Pax6-positive Müller glia cells express cell cycle markers but do not proliferate after photoreceptor injury in the mouse retina

In lower vertebrates, such as fish, Müller glia plays an essential role in the restoration of visual function after retinal degeneration by transdifferentiating into photoreceptors and other retinal neurons. During this process, Müller cells re-enter the cell cycle, proliferate, and migrate from the inner nuclear layer (INL) to the photoreceptor layer where they express photoreceptor-specific markers. This process of Müller cell transdifferentiation is absent in mammals, and the loss of photoreceptors leads to permanent vision deficits.The mechanisms underlying the failure of mammalian Müller cells to behave as stem cells after photoreceptor degeneration are poorly understood. In the present study, we show that photoreceptor injury induces migration of PAX6-positive Müller cell nuclei toward the outer part of the INL and into the inner part of the outer nuclear layer. These cells express markers of the cell cycle, suggesting an attempt to re-enter the cell cycle similarly to lower vertebrates.However, mouse Müller cells do not proliferate in response to photoreceptor injury implying a blockade of the S-phase transition. Our results suggest that a release of the S-phase blockade may be crucial for Müller cells to successfully transdifferentiate and replace injured photoreceptors in mammals.     

小鼠视网膜前体细胞的视网膜下腔移植

The development of cell replacement techniques is promising as a potential treatment for photoreceptor loss. However, the limited integration ability of donor and recipient cells presents a challenge following transplantation. In the present study, retinal progenitor cells (RPCs) were harvested from the neural retinas of enhanced green fluorescent protein mice on postnatal day 1, and expanded in a neurobasal medium supplemented with fetal bovine serum without endothelial growth factor. Using a confocal microscope, immunohistochemistry demonstrated that expanded RPCs in vitro maintain retinal stem cell properties and can be differentiated into photoreceptor cells. Three weeks after transplantation, subretinal transplanted RPCs were found to have migrated and integrated into the outer nuclear layer of recipient retinas with laser injury, some of the integrated cells had differentiated into photoreceptors, and a subpopulation of these cells expressed photoreceptor specific synaptic protein, appearing to form synaptic connections with bipolar cells. These results suggest that subretinal transplantation of RPCs may provide a feasible therapeutic strategy for the loss of retinal photoreceptor cells.     

Studies of host-graft interactions in vitro

Progenitor and stem cell transplantation represent therapeutic strategies for retinal disorders that are accompanied by photoreceptor degeneration. The transplanted cells may either replace degenerating photoreceptors or secrete beneficial factors that halt the processes of photoreceptor degeneration. The present study analyzes whether rat retinal progenitor cells differentiated into photoreceptor phenotypic cells in neurospheres have a potential to interact with rat retinal explants. Immunocytochemistry for rhodopsin and synaptophysin indicated photoreceptor cell-like differentiation in neurospheres that were stimulated by basic fibroblast growth factor and epidermal growth factor. Differentiation into neural phenotypes including photoreceptor cells was effectively blocked by an addition of leukemia inhibitory factor. Grafting of neurospheres onto retinal explants demonstrated a consistent penetration of glial cell processes into the explanted tissue. On the other hand, the incorporation of donor cells into explants was very low. A general finding was that neurospheres grafting was associated with local decrease in Müller cell activation in the explants. Further characterization of these effect(s) could provide further insight into progenitor cell-based therapies of retinal degenerative disorders.     

Paradoxical opsin expressing cells in the inner retina that are augmented following retinal degeneration

Here we reveal a population of cells that express cone photoreceptor opsins that are located in the inner retina, distant from outer retinal photoreceptors. These cells are present in rodents and human. They also express a range of key proteins critical in the cone phototransduction cascade and make contact with other retinal neurons. Their opsins are not generally confined to cellular specialized regions but are present throughout the plasma membrane, although their nuclear configurations are similar to those of outer retinal cones. This population is distinct from the ganglion cells that contain melanopsin and which are known to be inner retinal irradiance detectors regulating circadian behaviour. Surprisingly, the size of the population of short wavelength opsin positive cells in the ganglion cell layer is plastic. In normal animals their number declines with age. However, their numbers increase significantly in response to outer retinal photoreceptor loss, probably by drawing on a pool of inner retinal cells that express cone specific markers, but not opsins.     

Photoreceptor layer reconstruction in a rodent model of retinal degeneration.

We have examined the potential of retinal cell transplantation to dystrophic retinal degeneration mice as a way of replacing photoreceptors lost because of an intrinsic genetic defect. Early postnatal retinae which had been gently dissociated survived for at least 6 weeks after transplantation to the subretinal space. Over a significant area of distribution, transplanted cells formed outer segments which lay in close apposition to the host retinal pigment epithelial cell layer. The grafts integrated with the remaining host retina, sufficient at least to mediate a simple light-dark preference. A new synaptic layer was seen at the graft-host interface, which contained substantial numbers of photoreceptor synapses. This and the fact that the behavior could be elicited at low luminance levels argue for functional circuit reconstruction between grafted cells and host retina.     

Photoreceptor transplantation: anatomic, electrophysiologic, and behavioral evidence for the functional reconstruction of retinas lacking photoreceptors.

We have investigated the possibility of using transplantation of immature or mature rodent photoreceptors as well as mature human photoreceptors to reconstruct retinas in which photoreceptor degeneration is either inherited or environmentally induced. To this end, we have devised methods for isolating and transplanting the outer nuclear layer (ONL) (e.g., the photoreceptor layer) to the subretinal space of mature rodents. In addition we found that if portions of the inner retina are transplanted along with the intact photoreceptor sheet, photoreceptor organization is better maintained. In ultrastructural studies of the reconstructed retina an outer plexiform-like layer (OPL) is visible at the interface of the transplanted ONL and the host inner nuclear layer, with invaginating ribbon synapses characteristic of those formed by rod photoreceptors evident within this OPL. Ribbon synapses are found only rarely in unreconstructed retina. These results suggest that synaptic connections between transplanted photoreceptors and host cells may be made. Evidence for the potential recovery of function following photoreceptor transplantation is found in visually evoked cortical responses and behavioral responses (pupillary reflex) to light stimulation of the reconstructed eye. These findings suggest the possibility that neural transplantation can reconstruct a sensory end organ--in this case the retina--to restore evoked activity and an appropriate behavioral response to sensory stimulation.     

Müller glial cells induce stem cell properties in retinal progenitors in vitro and promote their further differentiation into photoreceptors

Using stem cells to replace lost neurons is a promising strategy for treating retinal neurodegenerative diseases. Among their multiple functions, Müller glial cells are retina stem cells, with a robust regenerative potential in lower vertebrates, which is much more restricted in mammals. In rodents, most retina progenitors exit the cell cycle immediately after birth, differentiate as neurons, and then cannot reenter the cell cycle. Here we demonstrate that, in mixed cultures with Müller glial cells, rat retina progenitor cells expressed stem cell properties, maintained their proliferative potential, and were able to preserve these properties and remain mitotically active after several consecutive passages. Notably, these progenitors retained the capacity to differentiate as photoreceptors, even after successive reseedings. Müller glial cells markedly stimulated differentiation of retina progenitors; these cells initially expressed Crx and then developed as mature photoreceptors that expressed characteristic markers, such as opsin and peripherin. Moreover, they were light responsive, insofar as they decreased their cGMP levels when exposed to light, and they also showed high-affinity glutamate uptake, a characteristic of mature photoreceptors. Our present findings indicate that, in addition to giving rise to new photoreceptors, Müller glial cells might instruct a pool of undifferentiated cells to develop and preserve stem cell characteristics, even after successive reseedings, and then stimulate their differentiation as functional photoreceptors. This complementary mechanism might contribute to enlarge the limited regenerative capacity of mammalian Müller cells.     

An Evaluation of Neurotoxicity Following Fluoride Exposure from Gestational Through Adult Ages in Long-Evans Hooded Rats

Antibiotics such as gentamicin (an aminoglycoside) and penicillin (a beta-lactam antibiotic) are routinely used in retinal cell and explant cultures. In many cases, these in vitro systems are testing parameters regarding photoreceptor transplantation or preparing cells for transplantation. In vivo, milligram doses of gentamicin are neurotoxic to the retina. However, little is known about the effects of antibiotics to retina in vitro and whether smaller doses of gentamicin are toxic to retinal cells. To test toxicity, retinal cells were dissociated from tiger salamander, placed in culture, and treated with either 20 μg/ml gentamicin, 100 μg/ml streptomycin, 100 U/ml antibiotic/antimycotic, 0.25 μg/ml amphotericin B, or 100 U/ml penicillin G. All dosages were within manufacturer’s recommended levels. Control cultures had defined medium only. Cells were fixed at 2 h or 7 days. Three criteria were used to assess toxicity: (1) survival of retinal neurons, (2) neuritic growth of photoreceptors assessed by the development of presynaptic varicosities, and (3) survival and morphology of Mueller cells. Rod cells were immunolabeled for rod opsin, Mueller cells for glial fibrillary acidic protein, and varicosities for synaptophysin. Neuronal cell density was reduced with all pharmacological treatments. The number of presynaptic varicosities was also significantly lower in both rod and cone photoreceptors in treated compared to control cultures; further, rods were more sensitive to gentamicin than cones. Penicillin G (100 U/ml) was overall the least inhibitory and amphotericin B the most toxic of all the agents to photoreceptors. Mueller cell survival was reduced with all treatments; reduced survival was accompanied by the appearance of proportionally fewer stellate and more rounded glial morphologies. These findings suggest that even microgram doses of antibiotic and antimycotic drugs can be neurotoxic to retinal cells and reduce neuritic regeneration in cell culture systems.     

PALS1 is essential for retinal pigment epithelium structure and neural retina stratification

The membrane-associated palmitoylated protein 5 (MPP5 or PALS1) is thought to organize intracellular PALS1-CRB-MUPP1 protein scaffolds in the retina that are involved in maintenance of photoreceptor-Müller glia cell adhesion. In humans, the Crumbs homolog 1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, especially PALS1, we generated and analyzed conditional knockdown mice for Pals1. Small irregularly shaped spots were detected throughout the PALS1 deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. The electroretinography a- and b-wave was severely attenuated in the aged mutant retinas, suggesting progressive degeneration of photoreceptors. The histological analysis showed abnormal retinal pigment epithelium structure, ectopic photoreceptor nuclei in the subretinal space, an irregular outer limiting membrane, half rosettes of photoreceptors in the outer plexiform layer, and a thinner photoreceptor synaptic layer suggesting improper photoreceptor cell layering during retinal development. The PALS1 deficient retinas showed reduced levels of Crumbs complex proteins adjacent to adherens junctions, upregulation of glial fibrillary acidic protein indicative of gliosis, and persisting programmed cell death after retinal maturation. The phenotype suggests important functions of PALS1 in the retinal pigment epithelium in addition to the neural retina.     

Photoreceptor morphology is severely affected in the beta,beta-carotene-15,15'-oxygenase (bcox) zebrafish morphant

The retinoic acid molecule, a vitamin A derivative, is of key importance for eye and photoreceptor development in vertebrates. Several studies have provided evidence that the ventral part of the retina is particularly susceptible to impairment in retinoid signalling during the period of its development. In zebrafish, targeted gene knockdown of beta,beta-carotene-15,15'-oxygenase (bcox), the key enzyme for vitamin A formation, provokes a loss of retinoid signalling during early eye development that results in microphthalmia at larval stages. Using this model, we analysed the consequences of this for the retinal morphology of the fish larvae in structural details. Our analyses revealed that rods and cones do not express photoreceptor specific proteins (rhodopsin, peanut agglutinin, zpr1) in the peripheral retina. The photoreceptors in the central retina showed shortened outer segments, and electron dense debris in their intermembranal space. The number of phagosomes was increased, and cell death was frequently observed in the outer nuclear layer. Furthermore, the number of Muller cells was significantly reduced in the inner nuclear layer. Thus, we found that the lack of retinoid signalling strongly effects photoreceptor development in the ventral and dorsal retina. In addition, shortened outer segments and cell death of the remaining photoreceptors in the central retina indicate that there is an ongoing need for retinoid signalling for photoreceptor integrity and survival at later developmental stages.     

Retinal pigment epithelial cells promote spatial reorganization and differentiation of retina photoreceptors

Retina differentiation involves the acquisition of a precise layered arrangement, with RPE cells in the first layer in intimate contact with photoreceptors in the second layer. Here, we developed an in vitro coculture model, to test the hypothesis that RPE cells play a pivotal role in organizing the spatial structure of the retina. We cocultured rat retinal neurons with ARPE-19 epithelial cells under various experimental conditions. Strikingly, when seeded over RPE cells, photoreceptors attached to their apical surfaces and proceeded with their development, including the increased synthesis of rhodopsin. Conversely, when we seeded RPE cells over neurons, the RPE cells rapidly detached photoreceptors from their substrata and positioned themselves underneath, thus restoring the normal in vivo arrangement. Treatment with the metalloproteinase inhibitor TIMP-1 blocked this reorganization, suggesting the involvement of metalloproteinases in this process. Reorganization was highly selective for photoreceptors because 98% of photoreceptors but very few amacrine neurons were found to redistribute on top of RPE cells. Interestingly, RPE cells were much more efficient than other epithelial or nonepithelial cells in promoting this reorganization. RPE cells also promoted the growth of photoreceptor axons away from them. An additional factor that contributed to the distal arrangement of photoreceptor axons was the migration of photoreceptor cell bodies along their own neurites toward the RPE cells. Our results demonstrate that RPE and photoreceptor cells interact in vitro in very specific ways. They also show that in vitro studies may provide important insights into the process of pattern formation in the retina.     

Lutein delays photoreceptor degeneration in a mouse model of retinitis pigmentosa

Retinitis pigmentosa is a retinal disease characterized by photoreceptor degeneration.There is currently no effective treatment for retinitis pigmentosa.Although a mixture of lutein and other antioxidant agents has shown promising effects in protecting the retina from degeneration,the role of lutein alone remains unclear.In this study,we administered intragastric lutein to Pde6brd10 model mice,which display degeneration of retinal photoreceptors,on postnatal days 17(P17)to P25,when rod apoptosis reaches peak.Lutein at the optimal protective dose of 200 mg/kg promoted the survival of photoreceptors compared with vehicle control.Lutein increased rhodopsin expression in rod cells and opsin expression in cone cells,in line with an increased survival rate of photoreceptors.Functionally,lutein improved visual behavior,visual acuity,and retinal electroretinogram responses in Pde6brd10 mice.Mechanistically,lutein reduced the expression of glial fibrillary acidic protein in Müller glial cells.The results of this study confirm the ability of lutein to postpone photoreceptor degeneration by reducing reactive gliosis of Müller cells in the retina and exerting anti-inflammatory effects.This study was approved by the Laboratory Animal Ethics Committee of Jinan University(approval No.LACUC-20181217-02)on December 17,2018.     

Adenosine triphosphate‐induced photoreceptor death and retinal remodeling in rats

Many common causes of blindness involve the death of retinal photoreceptors, followed by progressive inner retinal cell remodeling. For an inducible model of retinal degeneration to be useful, it must recapitulate these changes. Intravitreal administration of adenosine triphosphate (ATP) has recently been found to induce acute photoreceptor death. The aim of this study was to characterize the chronic effects of ATP on retinal integrity. Five‐week‐old, dark agouti rats were administered 50 mM ATP into the vitreous of one eye and saline into the other. Vision was assessed using the electroretinogram and optokinetic response and retinal morphology investigated via histology. ATP caused significant loss of visual function within 1 day and loss of 50% of the photoreceptors within 1 week. At 3 months, 80% of photoreceptor nuclei were lost, and total photoreceptor loss occurred by 6 months. The degeneration and remodeling were similar to those found in heritable retinal dystrophies and age‐related macular degeneration and included inner retinal neuronal loss, migration, and formation of new synapses; Müller cell gliosis, migration, and scarring; blood vessel loss; and retinal pigment epithelium migration. In addition, extreme degeneration and remodeling events, such as neuronal and glial migration outside the neural retina and proliferative changes in glial cells, were observed. These extreme changes were also observed in the 2‐year‐old P23H rhodopsin transgenic rat model of retinitis pigmentosa. This ATP‐induced model of retinal degeneration may provide a valuable tool for developing pharmaceutical therapies or for testing electronic implants aimed at restoring vision. J. Comp. Neurol. 522:2928–2950, 2014. © 2014 Wiley Periodicals, Inc.     

Inner retinal abnormalities in a mouse model of Leber's congenital amaurosis

Leber's congenital amaurosis (LCA) is the earliest and most severe form in the world of genetic retinal dystrophy causing blindness. An animal model of LCA was recently created in which the cone-rod homeobox (crx) gene was disrupted using homologous recombination. Crx-/- mice display abnormal development of photoreceptors followed by their degeneration. We analyzed the morphology of inner retinal cells in crx-/- mice in order to evaluate the effects of abnormal photoreceptor development and death upon other retinal neurons. The identification of a time window during which inner retinal cells are still viable could be very important in view of the possibilities that photoreceptor transplantation or gene therapy might be used to restore vision in LCA. We used a combination of immunocytochemical and confocal microscopy techniques to screen the crx-/- inner retina and verify its morphological integrity after photoreceptor degeneration. We found significant morphological alterations in second-order neurons in crx-/- animals. The appearance of mutant retinas after photoreceptor death is indistinguishable from that of the retinal degeneration (rd/rd) mouse, a different genetic model of a retinal disease characterized by photoreceptor degeneration. However, at early stages of photoreceptor degeneration the morphology of retinal cells in the crx-/- mutant is considerably well preserved. It is likely that different genetic mechanisms that cause abnormal photoreceptor development and/or degeneration lead to a common pathway that determines second-order neuron modifications. The severity of modifications is linked to the timing of onset of the degeneration and appears to increase with time.     

Widespread Integration and Survival of Adult-Derived Neural Progenitor Cells in the Developing Optic Retina

Adult rat hippocampus-derived neural progenitor cells (AHPC) show considerable adaptability following grafting to several brain regions. To evaluate the plasticity of AHPCs within the optic retina, retrovirally engineered AHPCs were grafted into the vitreous cavity of the adult and newborn rat eye. Within the adult eye, AHPCs formed a uniform nondisruptive lamina in intimate contact with the inner limiting membrane. Within 4 weeks of grafting to the developing eye, the AHPCs were well integrated into the retina and adopted the morphologies and positions of Müller, amacrine, bipolar, horizontal, photoreceptor, and astroglial cells. Although the cells expressed neuronal or glial markers, none acquired end-stage markers unique to retinal neurons. This suggests that the adult-derived stem cells can adapt to a wide variety of heterologous environments and express some but not all features of retinal cells when exposed to the cues present late in retinal development.     

Dopamine has a critical role in photoreceptor degeneration in the rd mouse

Photoreceptors receive paracrine input from dopaminergic interplexiform cells. Rod photoreceptors in the rd mouse degenerate rapidly due to a specific gene defect. We investigated the effects of dopamine on rd mouse photoreceptors in retinal organ culture. Retinas were harvested from rd or wild-type mice at postnatal day 2 and grown in organ culture for 27 days. When antagonists for either D(1)- or D(2)-family dopamine receptors were added to the media, photoreceptor degeneration was blocked. Furthermore, when dopamine was depleted by the addition of 6-hydroxydopamine and pargyline, photoreceptor survival appeared comparable to wild-type retinal cultures. The addition of a dopamine agonist induced photoreceptor degeneration in dopamine-depleted rd organ cultures. In all cases, photoreceptors maintained robust staining of opsin. These results demonstrate that dopamine antagonists or dopamine depletion blocks photoreceptor degeneration and that dopamine is necessary for photoreceptor degeneration in the rd mouse retinal organ culture model, indicating that dopamine antagonists may represent a therapeutic strategy in retinal degenerative disease.     

Pigment epithelium-derived factor delays the death of photoreceptors in mouse models of inherited retinal degenerations

Pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor superfamily produced by retinal pigment epithelial cells in the developing and adult retina. In vitro, it induces neuronal differentiation of retinoblastoma cells and promotes survival of cerebellar granule neurons. The pedf gene is closely linked to an autosomal-dominant locus for retinitis pigmentosa, suggesting that PEDF could be a survival factor for photoreceptors. We have investigated this possibility by injecting PEDF into the eyes of homozygous retinal degeneration (rd) and retinal degeneration slow (rds) mice, two mutants displaying apoptotic photoreceptor loss. This procedure resulted in a transient delay of photoreceptor loss in the rd mouse and a reduction in apoptotic photoreceptor profiles in the rds mouse. We conclude that PEDF can act as a survival-promoting factor for photoreceptors in vivo and could potentially be useful for the treatment of photoreceptor diseases.     

Light stimulation evokes two different calcium responses in Müller glial cells of the guinea pig retina

Intracellular calcium responses are a characteristic of glial activation upon neuronal activity. In acutely isolated preparations of the guinea pig retina, Müller glial cells displayed cytosolic calcium rises in response to repetitive light stimulation. The calcium rises consisted of two components, a slowly developing immediate response that occurred simultaneously over the whole length of all Müller cell fibers and a delayed fast response that originated in the ganglion cell layer and spread as a wave through the bodies of some Müller cells toward the outer processes in the photoreceptor layer. The slow calcium response was evoked by photoreceptor-to-glia signaling, resulting in a glutamate transporter- and zinc-mediated alteration in the membrane potential and an influx of calcium from the extracellular space. The fast calcium response was evoked by a release of calcium from intracellular stores, probably after activation of purinergic receptors. The data suggest that light stimulation of the retina causes glial activation by alterations in both the membrane potential and receptor-mediated mechanisms. The former may be implicated in glial support of the neuronal signal transfer from photoreceptors to ganglion cells (glial forward signaling), whereas the latter may constitute a glial feedback signaling from ganglion cells to photoreceptors.     

Location of CNTFRalpha on outer segments: evidence of the site of action of CNTF in rat retina

Ciliary neurotrophic factor (CNTF) is an important factor in the retina's mechanisms of self-protection. It is generated by retinal glial cells in response to stress, and has a significant protective effect on retinal neurones. In this study we have identified the location of the alpha component of the CNTF receptor complex (CNTFRalpha) in rat retina, using immunohistochemistry and high-resolution confocal microscopy. The major location of CNTFRalpha is on photoreceptor outer segments. More scattered, granular forms of CNTFRalpha were identified in association with Müller cell processes in other retinal layers. Colocalisation of CNTF with CNTFRalpha, suggestive of ligand-receptor binding, was detected on outer segments, and in both normal retinas and retinas stressed by light or oxygen. Results provide evidence that the principal site of CNTF action is the outer segments of photoreceptors. This confirms the known ability of CNTF to protect photoreceptors against stress, and suggest that it acts by modulating mechanisms specific to the outer segment, such as the phototransduction cascade or the membrane channels, which control dark current.