anterior horn were highly reactive. If overexpression of S100A6 is specific for ALS, it will be a valuable diagnostic marker for this disease.

International audience; Astrocytes play a predominant role in energy metabolism and in the catabolism of gamma-aminobutyric acid (GABA) and glutamate, neurotransmitters critically involved in epileptic processes. We show specific astrocytic alterations in the genetic absence epilepsy rats from Strasbourg (GAERS). Spontaneous absence seizures appear in this strain in the cortex and thalamus after the age of 1 month. In these brain structures, we demonstrate increased GFAP expression in both adult and young GAERS, suggesting that reactive astrocytes are already present before the onset of seizures. Glutamate dehydrogenase (GDH) and glutamine synthetase (GS), which are localized mainly in astrocytes and involved in glutamate catabolism, are shown to be differentially altered. GDH expression was increased in the thalamus of both young and adult GAERS and in the cortex of young GAERS. GS expression was slightly decreased in the thalamus of young GAERS. These astrocytic modifications are not adaptive responses to seizures, as the modifications appear before the development of absence seizures. Thus, astrocytes might be involved in the neuronal processes giving rise to epileptic seizures in this strain.

International audience; Tenascin-C is an extracellular matrix glycoprotein with trophic and repulsive properties on neuronal cells, involved in migratory processes of immature neurons. Previous reports demonstrated that this molecule is produced and secreted by astrocytes, in vitro after activation by bFGF or in vivo after CNS lesion. In injured brain the expression of tenascin-C has been correlated with the glial reaction since it was observed in regions suffering a dramatic glial proliferation and hypertrophy. In this report we show that the treatment of cultured hippocampal astrocytes with tenascin-C results in an increased fibronectin and NCAM immunoreactivities. In addition, treated astrocytes form longer extensions than control ones. The number of cells as well as the levels of GFAP mRNA and protein immunoreactivity are not modified after tenascin-C treatment. The present changes may, therefore, be related to the modification of the adhesive properties of astrocytes to the substrate. These observations are compatible with the hypothesis that tenascin-C may contribute to the glial scarring process.

International audience; PURPOSE: Changes in extracellular pH occur in the retina and directly affect retinal activity and phototransduction. The authors analyzed the expression in rodent retina of ASIC3, a sensor of extracellular acidosis, and used ASIC3 knockout mice to explore its role in retinal function and survival. METHODS: The expression and the role of ASIC3 were examined by immunolocalization and by comparing retinas from wild-type and knockout mice at different ages through electroretinography, retinal histology (light and electron microscopy), expression of glial fibrillary acidic protein (GFAP), analysis of cell apoptosis (TUNEL assay), and patch-clamp recordings in primary cultures of retinal ganglion cells (RGCs). RESULTS: ASIC3 is present in the rod inner segment of photoreceptors and in horizontal and some amacrine cells. ASIC3 is also detected in RGCs but does not significantly contribute to ASIC currents recorded in cultured RGCs. At 2 to 3 months, knockout mice experience a 19% enhancement of scotopic electroretinogram a-wave amplitude and a concomitant increase of b-wave amplitude without alteration of retinal structure. Older (8-month-old) knockout mice have 69% and 64% reductions in scotopic a- and b-waves, respectively, and reductions in oscillatory potential amplitudes associated with complete disorganization of the retina and degenerating rod inner segments. GFAP and TUNEL staining performed at 8 and 12 months of age revealed an upregulation of GFAP expression in Müller cells and the presence of apoptotic cells in the inner and outer retina. CONCLUSIONS: Inactivation of ASIC3 enhances visual transduction at 2 to 3 months but induces late-onset rod photoreceptor death, suggesting an important role for ASIC3 in maintaining retinal integrity.

International audience; Two clones encoding human glial fibrillary acidic protein (GFAP) were isolated from a human astrocytoma cDNA library. The clones pHGFAP1 and pHGFAP2 were selected by the combined use of differential colony hybridization and hybridization-selection technique with polyclonal anti GFAP antiserum. The longer one, pHGFAP1, encompasses 3.0 kb and includes the 1.8 kb long 3' untranslated region specific to the human mRNA. Sequence data disclosed an extensive homology within the coding region of human and mouse GFAP cDNAs even in the end domains. Blot hybridization analysis of RNAs from human, rat and mouse brain revealed a single GFAP mRNA species of 3.1, 2.8 and 2.7 kb respectively and Southern blot experiments indicated that this mRNA is most probably transcribed from a unique gene. In situ hybridization performed with biotinylated probes on cultured mouse brain cells suggests both the sorting and the transport of GFAP mRNA throughout the cytoplasm and processes of the astrocytes. As a model of reactive gliosis secondary to degenerative disorders, 6-hydroxydopamine (6-OHDA) lesion of the substantia nigra in the rat was performed. GFAP mRNA increased 1.4 fold in the ipsilateral striatum on day 10 after the lesion. It then declined to the control level 4 months later contrasting with the lower and more sustained increase in preproenkephalin (PPE) mRNA. The interspecies cross-reactivity of the HGFAP probes make them useful as a tool for the molecular analysis of reactive gliosis in various experimental models.

International audience; GFAP mRNA levels were quantified by Northern blot analysis using a human GFAP (glial fibrillary acidic protein) cDNA probe in association with immunocytochemistry. Ten days after a unilateral lesion of substantia nigra with 6-hydroxydopamine (6-OHDA), GFAP mRNA level is increased 1.4-fold in the ipsilateral striatum; thereafter it declined continuously to reach the control level 4 months later. This effect contrasted with the lower and more sustained increase of preproenkephalin (PPE) mRNA, a marker of neuronal target of nigrostriatal pathway. Following ibotenic acid-induced neuronal degeneration of the neostriatum in the rat, we observed a sharp elevation of the GFAP transcripts (4-fold) as soon as 2 days after the lesion both in the striatum and in the substantia nigra. Whereas in the striatum GFAP mRNA level already declined at 5 days postlesion, it remained stable in the substantia nigra. In comparison GFAP immunoreactivity was slightly delayed. No obvious modification was observed in the contralateral side to the lesion whatever the denervation condition studied. Implication of these results on the understanding and the therapeutic approach of glial scarring is discussed.

International audience; PURPOSE: To evaluate the potential delay of the retinal degeneration in rd1/rd1 mice using recombinant human glial cell line-derived neurotrophic factor (rhGDNF) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) microspheres. METHODS: rhGDNF-loaded PLGA microspheres were prepared using a water in oil in water (w/o/w) emulsion solvent extraction-evaporation process. In vitro, the rhGDNF release profile was assessed using radiolabeled factor. In vivo, rhGDNF microspheres, blank microspheres, or microspheres loaded with inactivated rhGDNF were injected into the vitreous of rd1/rd1 mice at postnatal day 11 (PN11). The extent of retinal degeneration was examined at PN28 using rhodopsin immunohistochemistry on whole flat-mount retinas, outer nuclear layer (ONL) cell counting on histology sections, and electroretinogram tracings. Immunohistochemical reactions for glial fibrillary acidic protein (GFAP), F4/80, and rhodopsin were performed on cryosections. RESULTS: Significant delay of rod photoreceptors degeneration was observed in mice receiving the rhGDNF-loaded microspheres compared to either untreated mice or to mice receiving blank or inactivated rhGDNF microspheres. The degeneration delay in the eyes receiving the rhGDNF microspheres was illustrated by the increased rhodopsin positive signals, the preservation of significantly higher number of cell nuclei within the ONL, and significant b-wave increase. A reduction of the subretinal glial proliferation was also observed in these treated eyes. No significant intraocular inflammatory reaction was observed after the intravitreous injection of the various microspheres. CONCLUSIONS: A single intravitreous injection of rhGDNF-loaded microspheres slows the retinal degeneration processes in rd1/rd1 mice. The use of injectable, biodegradable polymeric systems in the vitreous enables the efficient delivery of therapeutic proteins for the treatment of retinal diseases.

International audience; Hypoxic preconditioning has been shown to induce neuroprotection against a subsequent damaging insult. In order to study the underlying molecular and cellular mechanisms of hypoxic preconditioning, we investigated, in gerbil hippocampus, the effects in vivo of transient exposure to hypoxia (4% O(2) for 6 min followed by either 48 h or 7 days of reoxygenation) (i) on the induction of 72 kDa heat shock protein (HSP72), heme oxygenase-1 (HO-1) and manganese superoxide dismutase (Mn SOD) as assessed by Western immunoblotting and (ii) on the astroglial and microglial activation as detected by both immunohistochemistry and Western immunoblotting for GFAP, and histochemistry for isolectin B4, respectively. Our data show that, although hypoxia and subsequent reoxygenation led to neither neuronal damage nor HSP72 induction in gerbil hippocampus, it induced a progressive and sustained expression of HO-1 and Mn SOD. As expected from the absence of neuronal death, hypoxia was not associated with microglial activation but led to a significant astrocytic activation. These findings demonstrate that transient hypoxia enhances the antioxidative enzymatic defenses of the brain, which are susceptible to increased tolerance against a subsequent damaging insult.