Sildenafil Administration in Dogs Heterozygous for a Functional Null Mutation in Pde6a: Suppressed Rod-Mediated ERG Responses and Apparent Retinal Outer Nuclear Layer Thinning.

This study was designed to assess risk for retinal toxicity associated with administration of high-dose sildenafil citrate to dogs heterozygous for a functionally null mutation in Pde6a over a 4-month period. Three Pde6a +/- dogs were administered 14.3 mg/kg sildenafil per os and two Pde6a +/- dogs placebo once daily for 16 weeks. Three Pde6a +/+ dogs were administered sildenafil for 7 days. Ophthalmic examination, vision testing, and electroretinography (ERG) were regularly performed. At study termination, dogs were euthanized and globes collected. Retinal layer thickness and photoreceptor nuclei counts were determined from plastic sections. In both Pde6a +/- and Pde6a +/+ sildenafil-treated (ST) dogs, elevation of dark-adapted b-wave threshold and unmasking of the scotopic threshold response (STR) were observed. Sildenafil treated Pde6a +/- dogs had significantly thinner ONL (24.90 +/-1.88 µm, p = 0.004) and lower photoreceptor nuclei counts (273.6 +/- 29.3 cells/100 µm, p = 0.008) compared to measurements (35.90 +/- 1.63 µm) and counts (391.5 +/-27.0 cells/100 µm) from archived untreated Pde6a +/- dogs.

Targeted disruption of the endogenous zebrafish rhodopsin locus as models of rapid rod photoreceptor degeneration.

Purpose: Retinitis pigmentosa (RP) is a collection of genetic disorders that results in the degeneration of light-sensitive photoreceptor cells, leading to blindness. RP is associated with more than 70 loci that may display dominant or recessive modes of inheritance, but mutations in the gene encoding the visual pigment rhodopsin (RHO) are the most frequent cause. In an effort to develop precise mutations in zebrafish as novel models of photoreceptor degeneration, we describe the generation and germline transmission of a series of novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-induced insertion and deletion (indel) mutations in the major zebrafish rho locus, rh1-1. Methods: One- or two-cell staged zebrafish embryos were microinjected with in vitro transcribed mRNA encoding Cas9 and a single guide RNA (gRNA). Mutations were detected by restriction fragment length polymorphism (RFLP) and DNA sequence analyses in injected embryos and offspring. Immunolabeling with rod- and cone-specific antibodies was used to test for histological and cellular changes. Results: Using gRNAs that targeted highly conserved regions of rh1-1, a series of dominant and recessive alleles were recovered that resulted in the rapid degeneration of rod photoreceptors. No effect on cones was observed. Targeting the 5'-coding sequence of rh1-1 led to the recovery of several indels similar to disease-associated alleles. A frame shift mutation leading to a premature stop codon (T17*) resulted in rod degeneration when brought to homozygosity. Immunoblot and fluorescence labeling with a Rho-specific antibody suggest that this is indeed a null allele, illustrating that the Rho expression is essential for rod survival. Two in-frame mutations were recovered that disrupted the highly conserved N-linked glycosylation consensus sequence at N15. Larvae heterozygous for either of the alleles demonstrated rapid rod degeneration. Targeting of the 3'-coding region of rh1-1 resulted in the recovery of an allele encoding a premature stop codon (S347*) upstream of the conserved VSPA sorting sequence and a second in-frame allele that disrupted the putative phosphorylation site at S339. Both alleles resulted in rod death in a dominant inheritance pattern. Following the loss of the targeting sequence, immunolabeling for Rho was no longer restricted to the rod outer segment, but it was also localized to the plasma membrane. Conclusions: The efficiency of CRISPR/Cas9 for gene targeting, coupled with the large number of mutations associated with RP, provided a backdrop for the rapid isolation of novel alleles in zebrafish that phenocopy disease. These novel lines will provide much needed in-vivo models for high throughput screens of compounds or genes that protect from photoreceptor degeneration.

mTor signaling is required for the formation of proliferating Müller glia-derived progenitor cells in the chick retina.

We investigate the roles of mTor signaling in the formation of Müller glia-derived progenitor cells (MGPCs) in the chick retina. During embryonic development, pS6 (a readout of active mTor signaling) is present in early-stage retinal progenitors, differentiating amacrine and ganglion cells, and late-stage progenitors or maturing Müller glia. By contrast, pS6 is present at low levels in a few scattered cell types in mature, healthy retina. Following retinal damage, in which MGPCs are known to form, mTor signaling is rapidly activated in Müller glia. Inhibition of mTor in damaged retinas prevented the accumulation of pS6 in Müller glia and reduced numbers of proliferating MGPCs. Inhibition of mTor had no effect on MAPK signaling or on upregulation of the stem cell factor Klf4, whereas Pax6 upregulation was significantly reduced. Inhibition of mTor potently blocked the MGPC-promoting effects of Hedgehog, Wnt and glucocorticoid signaling in damaged retinas. In the absence of retinal damage, insulin, IGF1 and FGF2 induced pS6 in Müller glia, and this was blocked by mTor inhibitor. In FGF2-treated retinas, in which MGPCs are known to form, inhibition of mTor blocked the accumulation of pS6, the upregulation of Pax6 and the formation of proliferating MGPCs. We conclude that mTor signaling is required, but not sufficient, to stimulate Müller glia to give rise to proliferating progenitors, and the network of signaling pathways that drive the formation of MGPCs requires activation of mTor.

Comparative analysis of glucagonergic cells, glia, and the circumferential marginal zone in the reptilian retina.

Retinal progenitors in the circumferential marginal zone (CMZ) and Müller glia-derived progenitors have been well described for the eyes of fish, amphibians, and birds. However, there is no information regarding a CMZ and the nature of retinal glia in species phylogenetically bridging amphibians and birds. The purpose of this study was to examine the retinal glia and investigate whether a CMZ is present in the eyes of reptilian species. We used immunohistochemical analyses to study retinal glia, neurons that could influence CMZ progenitors, the retinal margin, and the nonpigmented epithelium of ciliary body of garter snakes, queen snakes, anole lizards, snapping turtles, and painted turtles. We compare our observations on reptile eyes to the CMZ and glia of fish, amphibians, and birds. In all species, Sox9, Pax6, and the glucocorticoid receptor are expressed by Müller glia and cells at the retinal margin. However, proliferating cells were found only in the CMZ of turtles and not in the eyes of anoles and snakes. Similar to eyes of chickens, the retinal margin in turtles contains accumulations of GLP1/glucagonergic neurites. We find that filamentous proteins, vimentin and GFAP, are expressed by Müller glia, but have different patterns of subcellular localization in the different species of reptiles. We provide evidence that the reptile retina may contain nonastrocytic inner retinal glial cells, similar to those described in the avian retina. We conclude that the retinal glia, glucagonergic neurons, and CMZ of turtles appear to be most similar to those of fish, amphibians, and birds.

Activation of glucocorticoid receptors in Müller glia is protective to retinal neurons and suppresses microglial reactivity.

Reactive microglia and macrophages are prevalent in damaged retinas. Glucocorticoid signaling is known to suppress inflammation and the reactivity of microglia and macrophages. In the vertebrate retina, the glucocorticoid receptor (GCR) is known to be activated and localized to the nuclei of Müller glia (Gallina et al., 2014). Accordingly, we investigated how signaling through GCR influences the survival of neurons using the chick retina in vivo as a model system. We applied intraocular injections of GCR agonist or antagonist, assessed microglial reactivity, and the survival of retinal neurons following different damage paradigms. Microglial reactivity was increased in retinas from eyes that were injected with vehicle, and this reactivity was decreased by GCR-agonist dexamethasone (Dex) and increased by GCR-antagonist RU486. We found that activation of GCR suppresses the reactivity of microglia and inhibited the loss of retinal neurons resulting from excitotoxicity. We provide evidence that the protection-promoting effects of Dex were maintained when the microglia were selectively ablated. Similarly, intraocular injections of Dex protected ganglion cells from colchicine-treatment and protected photoreceptors from damage caused by retinal detachment. We conclude that activation of GCR promotes the survival of ganglion cells in colchicine-damaged retinas, promotes the survival of amacrine and bipolar cells in excitotoxin-damaged retinas, and promotes the survival of photoreceptors in detached retinas. We propose that suppression of microglial reactivity is secondary to activation of GCR in Müller glia, and this mode of signaling is an effective means to lessen the damage and vision loss resulting from different types of retinal damage.

Reactive retinal microglia, neuronal survival, and the formation of retinal folds and detachments.

Reactive microglia and macrophages are prevalent in damaged retinas. Accordingly, we investigate how the activation or ablation of microglia/macrophages influences the survival of neurons in the chick retina in vivo. We applied intraocular injections of interleukin 6 (IL6) to stimulate the reactivity of microglia/macrophages and clodronate-liposomes to ablate microglia/macrophages. Activation of the microglia/macrophages with IL6 delays the death of retinal neurons from N-methyl-D-aspartate (NMDA) -induced excitotoxicity. In addition, activation of microglia/macrophages combined with colchicine-mediated retinal damage diminished the survival of ganglion cells. Application of IL6 after an excitotoxic insult greatly exacerbates the damage, and causes widespread retinal detachments and folds, accompanied by accumulation of microglia/macrophages in the subretinal space. Damage-induced retinal folds and detachments were significantly reduced by the ablation of microglia/macrophages. We conclude that microglial reactivity is detrimental to the survival of ganglion cells in colchicine-damaged retinas and detrimental to the survival of photoreceptors in retinal folds. In addition, we conclude that IL6-treatment transiently protects amacrine and bipolar cells against an excitotoxic insult. We propose that suppressing reactivity of microglia/macrophages may be an effective means to lessen the damage and vision loss resulting from damage, in particular during retinal detachment injuries.

Glucocorticoid receptors in the retina, Müller glia and the formation of Müller glia-derived progenitors.

Identification of the signaling pathways that influence the reprogramming of Müller glia into neurogenic retinal progenitors is key to harnessing the potential of these cells to regenerate the retina. Glucocorticoid receptor (GCR) signaling is commonly associated with anti-inflammatory responses and GCR agonists are widely used to treat inflammatory diseases of the eye, even though the cellular targets and mechanisms of action in the retina are not well understood. We find that signaling through GCR has a significant impact upon the ability of Müller glia to become proliferating Müller glia-derived progenitor cells (MGPCs). The primary amino acid sequence and pattern of GCR expression in the retina is highly conserved across vertebrate species, including chickens, mice, guinea pigs, dogs and humans. In all of these species we find GCR expressed by the Müller glia. In the chick retina, we find that GCR is expressed by progenitors in the circumferential marginal zone (CMZ) and is upregulated by Müller glia in acutely damaged retinas. Activation of GCR signaling inhibits the formation of MGPCs and antagonizes FGF2/MAPK signaling in the Müller glia. By contrast, we find that inhibition of GCR signaling stimulates the formation of proliferating MGPCs in damaged retinas, and enhances the neuronal differentiation while diminishing glial differentiation. Given the conserved expression pattern of GCR in different vertebrate retinas, we propose that the functions and mechanisms of GCR signaling are highly conserved and are mediated through the Müller glia. We conclude that GCR signaling directly inhibits the formation of MGPCs, at least in part, by interfering with FGF2/MAPK signaling.

Reactive microglia and macrophage facilitate the formation of Müller glia-derived retinal progenitors.

In retinas where Müller glia have been stimulated to become progenitor cells, reactive microglia are always present. Thus, we investigated how the activation or ablation of microglia/macrophage influences the formation of Müller glia-derived progenitor cells (MGPCs) in the retina in vivo. Intraocular injections of the Interleukin-6 (IL6) stimulated the reactivity of microglia/macrophage, whereas other types of retinal glia appear largely unaffected. In acutely damaged retinas where all of the retinal microglia/macrophage were ablated, the formation of proliferating MGPCs was greatly diminished. With the microglia ablated in damaged retinas, levels of Notch and related genes were unchanged or increased, whereas levels of ascl1a, TNFα, IL1ß, complement component 3 (C3) and C3a receptor were significantly reduced. In the absence of retinal damage, the combination of insulin and Fibroblast growth factor 2 (FGF2) failed to stimulate the formation of MGPCs when the microglia/macrophage were ablated. In addition, intraocular injections of IL6 and FGF2 stimulated the formation of MGPCs in the absence of retinal damage, and this generation of MGPCs was blocked when the microglia/macrophage were absent. We conclude that the activation of microglia and/or infiltrating macrophage contributes to the formation of proliferating MGPCs, and these effects may be mediated by components of the complement system and inflammatory cytokines.

The reactivity, distribution and abundance of Non-astrocytic Inner Retinal Glial (NIRG) cells are regulated by microglia, acute damage, and IGF1.

Recent studies have described a novel type of glial cell that is scattered across the inner layers of the avian retina and possibly the retinas of primates. These cells have been termed Non-astrocytic Inner Retinal Glial (NIRG) cells. These cells are stimulated by insulin-like growth factor 1 (IGF1) to proliferate, migrate distally into the retina, and become reactive. These changes in glial activity correlate with increased susceptibility of retinal neurons and Müller glia to excitotoxic damage. The purpose of this study was to further study the NIRG cells in retinas treated with IGF1 or acute damage. In response to IGF1, the reactivity, proliferation and migration of NIRG cells persists through 3 days after treatment. At 7 days after treatment, the numbers and distribution of NIRG cells returns to normal, suggesting that homeostatic mechanisms are in place within the retina to maintain the numbers and distribution of these glial cells. By comparison, IGF1-induced microglial reactivity persists for at least 7 days after treatment. In damaged retinas, we find a transient accumulation of NIRG cells, which parallels the accumulation of reactive microglia, suggesting that the reactivity of NIRG cells and microglia are linked. When the microglia are selectively ablated by the combination of interleukin 6 and clodronate-liposomes, the NIRG cells down-regulate transitin and perish within the following week, suggesting that the survival and phenotype of NIRG cells are somehow linked to the microglia. We conclude that the abundance, reactivity and retinal distribution of NIRG cells can be dynamic, are regulated by homoestatic mechanisms and are tethered to the microglia.

Vision-guided ocular growth in a mutant chicken model with diminished visual acuity.

Visual experience is known to guide ocular growth. We tested the hypothesis that vision-guided ocular growth is disrupted in a model system with diminished visual acuity. We examine whether ocular elongation is influenced by form-deprivation (FD) and lens-imposed defocus in the Retinopathy, Globe Enlarged (RGE) chicken. Young RGE chicks have poor visual acuity, without significant retinal pathology, resulting from a mutation in guanine nucleotide-binding protein ß3 (GNB3), also known as transducin ß3 or Gß3. The mutation in GNB3 destabilizes the protein and causes a loss of Gß3 from photoreceptors and ON-bipolar cells (Ritchey et al., 2010). FD increased ocular elongation in RGE eyes in a manner similar to that seen in wild-type (WT) eyes. By comparison, the excessive ocular elongation that results from hyperopic defocus was increased, whereas myopic defocus failed to significantly decrease ocular elongation in RGE eyes. Brief daily periods of unrestricted vision interrupting FD prevented ocular elongation in RGE chicks in a manner similar to that seen in WT chicks. Glucagonergic amacrine cells differentially expressed the immediate early gene Egr1 in response to growth-guiding stimuli in RGE retinas, but the defocus-dependent up-regulation of Egr1 was lesser in RGE retinas compared to that of WT retinas. We conclude that high visual acuity, and the retinal signaling mediated by Gß3, is not required for emmetropization and the excessive ocular elongation caused by FD and hyperopic defocus. However, the loss of acuity and Gß3 from RGE retinas causes enhanced responses to hyperopic defocus and diminished responses to myopic defocus.

The combination of IGF1 and FGF2 and the induction of excessive ocular growth and extreme myopia.

Different growth factors have been shown to influence the development of form-deprivation myopia and lens-induced ametropias. However, growth factors have relatively little effect on the growth of eyes with unrestricted vision. We investigate whether the combination of insulin-like growth factor 1 (IGF1) and fibroblast growth factor 2 (FGF2) influence ocular growth in eyes with unrestricted vision. Different doses of IGF1 and FGF2 were injected into the vitreous chamber of postnatal chicks. Measurements of ocular dimensions and intraocular pressure (IOP) were made during and at the completion of different treatment paradigms. Histological and immunocytochemical analyses were performed to assess cell death, cellular proliferation and integrity of ocular tissues. Treated eyes had significant increases in equatorial diameter and vitreous chamber depth. With significant variability between individuals, IGF1/FGF2-treatment caused hypertrophy of lens and ciliary epithelia, lens thickness was increased, and anterior chamber depth was decreased. Treated eyes developed myopia, in excess of 15 diopters of refractive error. Shortly after treatment, eyes had increased intraocular pressure (IOP), which was increased in a dose-dependent manner. Seven days after treatment with IGF1 and FGF2 changes to anterior chamber depth, lens thickness and elevated IOP were reduced, whereas increases in the vitreous chamber were persistent. Some damage to ganglion cells was detected in peripheral regions of the retina at 7 days after treatment. We conclude that the extreme myopia in IGF1/FGF2-treated eyes results from increased vitreous chamber depth, decreased anterior chamber depth, and changes in the lens. We propose that factor-induced ocular enlargement and myopia result from changes to the sclera, lens and anterior chamber depth.

The chicken cornea as a model of wound healing and neuronal re-innervation.

PURPOSE: The cornea is the major refractive component of the eye and serves as a barrier to the external environment. Understanding how the cornea responds to injury is important to developing therapies to treat vision disorders that affect the integrity and refractive properties of the cornea. Thus, investigation of the wound healing responses of the cornea to injury in a cost-effective animal model is a valuable tool for research. This study characterizes the wound healing responses in the corneas of White Leghorn chicken. METHODS: Linear corneal wounds were induced in post-natal day 7 (P7) chicks and cellular proliferation, apoptosis and regulation of structural proteins were assessed using immunohistochemical techniques. We describe the time course of increased expression of different scar-related markers, including vimentin, vinculin, perlecan and smooth muscle actin. RESULTS: We find evidence for acute necrotic cell death in the corneal region immediately surrounding cite of incision, whereas we failed to find evidence of delayed cell death or apoptosis. We find that the neuronal re-innervation of SV2-positive axon terminals within the corneal stroma and epithelium occurs very quickly after the initial scarring insult. We describe an accumulation of cells within the stroma immediately underlying the scar, which results, at least in part, from the local proliferation of keratocytes. Further, we provide evidence for scar-induced accumulations of CD45-positive monocytes in injured corneas. CONCLUSIONS: We conclude that the chick cornea is an excellent model system in which to study wound healing, formation of scar tissue, and neuronal re-innervation of sensory endings.

Heterogeneity of glia in the retina and optic nerve of birds and mammals.

We have recently described a novel type of glial cell that is scattered across the inner layers of the avian retina [1]. These cells are stimulated by insulin-like growth factor 1 (IGF1) to proliferate, migrate distally into the retina, and up-regulate the nestin-related intermediate filament transition. These changes in glial activity correspond with increased susceptibility of neurons to excitotoxic damage. This novel cell-type has been termed the Non-astrocytic Inner Retinal Glia-like (NIRG) cells. The purpose of the study was to investigate whether the retinas of non-avian species contain cells that resemble NIRG cells. We assayed for NIRG cells by probing for the expression of Sox2, Sox9, Nkx2.2, vimentin and nestin. NIRG cells were distinguished from astrocytes by a lack of expression for Glial Fibrilliary Acidic Protein (GFAP). We examined the retinas of adult mice, guinea pigs, dogs and monkeys (Macaca fasicularis). In the mouse retina and optic nerve head, we identified numerous astrocytes that expressed GFAP, S100beta, Sox2 and Sox9; however, we found no evidence for NIRG-like cells that were positive for Nkx2.2, nestin, and negative for GFAP. In the guinea pig retina, we did not find astrocytes or NIRG cells in the retina, whereas we identified astrocytes in the optic nerve. In the eyes of dogs and monkeys, we found astrocytes and NIRG-like cells scattered across inner layers of the retina and within the optic nerve. We conclude that NIRG-like cells are present in the retinas of canines and non-human primates, whereas the retinas of mice and guinea pigs do not contain NIRG cells.

Notch signaling influences neuroprotective and proliferative properties of mature Müller glia.

Notch signaling is known to play important roles during retinal development. Recently, Notch signaling has been shown to be active in proliferating Müller glia in acutely damaged chick retina (Hayes et al., 2007). However, the roles of Notch in mature, undamaged retina remain unknown. Thus, the purpose of this study was to determine the role of the Notch-signaling pathway in the postnatal retina. Here we show that components of the Notch-signaling pathway are expressed in most Müller glia at low levels in undamaged retina. The expression of Notch-related genes varies during early postnatal development and across regions, with higher expression in peripheral versus central retina. Blockade of Notch activity with a small molecule inhibitor before damage was protective to retinal interneurons (amacrine and bipolar cells) and projection neurons (ganglion cells). In the absence of damage, Notch is upregulated in retinas treated with insulin and FGF2; the combination of these factors is known to stimulate the proliferation and dedifferentiation of Müller glia (Fischer et al., 2002b). Inhibition of Notch signaling during FGF2 treatment reduces levels of the downstream effectors of the MAPK-signaling pathway-p38 MAPK and pCREB in Müller glia. Further, inhibition of Notch activity potently inhibits FGF2-induced proliferation of Müller glia. Together, our data indicate that Notch signaling is downstream of, and is required for, FGF2/MAPK signaling to drive the proliferation of Müller glia. In addition, our data suggest that low levels of Notch signaling in Müller glia diminish the neuroprotective activities of these glial cells.

A novel type of glial cell in the retina is stimulated by insulin-like growth factor 1 and may exacerbate damage to neurons and Müller glia.

Recent studies have demonstrated that insulin can have profound affects on the survival of neurons within the retina. The purpose of this study was to determine how insulin-like growth factor 1 (IGF1) influences retinal cells; in particular, the glial cells. We identify a novel type of glial cell in the avian retina and provide evidence that these cells can respond to acute damage and IGF1. In normal retinas, we found a distinct cell-type, scattered across the ganglion cell and inner plexiform layers that express Sox2, Sox9, Nkx2.2, vimentin, and transitin, the avian homologue of mammalian nestin. These glial cells have a unique immunohistochemical profile, morphology, and distribution that are distinct among other known types of retinal glia, including microglia, oligodendrocytes, astrocytes, and Muller glia. We termed these cells nonastrocytic inner retinal glia-like (NIRG) cells. We found that the NIRG cells may express the IGF1 receptor and respond to IGF1 by proliferating, migrating distally into the retina, and upregulating transitin. In addition, IGF1 stimulated microglia to become reactive and upregulate lysosomal membrane glycoprotein and CD45. With microglia and NIRG cells stimulated by IGF1 there were elevated levels of cell death and numerous focal detachments across the retina in response to excitotoxic damage. Cell death was prominent within the areas of detachment coinciding with a stark loss of Müller glia and accumulation of NIRG cells. We conclude that NIRG cells are a novel type of retinal glia that is sensitive to IGF1 and whose activity may impact the survival of neurons and Müller glia.

Serotonin released from amacrine neurons is scavenged and degraded in bipolar neurons in the retina.

The neurotransmitter serotonin is synthesized in the retina by one type of amacrine neuron but accumulates in bipolar neurons in many vertebrates. The mechanisms, functions and purpose underlying serotonin accumulation in bipolar cells remain unknown. Here, we demonstrate that exogenous serotonin transiently accumulates in a distinct type of bipolar neuron. KCl-mediated depolarization causes the depletion of serotonin from amacrine neurons and, subsequently, serotonin is taken-up by bipolar neurons. The accumulation of endogenous and exogenous serotonin by bipolar neurons is blocked by selective reuptake inhibitors. Exogenous serotonin is specifically taken-up by bipolar neurons even when serotonin-synthesizing amacrine neurons are destroyed; excluding the possibility that serotonin diffuses through gap junctions from amacrine into bipolar neurons. Further, inhibition of monoamine oxidase A prevents the degradation of serotonin in bipolar neurons, suggesting that monoamine oxidase A is present in these neurons. However, the vesicular monoamine transporter 2 is present only in amacrine cells suggesting that serotonin is not transported into synaptic vesicles and reused as a transmitter in the bipolar neurons. We conclude that the serotonin-accumulating bipolar neurons perform glial functions in the retina by actively transporting and degrading serotonin that is synthesized in neighboring amacrine cells.