Microglial response to light‐induced photoreceptor degeneration in the mouse retina

The microglial response elicited by degeneration of retinal photoreceptor cells was characterized in BALB/c mice exposed to bright light for 7 hours and then kept in complete darkness for survival times ranging from 0 hours to 10 days. Photodegeneration resulted in extensive cell death in the retina, mainly in the outer nuclear layer (ONL), where the photoreceptor nuclei are located. Specific immunolabeling of microglial cells with anti‐CD11b, anti‐CD45, anti‐F4/80, anti‐SRA, and anti‐CD68 antibodies revealed that microglial cells were activated in light‐exposed retinas. They migrated to the ONL, changed their morphology, becoming rounded cells with short and thick processes, and, finally, showed immunophenotypic changes. Specifically, retinal microglia began to strongly express antigens recognized by anti‐CD11b, anti‐CD45, and anti‐F4/80, coincident with cell degeneration. In contrast, upregulation of the antigen recognized by anti‐SRA was not detected by immunocytochemistry until 6 hours after light exposure. Differences were also observed at 10 days after light exposure: CD11b, CD45, and F4/80 continued to be strongly expressed in retinal microglia, whereas the expression of CD68 and SRA had decreased to near‐normal values. Therefore, microglia did not return to their original state after photodegeneration and continued to show a degree of activation. The accumulation of activated microglial cells in affected regions simultaneously with photoreceptor degeneration suggests that they play some role in photodegeneration. J. Comp. Neurol. 518:477–492, 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Downregulation of STAT3 activation is required for presumptive rod photoreceptor cells to differentiate in the postnatal retina

Ciliary neurotrophic factor (CNTF) has been known to inhibit the differentiation of presumptive rod photoreceptor cells; however, the underlying mechanisms have remained to be elucidated. We demonstrated that STAT3 activation, but not SHP2 activation, is responsible for the CNTF/gp130 signaling that inhibits expression of Rhodopsin and its upstream activator, crx, in the retinal explants derived from P0 mice (P0 retinal explants), utilizing STAT3-deficient retina and electroporation of dominant-negative form of STAT3 (STAT3F). We also demonstrated that STAT3 activation in presumptive rod photoreceptor cells at E18.5 is rapidly downregulated at P0, when Rhodopsin expression starts during retinal development. Persistent STAT3 activation in the P0 retinal explants prevented Rhodopsin expression and rapid upregulation of crx expression. STAT3-deficient retinas did not exhibit precocious rod photoreceptor cell differentiation as a whole, although they occasionally exhibited precocious upregulation of crx mRNA. Thus, we conclude that downregulation of STAT3 activation is required, but insufficient, for rod photoreceptor cell differentiation in the postnatal retina.     

Generation of cells committed towards the photoreceptor fate for retinal transplantation

Cell transplantation is an active field of research to replace lost cells in retinal dystrophies to potentially restore visual function. We hypothesized that in-vitro differentiated retinal stem cells would integrate the appropriate retinal layer and differentiate into photoreceptors when transplanted during development. Here we show that retinal stem cells driven to the photoreceptor fate start to incorporate the retina and express photoreceptor markers but do not survive. Nevertheless surviving grafted cells express the glial marker glial fibrillary acidic protein and incorporate the ganglion cell layer as well as the inner plexiform layer. These results suggest that the maturation state of the photoreceptors is primordial to obtain robust incorporation and that a fine tuning of retinal stem cells differentiation should provide adequate cells for transplantation.     

Melanopsin ganglion cell outer retinal dendrites: Morphologically distinct and asymmetrically distributed in the mouse retina

A small population of retinal ganglion cells expresses the photopigment melanopsin and function as autonomous photoreceptors. They encode global luminance levels critical for light‐mediated non‐image forming visual processes including circadian rhythms and the pupillary light reflex. There are five melanopsin ganglion cell subtypes (M1–M5). M1 and displaced M1 (M1d) cells have dendrites that ramify within the outermost layer of the inner plexiform layer. It was recently discovered that some melanopsin ganglion cells extend dendrites into the outer retina. Outer Retinal Dendrites (ORDs) either ramify within the outer plexiform layer (OPL) or the inner nuclear layer, and while present in the mature retina, are most abundant postnatally. Anatomical evidence for synaptic transmission between cone photoreceptor terminals and ORDs suggests a novel photoreceptor to ganglion cell connection in the mammalian retina. While it is known that the number of ORDs in the retina is developmentally regulated, little is known about the morphology, the cells from which they originate, or their spatial distribution throughout the retina. We analyzed the morphology of melanopsin‐immunopositive ORDs in the OPL at different developmental time points in the mouse retina and identified five types of ORDs originating from either M1 or M1d cells. However, a pattern emerges within these: ORDs from M1d cells are generally longer and more highly branched than ORDs from conventional M1 cells. Additionally, we found ORDs asymmetrically distributed to the dorsal retina. This morphological analysis provides the first step in identifying a potential role for biplexiform melanopsin ganglion cell ORDs.     

The effect of photoreceptor degeneration on ganglion cell morphology

Retinitis pigmentosa refers to a family of inherited photoreceptor degenerations resulting in blindness. During and after photoreceptor loss, neurons of the inner retina are known to undergo plastic changes. Here, we have investigated in detail whether ganglion cells are altered at late stages of degeneration, well after the total loss of photoreceptors. We used mice, rd1‐Thy1, that carry a mutation in the β‐subunit of phosphodiesterase 6 and a fluorescent protein that labels a subset of ganglion cells and B6‐Thy1 control mice. Retinal wholemounts from mice aged 3–11 months were processed for immunohistochemistry and analyzed. Ganglion cells were classified based on soma area, dendritic field size, and branching of dendrites. The dendritic fields of some ganglion cells were further analyzed for their length, area and quantity of branching points. There was a decrease in size and level of branching of A2, B1, and D type ganglion cells in the degenerated retina at 11 months of age. In contrast, C1 ganglion cells remained unchanged. In addition, there was a shift in the proportion of ganglion cells ramifying in the different layers of the inner plexiform layer. Careful analysis of the dendrites of ganglion cells revealed some projecting to new, more distal regions of the inner plexiform layer. We propose that these changes in ganglion cell morphology could impact the function of individual cells as well as the retinal circuitry in the degenerated retina. J. Comp. Neurol. 522:1155–1170, 2014. © 2013 Wiley Periodicals, Inc.     

Retinal remodeling triggered by photoreceptor degenerations

Many photoreceptor degenerations initially affect rods, secondarily leading to cone death. It has long been assumed that the surviving neural retina is largely resistant to this sensory deafferentation. New evidence from fast retinal degenerations reveals that subtle plasticities in neuronal form and connectivity emerge early in disease. By screening mature natural, transgenic, and knockout retinal degeneration models with computational molecular phenotyping, we have found an extended late phase of negative remodeling that radically changes retinal structure. Three major transformations emerge: 1) Müller cell hypertrophy and elaboration of a distal glial seal between retina and the choroid/retinal pigmented epithelium; 2) apparent neuronal migration along glial surfaces to ectopic sites; and 3) rewiring through evolution of complex neurite fascicles, new synaptic foci in the remnant inner nuclear layer, and new connections throughout the retina. Although some neurons die, survivors express molecular signatures characteristic of normal bipolar, amacrine, and ganglion cells. Remodeling in human and rodent retinas is independent of the initial molecular targets of retinal degenerations, including defects in the retinal pigmented epithelium, rhodopsin, or downstream phototransduction elements. Although remodeling may constrain therapeutic intervals for molecular, cellular, or bionic rescue, it suggests that the neural retina may be more plastic than previously believed.     

Role of nitric oxide in photoreceptor survival in embryonic chick retinal cell culture

The presence of nitric oxide synthase (NOS) in chick retina during development has allowed us to study the role of nitric oxide (NO) during retinal differentiation in dissociated chick retinal cell culture from embryonic day 6. We have demonstrated the presence of nicotinamide adenine dinucleotide phosphate diaphorase staining in these cultures after 3 days in vitro (Div), with a maximal intensity after 8 Div, corresponding to embryonic day 14. Immunohistochemistry studies confirmed the presence of the two isoforms of NOS, NOS-I and -III, in dissociated retinal cell cultures at 8 Div. Addition of NG-monomethyl-L-arginine, a NOS inhibitor, to retinal cell cultures prevented NO production but did not modify the appearance and the survival of ganglion and amacrine cells. However, immunohistochemical analysis with distinct markers for photoreceptor cells (rods and cones) showed that inhibition of endogenous NOS in retinal cell cultures prevented the developmental decrease of rod number between 5 and 8 Div, thus supporting the hypothesis that NO may be involved in the cell death of rods during the development of the retina.     

Neural differentiation and synaptogenesis in retinal development

To investigate the pattern of neural differentiation and synaptogenesis in the mouse retina, immunolabeling, Brd U assay and transmission electron microscopy were used. We show that the neuroblastic cell layer is the germinal zone for neural differentiation and retinal lamination. Ganglion cells differentiated initially at embryonic day 13(E13), and at E18 horizontal cells appeared in the neuroblastic cell layer. Neural stem cells in the outer neuroblastic cell layer differentiated into photoreceptor cells as early as postnatal day 0(P0), and neural stem cells in the inner neuroblastic cell layer differentiated into bipolar cells at P7. Synapses in the retina were mainly located in the outer and inner plexiform layers. At P7, synaptophysin immunostaining appeared in presynaptic terminals in the outer and inner plexiform layers with button-like structures. After P14, presynaptic buttons were concentrated in outer and inner plexiform layers with strong staining. These data indicate that neural differentiation and synaptogenesis in the retina play important roles in the formation of retinal neural circuitry. Our study showed that the period before P14, especially between P0 and P14, represents a critical period during retinal development. Mouse eye opening occurs during that period, suggesting that cell differentiation and synaptic formation lead to the attainment of visual function.     

A new model of retinal photoreceptor cell degeneration induced by a chemical hypoxia-mimicking agent, cobalt chloride

Retinal photoreceptor cell degeneration was induced by cobalt chloride, a chemical hypoxia-mimicking agent in rodents. Time course and dose-response of photoreceptor cell degeneration in mouse retina after intravitreal injection of cobalt chloride were examined by conventional histological analysis by hematoxylin and eosin staining and in situ terminal dUTP-biotin nick end labeling of DNA fragments (TUNEL) method with the use of paraffin-embedded sections. The dose-response of photoreceptor cell degeneration in rat retina was also examined. Photoreceptor cells progressively degenerated with time and under dose-response relationship. The suitable dose of cobalt chloride for the selective photoreceptor cell degeneration in mice is 10-12 nmol intravitreal injection at the volume of 2 microl. The retinal morphology of the mice 2 weeks after the 10-12 nmol intravitreal injection was similar to that of retinal degeneration in the mutant rd mouse. Retinal damage of total retinal layers was induced by an excessive dose of cobalt chloride. The progression of retinal damage after cobalt chloride injection, measured morphologically, was completed at 1 week. However, nuclear DNA fragmentation, mainly detected at outer nuclear layer by TUNEL, peaked at 48 h after 12 nmol cobalt chloride injection. Thus, the selective photoreceptor cell degeneration induced by cobalt chloride follows DNA fragmentation at outer nuclear layer. The photoreceptor cell degeneration is established optionally by cobalt chloride without use of the retinal degeneration mutant animals. Thus, we have described the development of a new model of retinal photoreceptor cell degeneration induced by a chemical hypoxia-mimicking agent.     

Recoverin negative photoreceptor cells

Recoverin, a calcium-binding protein, is unique with respect to its cellular regulation. It is present in retinal rods, cones, cone bipolar cells, and in a rare population of cells in the ganglion cell layer. Inappropriate turn-on or turn-off of recoverin expression has been reported both in small cell lung carcinoma cells from patients with cancer-associated retinopathy (Matsusara et al. [1996] Br. J. Cancer 74:1419-1422; Adamus et al. [1998] J. Autoimmun. 11: 523-533; Ohguro et al. [1999] Invest. Ophthalmol. Vis. Sci. 40:82-89) and in cultured retinal neurons (McGinnis et al. [1999] J. Neursci. Res. 55:252-260). In a recent report using double labeling immunofluorescence microscopy methods with antibodies against either rhodopsin and recoverin or arrestin and recoverin, two unique cell phenotypes, rhodopsin-positive and recoverin-negative, and arrestin-positive, and recoverin-negative were observed in vitro. These two unique cell types could be nonphotoreceptor cells in which rhodopsin and arrestin are inappropriately turned on or they are photoreceptor cells in which the recoverin gene is inappropriately turned off. In this study, multiple antibodies were used to study, on a single-cell basis, whether the photoreceptor cell-specific marker, rhodopsin, is inappropriately expressed in nonphotoreceptor cells in our retinal neuronal culture system. We also examined the hypothesis that the two unique cell phenotypes represent the same population of cells. A triple labeling method has been established to visualize recoverin, rhodopsin, and arrestin protein expression simultaneously in cultured retinal neurons. Our data clearly and directly demonstrate that the previously described unique cell phenotypes are the same population of cells, rod photoreceptors. The existence of recoverin-negative photoreceptors demonstrates that the recoverin gene can be regulated independently of other photoreceptor cell-specific proteins and suggests that this primary cell culture may be useful as a model system for investigating the illicit expression of the recoverin gene in cancer associated retinopathy.     

Morphological changes in the Royal College of Surgeons rat retina during photoreceptor degeneration and after cell-based therapy

There are concomitant morphological and functional changes in the inner retina during the course of photoreceptor degeneration in a range of animal models of retina degeneration and in humans with eye disease. One concern that has been raised is that the changes occurring in the inner retina might compromise attempts to rescue or restore visual input by various interventional approaches. It is known that cell-based therapy can preserve significant visual capability for many months. In this study, we examine the overall changes in the Royal College of Surgeons (RCS) rat during degeneration and the effects of cell transplantation by means of immunohistochemistry and confocal microscopy. The degenerative changes are complex, and they progress with age. They involve the neurons with which both rods and cones interconnect--retinal second- and third-order neurons underwent dramatic modification, including sprouting, retraction as photoreceptor loss progressed--as well as Müller glia and secondary vascular changes, which were associated at later times with neuronal migration. The pathological vascular changes led to major disruption of inner retina. After introducing a retinal pigment epithelial cell line to the subretinal space early in the progress of photoreceptor degeneration, most inner retinal changes were held in abeyance for up to at least 10 months of age. Given the concern that has been raised regarding whether inner retinal changes might compromise any graft-related benefit, this is an encouraging finding.     

Contribution of calpains to photoreceptor cell death in N-methyl-N-nitrosourea-treated rats

The purpose of the present study was to determine if proteolysis by the calcium-dependent enzyme calpains (EC 3.4.22.17) contributed to retinal cell death in a rat model of photoreceptor degeneration induced by intraperitoneal injection of N-methyl-N-nitrosourea (MNU). Retinal degeneration was evaluated by H&E staining, and cell death was determined by TUNEL assay. Total calcium in retina was measured by atomic absorption spectrophotometry. Activation of calpains was determined by casein zymography and immunoblotting. Proteolysis of alpha-spectrin and p35 (regulator of Cdk5) were evaluated by immunoblotting. Calpain inhibitor SNJ-1945 was orally administrated to MNU-treated rats to test drug efficacy. MNU decreased the thickness of photoreceptor cell layer, composed of the outer nuclear layer (ONL) and outer segment (OS). Numerous cells in the ONL showed positive TUNEL staining. Total calcium was increased in retina after MNU. Activation of calpains and calpain-specific proteolysis of alpha-spectrin were observed after MNU injection. Oral administration of SNJ-1945 to MNU-treated rats showed a significant protective effect against photoreceptor cell loss, confirming involvement of calpains in photoreceptor degeneration. Conversion of p35 to p25 was well correlated with calpain activation, suggesting prolonged activation of Cdk5/p25 as a possible downstream mechanism for MNU-induced photoreceptor cell death. SNJ-1945 reduced photoreceptor cells death, even though MNU is one of the most severe models of photoreceptor cell degeneration. Oral calpain inhibitor SNJ-1945 may be a candidate for testing as a medication against retinal degeneration in retinitis pigmentosa.     

Establishment of photoreceptor cell polarity in culture revealed by mushroom lectin binding.

Differentiation of photoreceptor cells dissociated from chick embryo was investigated in a monolayer culture at low cell density and low serum concentration. The precursor cells were initially round and finally adopted the elongated and monopolar shape characteristic of such photoreceptor cells in vivo. They exhibited the primitive inner segment, and on its distal side showed a crest-like membrane expansion instead of the developed outer segment. The cell polarity of these cultured photoreceptor cells was examined using mushroom lectin, which has specific affinity for the D-galactose-beta(1,3)-N-acetylgalactosamine moiety. Rhodamine-labeled mushroom lectin uniformly initially stained the surface of early round photoreceptor cells, but with time the staining became restricted to the inner segment and its distal membrane expansion in all elongated photoreceptor cells at 12 days from the beginning of development. In semithin sections of 12-day-old chick embryo retina, mushroom lectin also stained the immature inner segment that had emerged at this stage. After hatching, light and electron microscopic observations revealed that the entire scleral surface of retinal plasma membranes as far as the outer limiting membrane, i.e. the inner and outer segments of all types of photoreceptor cells and also Müller cell processes, were stained with the mushroom lectin. These results clearly show that all photoreceptor cells can establish their inherent cell polarity even in culture, and that mushroom lectin is a valuable marker, for the investigation of photoreceptor cell polarity.     

Müller cells are a preferred substrate for in vitro neurite extension by rod photoreceptor cells

To define the factors important in photoreceptor cell morphogenesis, we have examined the ability of rods to extend neurites in vitro. Retinas from neonatal rats were dissociated and plated onto substrate-bound extracellular matrix (ECM) components or cell monolayers. When rods, identified with monoclonal antibodies to opsin, were in contact exclusively with purified ECM (e.g., laminin, fibronectin, type I collagen, or Matrigel), neurite outgrowth was extremely limited. By contrast, rods extended long neurites on Müller cells. Retinal or brain astrocytes, endothelial cells, 3T3 fibroblasts, or other retinal neurons were less supportive of rod process outgrowth. These data demonstrate regional specificity in the promotion of neurite outgrowth by glia and suggest that not all neurons within the retina require the same morphogenic factors.     

Autoimmune responses against photoreceptor antigens during retinal degeneration and their role in macrophage recruitment into retinas of RCS rats

Autoimmunity may contribute to retinal degeneration. The studies examined the evolution of autoimmune responses against retina in naïve dystrophic RCS rats over the course of retinal degeneration. We showed that anti-retinal autoantibodies and T cells are generated in response to the availability of antigenic material released from dying photoreceptor cells during retinal degeneration but with distinctive activation trends. Passive transfer of anti-retinal antibodies enhanced disease progression by disrupting the BRB, upregulating MCP-1, attracting blood macrophages into retina, and augmenting apoptotic photoreceptor cell death. Our findings directly link anti-retinal autoantibodies to activated macrophage entry and their possible role in neurodegeneration.