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Several signaling pathways and transcription factors control the cell fate in its in vitro development and differentiation. The orchestrated use of these factors results in cell specification. In coculture methods, many of these factors secrete from host cells but control the process. Today, transcription factors required for retinal progenitor cells are well known, but the generation of these cells from mesenchymal stem cells is an ideal goal. The purpose of the paper is to review novel methods for retinal progenitor cell production and selecting a set of signaling molecules in the presence of adult retinal pigment epithelium and extraocular mesenchyme acting as inducers of retinal cell differentiation.

There are limited treatments currently available for retinal diseases such as age-related macular degeneration (AMD). Cell-based therapy holds great promise in treating these degenerative diseases. Three-dimensional (3D) polymeric scaffolds have gained attention for tissue restoration by mimicking the native extracellular matrix (ECM). The scaffolds can deliver therapeutic agents to the retina, potentially overcoming current treatment limitations and minimizing secondary complications. In the present study, 3D scaffolds made up of alginate and bovine serum albumin (BSA) containing fenofibrate (FNB) were prepared by freeze-drying technique. The incorporation of BSA enhanced the scaffold porosity due to its foamability, and the Maillard reaction increased crosslinking degree between ALG with BSA resulting in a robust scaffold with thicker pore walls with a compression modulus of 13.08 KPa suitable for retinal regeneration. Compared with ALG and ALG-BSA physical mixture scaffolds, ALG-BSA conjugated scaffolds had higher FNB loading capacity, slower release of FNB in the simulated vitreous humour and less swelling in water and buffers, and better cell viability and distribution when tested with ARPE-19 cells. These results suggest that ALG-BSA MR conjugate scaffolds may be a promising option for implantable scaffolds for drug delivery and retinal disease treatment.

Retinal Müller glial cells have already been implicated in age-related macular degeneration (AMD). AMD is characterized by accumulation of toxic amyloid-β peptide (Aβ); the question we raise is as follows: is P2X7 receptor, known to play an important role in several degenerative diseases, involved in Aβ toxicity on Müller cells? Retinal Müller glial cells were incubated with Aβ for 48 h. Cell viability was assessed using the alamarBlue assay and cytotoxicity using the lactate dehydrogenase (LDH) release assay. P2X7 receptor expression was highlighted by immunolabeling observed on confocal microscopy and its activation was evaluated by YO-PRO-1 assay. Hoechst 33342 was used to evaluate chromatin condensation, and caspases 8 and 3 activation was assessed using AMC assays. Lipid formulation rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) used in Age-Related Eye Disease Study 2 was incubated on cells for 15 min prior to Aβ incubation. For the first time, we showed that Aβ induced caspase-independent apoptosis through P2X7 receptor activation on our retinal model. DHA and EPA are polyunsaturated fatty acids recommended in food supplement to prevent AMD. We therefore modulated Aβ cytotoxicity using a lipid formulation rich in DHA and EPA to have a better understanding of the results observed in clinical studies. We showed that fish oil rich in EPA and DHA, in combination with a potent P2X7 receptor antagonist, represents an efficient modulator of Aβ toxicity and that P2X7 could be an interesting therapeutic target to prevent AMD. Graphical Abstract ᅟ

Aim To investigate whether the human olfactory mucosa mesenchymal stem cells (OM-MSCs) can differentiate into photoreceptor cells in vitro. Methods Through the olfactory mucosa adherent method, olfactory mucosa was isolated, cultured and identified in vitro among mesenchymal stem cells. The cell surface markers were analyzed by flow cytometry, induced to differentiate into retinal photoreceptor cells in vitro, and the expression of rhodopsin was observed and identified by Immunofluorescence and Western blot methods. Results OM-MSCs from human were spindle cell-based, and showing radial colony arrangement. OM-MSCs were negative for CD34, CD45 and CD105, but positive for CD73 and CD90. Following induction, a strong positive reaction was produced by photoreceptor specific marker rhodopsin in the cells. Conslusion This novel finding demonstrates that OM-MSCs can be cultured and expanded in vitro. They possess biological characteristics of mesenchymal stem cells, and have the ability to be induced into retinal cells.

The retina is theater of a number of biochemical reactions allowing, within its layers, the conversion of light impulses into electrical signals. The axons of the last neuronal elements, the ganglion cells, form the optic nerve and transfer the signals to the brain. Therefore, an appropriate cellular communication, not only within the different retinal cells, but also between the retina itself and the other brain structures, is fundamental. One of the most diffuse pathologies affecting retinal function and communication, which thus reverberates in the whole visual system, is glaucoma. This insidious disease is characterized by a progressive optic nerve degeneration and sight loss which may finally lead to irreversible blindness. Nevertheless, the progressive nature of this pathology offers an opportunity for therapeutic intervention. To better understand the cellular processes implicated in the development of glaucoma useful to envision a targeted pharmacological strategy, this manuscript first examines the complex cellular and functional organization of the retina and subsequently identifies the targets sensitive to neurodegeneration. Within this context, high ocular pressure represents a key risk factor. However, recent literature findings highlight the concept that lowering ocular pressure is not enough to prevent/slow down glaucomatous damage, suggesting the importance of combining the hypotensive treatment with other pharmacological approaches, such as the use of neuroprotectants. Therefore, this important and more novel aspect is extensively considered in this review, also emphasizing the idea that the neuroprotective strategy should be extended to the entire visual system and not restricted to the retina.

Glutamate is an excitotoxin responsible for causing neuronal damage associated with mitochondria dysfunction. We have analyzed the relationship between the mitochondrial respiratory rate, the membrane potential ([Delta][Psi]) and the activity of mitochondrial complexes in retinal cells in culture, used as neuronal models. Glutamate (10 [mu]M-10 mM) dose-dependently decreased the O2 consumption and the membrane potential. A linear correlation was found between these parameters, suggesting that the mitochondrial respiratory function was affected. Exposure to glutamate (100 [mu]M) for 10 min, in the absence of Mg2+, inhibited the activity of complex I (26.3%), complexes II/III (22.2%) and complex IV (26.7%). MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), a non-competitive antagonist of the NMDA (N-methyl--aspartate) receptors, completely reversed the effect exerted by 100 [mu]M glutamate at the level of complexes I, II/III and IV. These results suggest that NMDA receptor-mediated inhibition of mitochondrial respiratory chain complexes may be responsible for the alteration in the respiratory rate of chick retinal cells submitted to glutamate. http://www.sciencedirect.com/science/article/B6T0B-43MK6KX-9/1/6efac4a526cb12bdb79436e4c10b9294

The somatostatinergic system of the retina has been investigated in a variety of studies. A considerable amount of experimental evidence is available concerning the patterns of expression of somatostatin (SRIF) and its receptors in vertebrate retinas. However the functional roles of this peptidergic system in retinal physiology are far from being elucidated. Nonetheless, data have been provided concerning the regulatory action of SRIF on the excitability of different retinal cell types and on the modulation of ion channels in different vertebrate retinas. The present review is focused on recent and unpublished investigations of the mouse retina relative to the involvement of specific SRIF receptors in the regulation of ion channels and transmitter release, the transduction pathways coupled to SRIF receptors, and the mechanisms regulating the expression of SRIF and its receptors as derived from studies in transgenic animal models. In these models, altered expression levels of SRIF or of specific SRIF receptors have also been found to affect the morphology of retinal cell types (namely the rod bipolar cells) and to result in functional alterations at the level of both ion channel regulation and transmitter release. These new pieces of evidence constitute an important step forward in the understanding of the functional actions of the retinal somatostatinergic system, although our current knowledge is far from being exhaustive. The ultimate goal of understanding SRIF functional actions in the retina is concerned with the possibility of using SRIF or its analogs as therapeutic agents to cure retinal diseases. Indeed, encouraging results are being obtained in clinical investigations focused on the use of SRIF analogs to treat diabetic retinopathy, a retinal disease with high social impact and originating as a complication of diabetes. The closing part of the present paper examines the evidence supporting SRIF as a promising therapeutic agent in this disease.

In the mammalian retina, sparse amacrine cells contain somatostatin-14 (SRIF) which acts at multiple levels of neuronal circuitry through distinct SRIF receptors (sst1-5). Among them, the sst1 receptor has been localized to SRIF-containing amacrine cells in the rat and rabbit retina. Little is known about sst1 receptor localization and function in the mouse retina. We have addressed this question in the retina of mice with deletion of sst1 receptors (sst1 KO mice). In the retina of wild type (WT) mice, sst1 receptors are localized to SRIF-containing amacrine cells whereas in the retina of sst1 KO mice, sst1 receptors are absent. sst1 receptor loss causes a significant increase in retinal levels of SRIF whereas it does not affect SRIF messenger RNA indicating that sst1 receptors play a role in limiting retinal SRIF at the post-transcriptional level. As another consequence of sst1 receptor loss, levels of expression of sst2 receptors are significantly higher than in control retinas. Together, these findings provide the first demonstration of prominent compensatory regulation in the mouse retina as a consequence of a distinct SRIF receptor deletion. The fact that in the absence of the sst1 receptor, retinal SRIF increases in concomitance with an increase in sst2 receptors suggests that SRIF may regulate sst2 receptor expression and that this regulatory process is controlled upstream by the sst1 receptor. This finding can be important in the design of drugs affecting SRIF function, not only in the retina, but also elsewhere in the brain. L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com/

The present review examines various aspects of the developmental expression of neuropeptides and of their receptors in mammalian retinas, emphasizing their possible roles in retinal maturation. Different peptidergic systems have been investigated with some detail during retinal development, including substance P (SP), somatostatin (SRIF), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP), neuropeptide Y (NPY), opioid peptides and corticotrophin-releasing factor (CRF). Overall, the developmental expression of most peptides is characterized by early appearance, transient features and achievement of the mature pattern at the time of eye opening. Concerning possible developmental actions of neuropeptides, recent studies imply a role of SP in the modulation of cholinergic neurotransmission in early postnatal rabbit retinas, when cholinergic cells participate in the retinal spontaneous waves of activity. In addition, the presence of transient SRIF expressing ganglion cells and recent observations in SRIF receptor knock-out mice indicate variegated roles of this peptide in the development of the retina and of retinofugal projections. Furthermore, VIP and PACAP exert protective and growth-promoting actions that may sustain retinal neurons during their development, and opioid peptides may control cell proliferation in the developing retina. Finally, a peak in the expression of certain peptides, including VIP, NPY and CRF, is present around the time of eye opening, when the retina begins the analysis of structured visual information, suggesting important roles of these peptides during this delicate phase of retinal development. In summary, although the physiological actions of peptides during retinal development are far from being clarified, the data reviewed herein indicate promising perspectives in this field of study.

For an understanding of the basis for psychophysical measurement of visual resolution, quantitative morphological studies of retinal neuronal architecture are needed. Here we report on cell densities and retinal ganglion cell:cone ratio (RGC:C) from the foveal border to the peripheral retina (34 degrees eccentricity). Quantitative estimates of RGC and C densities were made using a modified disector method in three vertically sectioned human retinae and were adjusted for RGC displacement. In agreement with our previous data on humans, we found an RGC:C ratio close to 3 at 2-3 degrees eccentricity. Outside the foveal border, the ratio declined to 1.0 at 7.5 degrees eccentricity and to 0.5 at eccentricities larger than 19 degrees. Center-to-center separation of C and RGC in addition to center-to-center separation of estimated 'receptive fields' was calculated at corresponding locations along the superior and inferior hemimeridians. The center-to-center separation of estimated 'receptive fields' was found to be more closely related to resolution thresholds from the fovea to 19 degrees eccentricity than was the separation of RGC and C. On the basis of these quantitative estimates, models for neural circuitry involved in central and peripheral spatial vision can be discussed.

Purpose: To examine retinal horizontal and bipolar cells cultured from goldfish (Carassius auratus), black crappie (Pomoxis nigromaculatus) and bluegill (Lepomis macrochirus) on the light and electron microscope levels; and to compare them with cells from a more established model, the wild white bass (Roccus chrysops). Methods: Retinas from all species were dissociated by methods developed with wild white bass (Dowling et al., 1985). Cell type was identified within the first 3 hr and cells were cultured for up to 2 wk in L-15 medium. At various intervals, cells were fixed in glutaraldehyde, photographed with phase LM and processed for SEM by either the ethanol dehydration method or the OTOTO method (Thompson et al., 1992 and modified Gabriel, 1982). Specimens were viewed with an Hitachi SEM. Each technique was evaluated for safety, simplicity, and image quality. Results: Bluegill and goldfish retina yielded few cells of inferior quality. Neurite outgrowth was limited. In contrast, crappie retina yielded adequate number of recognizable cell morphology, including: rods, cones, horizontal cells (H1s, H2s, H3s, H4s), and bipolar cells (BPs). These cells closely resembled the same cell types obtained from white bass (Vaughan & Lasater, 1990). The most numerous cells were H2s and H4s. Neurite outgrowth was acceptable and long-term survival of HCs and BPs was routine. H1s were observed still in contact with one another and presumably still connected by gap junctions. Neither of the two SEM techniques applied yielded superior results; rather both resulted in distracting artifacts. Conclusion: Crappie are easier to capture and maintain in the laboratory than wild white bass and their retinal cells perform in culture more like those from wild white bass than the other species tested. SEM observations confirm some axon-like and dendrite-like process growth of the bipolar cells and marginal growth in the H1s. The ethanol dehydration method for preparing cells for SEM is quick, simple and cost effective although it lacks the contrast and high resolution provided by the OTOTO method.

As developmental biologists we study the role of fetal movements in providing continuity between prenatal and postnatal life. There are two major categories of fetal motility. The first category consists of movements that have an obvious effect on the survival or development of the fetus (e.g., changes of position, sucking and swallowing). The second category consists of fetal movements that anticipate postnatal functions. For example, fetal ocular movements (FOMs) predict postnatal eye function (e.g., motion vision) of the newborn and therefore represent an important indicator of fetal health. However, while the clinical significance of fetal motility is obvious, its biological significance is elusive. We propose to use retina of genetically modified mouse embryos to study the biological role of FOMs in the genesis of cell diversity and organ functional maturation. Our results have already demonstrated the importance of fetal eye motility in the differentiation of cholinergic amacrine cells (CACs) in the retina (Kablar, 2003). Apparently, these cells are sensitive to motion and also responsible for motion vision. In the current report, we suggest employing the unique opportunity provided by the mouse Myf5-/-:MyoD-/- knock-outs that lack skeletal musculature and FOMs, microarray analysis and the follow-up experiments to identify a group of candidate genes that are essential for the molecular regulation of CAC differentiation and in turn for the functional maturation of the visual system towards its ability to perform motion vision. Finally, the molecules identified via this approach may be important in the mechanochemical signal transduction pathways employed during the process of conversion of a mechanical stimulus into an instruction understandable by the developing retinal neurons and glia cells.

The relationship between bioenergetics and the glutamate system was analyzed in a neuronal model of retinal cells in culture, submitted to glucose deprivation and exposed to glutamate for 2 h, and compared with exposure to glutamate in the presence of glucose. Under glucose deprivation, a reduction (about 1.1-fold) in the energy charge of the cells occurred, probably as a result of a decrement (by about 75%) in the cellular redox efficacy, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. In the absence of glucose, exposure of retinal cells to 10 [mu]M glutamate potentiated the reduction in the energy charge (by about 1.2-fold) and induced a significant increase in the uptake of45Ca2+by the cells (1.3-fold), although no significant changes were observed in the presence of glucose. Under glucose deprivation, 100 [mu]M glutamate caused an irreversible cell membrane damage, as shown by the significant increase in lactate dehydrogenase (LDH) leakage (about 1.8-fold). A significant increase in membrane depolarization, measured by the reduction of [3H]tetraphenylphosphonium+([3H]TPP+) uptake, was also observed after glutamate exposure in the absence of glucose. In the presence of glucose, high glutamate concentrations (10 mM) induced a major increase in Ca2+entry into the cells and membrane depolarization, without affecting the energy charge or cell survival. In contrast, in the absence of glucose, 10 mM glutamate did not alter Ca2+accumulation by the cells and a smaller decrease in membrane potential occurred, as compared to 100 [mu]M glutamate exposure. Data shown in this study suggest that during a prolonged (2 h) and acute exposure to high glutamate (10 mM), under glucose deprivation conditions, the neuronal systems have "adaptive" mechanisms that allow the survival of cells. These findings may have implications in neuronal degeneration occurring during brain ischemia. http://www.sciencedirect.com/science/article/B6WFG-45K0YG1-1F/1/f72f07d2f8f34513657ace7d1febf7ca