Resveratrol, a polyphenol presents in grapes and wine, displays antioxidant and anti-inflammatory properties and cytoprotective effect in brain pathologies associated to oxidative stress and neurodegeneration. In previous work, we demonstrated that resveratrol exerts neuroglial modulation, improving glial functions, mainly related to glutamate metabolism. Astrocytes are a major class of glial cells and regulate neurotransmitter systems, synaptic processing, energy metabolism and defense against oxidative stress. This study sought to determine the protective effect of resveratrol against hydrogen peroxide (H2O2)-induced cytotoxicity in C6 astrocyte cell line, an astrocytic lineage, on neurochemical parameters and their cellular and biochemical mechanisms. H2O2 exposure increased oxidative-nitrosative stress, iNOS expression, cytokine proinflammatory release (TNFα levels) and mitochondrial membrane potential dysfunction and decreased antioxidant defenses, such as SOD, CAT and creatine kinase activity. Resveratrol strongly prevented C6 cells from H2O2-induced toxicity by modulating glial, oxidative and inflammatory responses. Resveratrol per se increased heme oxygenase 1 (HO1) expression and extracellular GSH content. In addition, HO1 signaling pathway is involved in the protective effect of resveratrol against H2O2-induced oxidative damage in astroglial cells. Taken together, these results show that resveratrol represents an important mechanism for protection of glial cells against oxidative stress.

To determine if exogenous addition of tumor necrosis factor alpha (TNFα) exacerbates retinal reactive gliosis in an organotypic culture of porcine neuroretina and to evaluate if concomitant adalimumab, a TNF-blocker, diminishes it.Porcine retinal explants from 20 eyeballs were cultured. Cultures with 100 pg/ml TNFα, 10 µg/ml adalimumab, 100 pg/ml TNFα plus 10 µg/ml adalimumab, or controls without additives were maintained for 9 days. Freshly detached retinas were processed in parallel. TNFα levels in control culture supernatants were quantified with enzyme-linked immunosorbent assay. Cryostat sections were doubly immunostained for glial fibrillary acidic protein (GFAP), a marker for reactive gliosis, and cellular retinaldehyde-binding protein (CRALBP), a marker for Müller cells. Sections were also labeled with the isolectin IB4, a label for microglia/macrophages.TNFα in control culture supernatants was detected only at day 1. Compared to the fresh neuroretinal samples, upregulation of GFAP and downregulation of CRALBP occurred during the 9 days of culture. Exogenous TNFα stimulated glial cells to upregulate GFAP and downregulate CRALBP immunoreactivity. TNFα-treated cultures also initiated the growth of gliotic membranes and underwent retinal disorganization. Adalimumab inhibited the spontaneous increases in GFAP and maintained CRALBP. In combination with TNFα, adalimumab reduced GFAP expression and conserved CRALBP, with only slight retinal disorganization. No appreciable changes in IB4 labeling were observed under the different culture conditions.In cultured porcine neuroretina, spontaneous reactive gliosis and retinal disorganization were exacerbated by exogenous TNFα. Adalimumab reduced spontaneous changes and those induced by TNFα. Therefore, inhibiting TNFα may represent a novel approach to controlling retinal fibrosis observed in some human diseases.

Pannexons are membrane channels formed by pannexins and are permeable to ATP. They have been implicated in various physiological and pathophysiological processes. Innexins, the invertebrate homologues of the pannexins, form innexons. Nerve injury induces calcium waves in glial cells, releasing ATP through glial pannexon/innexon channels. The ATP then activates microglia. More slowly, injury releases arachidonic acid (ArA). The present experiments show that ArA itself reduced the macroscopic membrane currents of innexin- and of pannexin-injected oocytes; ArA also blocked K(+) -induced release of ATP. In leeches, whose large glial cells have been favorable for studying control of microglia migration, ArA blocked glial dye-release and, evidently, ATP-release. A physiological consequence in the leech was block of microglial migration to nerve injuries. Exogenous ATP (100 µM) reversed the effect, for ATP causes activation and movement of microglia after nerve injury, but nitric oxide directs microglia to the lesion. It was not excluded that metabolites of ArA may also inhibit the channels. But for all these effects, ArA and its non-metabolizable analogue eicosatetraynoic acid (ETYA) were indistinguishable. Therefore, ArA itself is an endogenous regulator of pannexons and innexons. ArA thus blocks release of ATP from glia after nerve injury and thereby, at least in leeches, stops microglia at lesions. © 2013 Wiley Periodicals, Inc. Develop Neurobiol, 2013.

In retinal proliferative diseases, Müller glial cells (MGCs) acquire migratory abilities. However, the mechanisms that regulate this migration remain poorly understood. In addition, proliferative disorders associated with enhanced activities of matrix metalloprotease 2 (MMP-2) and MMP-9 also present increased levels of the protease inhibitor α2-macroglobulin (α2M) and its receptor, the low-density lipoprotein receptor-related protein 1 (LRP1). In the present work, we investigated whether the protease activated form of α2M, α2M*, and LRP1 are involved with the MGC migratory process. By performing wound-scratch migration and zymography assays, we demonstrated that α2M* induced cell migration and proMMP-2 activation in the human Müller glial cell line, MIO-M1. This induction was blocked when LRP1 and MT1-MMP were knocked down with siRNA techniques. Using fluorescence microscopy and biochemical procedures, we found that α2M* induced an increase in LRP1 and MT1-MMP accumulation in early endosomes, followed by endocytic recycling and intracellular distribution of MT1-MMP toward cellular protrusions. Moreover, Rab11-dominant negative mutant abrogated MT1-MMP recycling pathway, cell migration, and proMMP-2 activation induced by α2M*. In conclusion, α2M*, through its receptor LRP1, induces cellular migration of Müller glial cells by a mechanism that involves MT1-MMP intracellular traffic to the plasma membrane by a Rab11-dependent recycling pathway.-Barcelona, P. F., Jaldín-Fincati, J. R., Sánchez, M. C., Chiabrando, G. A. Activated α2-macroglobulin induces Müller glial cell migration by regulating MT1-MMP activity through LRP1.

Optical coherence tomography (OCT) provides a new dimenstion in ophthalmology because it allows evaluation of the pathology in vivo, and provides information to assist the management of macular disease and glaucoma. It is necessary to differentiate the diagnosis of glaucoma from diseases of the optic nerve and of the visual pathway. This study evaluates the usefulness of OCT in detecting disorders of the optic nerve and visual pathway. In addition, the pathogenesis of glaucomatous optic neuropathy (GON), the most common optic neuropathy, was investigated by focusing on the dynamics of aquaporin. I. Evaluation of optic nerve and visual pathway disorders by optical coherence tomography. The swinging flashlight test is an easy, sensitive, objective test to detect relative afferent pupillary defects (RAPD). The number of RAPD detected by the swinging flashlight test was closely correlated with the ratio of retinal nerve fiber layer thickness (RNFLT) between the two eyes of 20 cases of unilateral optic atrophy. OCT could assess the amount of RAPD that reflected an asymmetrical functional disturbance of the optic nerves, as a structural difference. The time courses of RNFLT and ganglion cell complex (GCC) changes' were observed immediately following the time of injury in 4 cases of traumatic optic neuropathy. OCT revealed that both the RNFLT and GCC decreased rapidly from 2 weeks after the injury until 20 weeks later. The RNFLT decreased significantly in the horizontal direction in comparison to the perpendicular direction in 34 eyes from the cases of optic chiasm syndrome. This means that OCT could quantitatively detect the band atrophy of the optic disc in optic chiasm syndrome. Measuring the RNFLT showed a thinning of RNFLT in the perpendicular direction in comparison to the horizontal direction in ipsilateral eyes and thinning in the horizontal direction in comparison to the perpendicular direction in the contralateral eyes in optic tract syndrome. Measuring the GCC showed a thinning of the GCC in the temporal hemifield to the central fovea of the ipsilateral eyes, and thinning of the GCC in the nasal hemifield of the contralateral eyes. This means that OCT could detect the structural changes of hourglass atrophy in the ipsilateral eye and band atrophy in the contralateral eye at the optic disc as well as the homonymous hemianopia in the visual field. OCT was useful in evaluating the optic nerve and visual pathway disorders, but there were also some limitations. The thinning area of RNFLT measured by OPTVue and Cirrus were in entirely opposite directions in cases of optic chasm syndrome. The reason was attributed to the better performance of RTVue in measuring a thin RNFLT on the nasal side of the optic disc in comparison to Cirrus. The specific characteristics of the instruments should be considered when the results of OCT are evaluated. II. Dynamics of aquaporin in the optic nerve Aquaporin (AQP) is a membrane protein that forms a water channel to facilitate water crossing the plasma membrane. AQP-4 was originally thought to be expressed in the optic nerve, but it is expressed only in the retrobulbar medullated region of the optic nerve and the expression of AQPs in the optic disc has not been detected. This study investigated the expressions of AQPs in the optic nerve in rat, monkey and human. The results demonstrate that only AQP-9 was expressed at the unmedullated pre-lamina cribrosa and lamina cribrosa regions, and both AQP-4 and AQP-9 were expressed at the medullated retrobulbar region. Astrocytes were observed to express AQP-9, because AQP-9 immunoreactivity was identical to that of glial fibrillary acidic protein. Elevated intraocular pressure substantially reduced AQP-9 expression in the optic nerve, whereas expression of AQP-4 was not changed in rat eyes. The same phenomena were also observed in the monkey eye with ocular hypertension as well as human eye with glaucoma. AQP-9 is an aquaglyceroporin that allows solutes such as lactate rather than water to cross the cell membrane. The astrocyte-to-neuron lactate shuttle hypothesis has been proposed, in which lactate transported from astrocytes is used by neurons as an energy substrate. Reduction of AQP-9 expression in the optic nerve head under elevated intraocular pressures might be closely related to the pathogenesis of GON.

Despite the advances made in the field of regenerative medicine, the progress in cutting-edge technologies for separating target therapeutic cells are still at early stage of development. These cells are often rare, such as stem cells or progenitor cells that their overall properties should be maintained during the separation process for their subsequent application in regenerative medicine. This work, presents separation of oligodendrocyte progenitor cells (OPCs) from rat brain primary cultures using an integrated thermoplastic elastomeric (TPE)- based multilayer microfluidic device fabricated using hot-embossing technology. OPCs are frequently used in recovery, repair and regeneration of central nervous system after injuries. Indeed, their ability to differentiate in vitro into myelinating oligodendrocytes, are extremely important for myelin repair. OPCs form 5-10% of the glial cells population. The traditional macroscale techniques for OPCs separation require pre-processing of cells and/or multiple time consuming steps with low efficiency leading very often to alteration of their properties. The proposed methodology implies to separate OPCs based on their smaller size compared to other cells from the brain tissue mixture. Using aforementioned microfluidic chip embedded with a 5 μm membrane pore size and micropumping system, a separation efficiency more than 99% was achieved. This microchip was able to operate at flow rates up to 100 μl/min, capable of separating OPCs from a confluent 75 cm(2) cell culture flask in less than 10 min, which provides us with a high-throughput and highly efficient separation expected from any cell sorting techniques.

In mammals, the biological activity of the stem/progenitor compartment sustains production of mature gametes through spermatogenesis. Spermatogonial stem cells and their progeny belong to the class of undifferentiated spermatogonia, a germ cell population found on the basal membrane of the seminiferous tubules. A large body of evidence has demonstrated that glial cell line-derived neurotrophic factor (GDNF), a Sertoli-derived factor, is essential for in vivo and in vitro stem cell self-renewal. However, the mechanisms underlying this activity are not completely understood. In this study, we show that GDNF induces dose-dependent directional migration of freshly selected undifferentiated spermatogonia, as well as germline stem cells in culture, using a Boyden chamber assay. GDNF-induced migration is dependent on the expression of the GDNF co-receptor GFRA1, as shown by migration assays performed on parental and GFRA1-transduced GC-1 spermatogonial cell lines. We found that the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) is specifically expressed in undifferentiated spermatogonia. VASP belongs to the ENA/VASP family of proteins implicated in actin-dependent processes, such as fibroblast migration, axon guidance, and cell adhesion. In intact seminiferous tubules and germline stem cell cultures, GDNF treatment up-regulates VASP in a dose-dependent fashion. These data identify a novel role for the niche-derived factor GDNF, and they suggest that GDNF may impinge on the stem/progenitor compartment, affecting the actin cytoskeleton and cell migration.

Perivascular cells of microvascular niches are the prime candidates for being a reservoire of mesenchymal stem cell (MSC)-like cells in many tissues and organs that could serve as a potential source of cells and a target of novel cell-based therapeutic approaches. In the present study, by utilising typical markers of pericytes (neuronal-glial antigen 2, NG2, a chondroitin sulphate proteoglycan) and those of MSCs (CD146 and CD105) and primitive pluripotent cells (sex-determining region Y-box 2, Sox2), the phenotypic traits and the distribution of murine and rat retinal perivascular cells were investigated in situ. Our findings indicate that retinal microvessels of juvenile rodents are highly covered by NG2-positive branching processes of pericytic (perivascular) cells that are less prominent in mature capillary networks of the adult retina. In the adult rodent retinal vascular bed, NG2 labeling is mainly confined to membranes of the cell body resulting in a pearl-chain-like distribution along the vessels. Retinal pericytes, which were identified by their morphology and NG2 expression, simultaneously express CD146. Furthermore, CD146-positive cells located at small arteriole-to-capillary branching points appear more intensely stained than elsewhere. Evidence for a differential expression of the two markers around capillaries that would hint at a clonal heterogeneity among pericytic cells, however, is lacking. In contrast, the expression of CD105 is exclusively restricted to vascular endothelial cells and Sox2 is detected neither in perivascular nor in endothelial cells. In dissociated retinal cultures, however, simultaneous expression of NG2 and CD105 was observed. Collectively, our data indicate that vascular wall resident retinal pericytes share some phenotypic features (i.e. CD146 expression) with archetypal MSCs, which is even more striking in dissociated retinal cultures (i.e. CD105 expression). These findings might have implications for the treatment of retinal pathologies.

The potential nanocarrier polyamidoamine (PAMAM) generation 5 (G5-NH2) dendrimer has been shown to evoke lasting neuronal depolarization and cell death in a concentration-dependent manner. In this study we explored the early progression of G5-NH2 action in brain tissue on neuronal and astroglial cells.In order to describe early mechanisms of G5-NH2 dendrimer action in brain tissue we assessed G5-NH2 trafficking, free intracellular Ca2+ and mitochondrial membrane potential (ΨMITO) changes in the rat hippocampal slice by microfluorimetry. With the help of fluorescent dye conjugated G5-NH2, we observed predominant appearance of the dendrimer in the plasma membrane of pyramidal neurons and glial cells within 30 min. Under this condition, G5-NH2 evoked robust intracellular Ca2+ enhancements and ΨMITO depolarization both in pyramidal neurons and astroglial cells. Intracellular Ca2+ enhancements clearly preceded ΨMITO depolarization in astroglial cells. Comparing activation dynamics, neurons and glia showed prevalence of lasting and transient ΨMITO depolarization, respectively. Transient as opposed to lasting ΨMITO changes to short-term G5-NH2 application suggested better survival of astroglia, as observed in the CA3 stratum radiatum area. We also showed that direct effect of G5-NH2 on astroglial ΨMITO was significantly enhanced by neuron-astroglia interaction, subsequent to G5-NH2 evoked neuronal activation.These findings indicate that the interaction of the PAMAM dendrimer with the plasma membrane leads to robust activation of neurons and astroglial cells, leading to mitochondrial depolarization. Distinguishable dynamics of mitochondrial depolarization in neurons and astroglia suggest that the enhanced mitochondrial depolarization followed by impaired oxidative metabolism of neurons may be the primary basis of neurotoxicity.

Optogenetic manipulation of the neuronal activity enables one to analyze the neuronal network both in vivo and in vitro with precise spatio-temporal resolution. Channelrhodopsins (ChRs) are light-sensitive cation channels that depolarize the cell membrane, whereas halorhodopsins and archaerhodopsins are light-sensitive Cl(-) and H(+) transporters, respectively, that hyperpolarize it when exogenously expressed. The cause-effect relationship between a neuron and its function in the brain is thus bi-directionally investigated with evidence of necessity and sufficiency. In this review we discuss the potential of optogenetics with a focus on three major requirements for its application: (i) selection of the light-sensitive proteins optimal for optogenetic investigation, (ii) targeted expression of these selected proteins in a specific group of neurons, and (iii) targeted irradiation with high spatiotemporal resolution. We also discuss recent progress in the application of optogenetics to studies of non-neural cells such as glial cells, cardiac and skeletal myocytes. In combination with stem cell technology, optogenetics may be key to successful research using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) derived from human patients through optical regulation of differentiation-maturation, through optical manipulation of tissue transplants and, furthermore, through facilitating survival and integration of transplants.