Quantitative and morphological studies of retinal development in mice with trisomy 19

To investigate the development of the retinal layers, the eyes of mice with trisomy 19 have been examined by light microscopy between the 2nd and 15th postnatal day. The diameter of the eye, thickness of the entire retina and both relative thickness and nuclear density of each of the retinal layers have been measured and compared to those of chromosomally balanced control animals. Malformations of the eye, alterations of cell morphology or disturbed lamination can not be observed. Retinal differentiation of trisomy 19 mice is delayed by approximately two days. The development of all cellular constituents, i.e., of both neuroectodermal and mesenchymal origin, is retarded accordingly. The eyes of trisomy 19 mice are of reduced size. The relative thickness of each retinal layer follows a normal growth pattern; there is no indication for a selective impairment of the development of one particular layer. With the exception of the ganglion cell layer, nuclear densities of each retinal layer do not differ from those of control mice. The comparison of nuclear densities in the ganglion cell layer suggests that in trisomy 19 mice fewer postmitotic cells differentiate into mature retinal cells.     

Metabotropic glutamate receptors are differentially regulated under elevated intraocular pressure

Glaucoma is a leading cause of blindness, ultimatively resulting in the apoptotic death of retinal ganglion cells. However, molecular mechanisms involved in ganglion cell death are poorly understood. While the involvement of ionotropic glutamate receptors has been extensively studied, virtually nothing is known about its metabotropic counterparts. Here, we compared the retinal gene expression of metabotropic glutamate receptors (mGluR) in eyes with normal and elevated intraocular pressure (IOP) of DBA/2J mice, a model for secondary angle-closure glaucoma using RT-PCR and immunohistochemistry. Elevated IOP in DBA/2J mice significantly increased retinal gene expression of mGluR1a, mGluR2, mGluR4a, mGluR4b, mGluR6 and mGluR7a when compared to C57BL/6 control animals, while mGluR5a/b and mGluR8a were decreased and no difference was observed for mGluR3 and mGluR8b. Specific antibodies detected an increase of mGluR1a and mGluR5a/b in both synaptic layers and in the ganglion cell layer of the retina under elevated IOP. Because ganglion cell death in DBA/2J mice occurs most likely by apoptotic mechanisms, we demonstrated up-regulation of mGluRs in neurons undergoing apoptosis. In summary, we support the idea that the specific gene regulation of mGluRs is a part of the glaucoma-like pathological process that develops in the eyes of DBA/2J mice.     

Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma

Glaucoma is one of the leading eye diseases resulting in blindness due to the death of retinal ganglion cells. This study aimed to develop novel protocol to promote the differentiation of retinal Müller cells into ganglion cells in vivo in a rat model of glaucoma. The stem cells dedifferentiated from rat retinal Müller cells were randomized to receive transfection with empty lentivirus PGC-FU-GFP or lentivirus PGC-FU-Atoh7-GFP, or no transfection. The stem cells were induced further to differentiate. Ocular hypertension was induced using laser photocoagulation. The eyes were injected with Atoh7 expression vector lentivirus PGC-FU-Atoh7-GFP. Eyeball frozen sections, immunohistochemistry, RT-PCR, Western bolt, and apoptosis assay were performed. We found that the proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of the other two groups. The mean intraocular pressure of glaucomatous eyes was elevated significantly compared with those of contralateral eyes. Some retinal Müller cells in the inner nuclear layer entered the mitotic cell cycle in rat chronic ocular hypertension glaucoma model. Atoh7 contributes to the differentiation of retinal Müller cells into retinal ganglion cells in rat model of glaucoma. In conclusion, Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma, thus opening up a new avenue for gene therapy and optic nerve regeneration in glaucoma.     

Aquaporins in complex tissues: distribution of aquaporins 1-5 in human and rat eye

Multiple physiological fluid movements areinvolved in vision. Here we define the cellular and subcellular sitesof aquaporin (AQP) water transport proteins in human and rat eyes byimmunoblotting, high-resolution immunocytochemistry, and immunoelectronmicroscopy. AQP3 is abundant in bulbar conjunctival epithelium andglands but is only weakly present in corneal epithelium. In contrast, AQP5 is prominent in corneal epithelium and apical membranes of lacrimal acini. AQP1 is heavily expressed in scleral fibroblasts, corneal endothelium and keratocytes, and endothelium covering thetrabecular meshwork and Schlemm's canal. Although AQP1 is plentiful inciliary nonpigmented epithelium, it is not present in ciliary pigmentedepithelium. Posterior and anterior epithelium of the iris and anteriorlens epithelium also contain significant amounts of AQP1, but AQP0(major intrinsic protein of the lens) is expressed in lens fiber cells.Retinal Müller cells and astrocytes exhibit notableconcentrations of AQP4, whereas neurons and retinal pigment epitheliumdo not display aquaporin immunolabeling. These studies demonstrateselective expression of AQP1, AQP3, AQP4, and AQP5 in distinct ocularepithelia, predicting specific roles for each in the complex networkthrough which water movements occur in the eye.

     

Vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGFbeta), and interleukin-6 (IL-6) in experimental herpesvirus retinopathy: association with inflammation and viral infection

Experimental herpesvirus retinopathy presents a unique model of a transient inflammatory response in the virus-injected eye and subsequent acute retinal necrosis and chronic inflammation in the contralateral eye. For 6 days after infection, VEGF, TGFbeta1, and TGFbeta2 were associated only with inflammatory cells in the injected eye. By 6 days (after viral antigens were no longer detected), VEGF and TGFbeta2 were upregulated in retinas of injected eyes until 8-10 days. In contralateral eyes, VEGF was first demonstrated in the retina at 6-7 days (prior to the appearance of viral antigens) and TGFbeta2 at 7-8 days. Staining for these factors was also evident around areas of necrosis. The VEGF receptor, flt-1, was associated with ganglion cells and the inner nuclear layer of normal and experimental mice and it was also demonstrated around areas of necrosis. Another VEGF receptor, flk-1, was localized to Muller cell processes and the outer plexiform layer in normal and experimental mice. Coincident with VEGF upregulation in the retinas of herpesvirus-1 injected mice, there was increased flk-1 in ganglion cells and the inner and outer nuclear layers. IL-6 was associated with Muller cell endfeet in normal mice. Following unilateral intraocular inoculation, IL-6 spread along the MUller cell processes and some astrocytes demonstrated IL-6 in both eyes at 6-8 days. The present study demonstrates that intraocular inoculation of herpesvirus is sufficient to induce VEGF, flk-1, TGFbeta2, and IL-6 in the retinas of injected and contralateral eyes. Further investigation of common signaling pathways for these factors during responses to viral infection and the development of acute retinal necrosis could provide information useful for therapeutic intervention in human herpesvirus retinopathy.     

Lycium barbarum polysaccharides reduce neuronal damage, blood-retinal barrier disruption and oxidative stress in retinal ischemia/reperfusion injury

Neuronal cell death, glial cell activation, retinal swelling and oxidative injury are complications in retinal ischemia/reperfusion (I/R) injuries. Lycium barbarum polysaccharides (LBP), extracts from the wolfberries, are good for "eye health" according to Chinese medicine. The aim of our present study is to explore the use of LBP in retinal I/R injury. Retinal I/R injury was induced by surgical occlusion of the internal carotid artery. Prior to induction of ischemia, mice were treated orally with either vehicle (PBS) or LBP (1 mg/kg) once a day for 1 week. Paraffin-embedded retinal sections were prepared. Viable cells were counted; apoptosis was assessed using TUNEL assay. Expression levels of glial fibrillary acidic protein (GFAP), aquaporin-4 (AQP4), poly(ADP-ribose) (PAR) and nitrotyrosine (NT) were investigated by immunohistochemistry. The integrity of blood-retinal barrier (BRB) was examined by IgG extravasations. Apoptosis and decreased viable cell count were found in the ganglion cell layer (GCL) and the inner nuclear layer (INL) of the vehicle-treated I/R retina. Additionally, increased retinal thickness, GFAP activation, AQP4 up-regulation, IgG extravasations and PAR expression levels were observed in the vehicle-treated I/R retina. Many of these changes were diminished or abolished in the LBP-treated I/R retina. Pre-treatment with LBP for 1 week effectively protected the retina from neuronal death, apoptosis, glial cell activation, aquaporin water channel up-regulation, disruption of BRB and oxidative stress. The present study suggests that LBP may have a neuroprotective role to play in ocular diseases for which I/R is a feature.     

Expression pattern of acidic and basic fibroblast growth factor genes in adult rat eyes

Although the retinal angiogenic and mitogenic factors have been identified to be acidic and basic fibroblast growth factors (aFGF and bFGF), little information has so far been available about the cells producing them and their function in retinal tissues. We found, by in situ hybridization, that the expression pattern of the aFGF gene differed remarkably from that of the bFGF gene in adult rat eyes. Our results demonstrated that the aFGF gene was produced by photoreceptor visual cells, neuronal cells in the inner nuclear layer and ganglion cells of the retina, in addition to pigment epithelial cells of the choroid, iris and ciliary body, and epithelial cells of the cornea, conjunctiva and lens, while bFGF was synthesized solely by the photoreceptor visual cells.     

Vertebrate features revealed in the rudimentary eye of the Pacific hagfish (Eptatretus stoutii)

Hagfish eyes are markedly basic compared to the eyes of other vertebrates, lacking a pigmented epithelium, a lens and a retinal architecture built of three cell layers: the photoreceptors, interneurons and ganglion cells. Concomitant with hagfish belonging to the earliest-branching vertebrate group (the jawless Agnathans), this lack of derived characters has prompted competing interpretations that hagfish eyes represent either a transitional form in the early evolution of vertebrate vision, or a regression from a previously elaborate organ. Here, we show the hagfish retina is not extensively degenerating during its ontogeny, but instead grows throughout life via a recognizable PAX6+ ciliary marginal zone. The retina has a distinct layer of photoreceptor cells that appear to homogeneously express a single opsin of the RH1 rod opsin class. The epithelium that encompasses these photoreceptors is striking because it lacks the melanin pigment that is universally associated with animal vision; notwithstanding, we suggest this epithelium is a homologue of gnathosome retinal pigment epithelium (RPE) based on its robust expression of RPE65 and its engulfment of photoreceptor outer segments. We infer that the hagfish retina is not entirely rudimentary in its wiring, despite lacking a morphologically distinct layer of interneurons: multiple populations of cells exist in the hagfish inner retina and subsets of these express markers of vertebrate retinal interneurons. Overall, these data clarify Agnathan retinal homologies, reveal characters that now appear to be ubiquitous across the eyes of vertebrates, and refine interpretations of early vertebrate visual system evolution.     

Therapeutic benefit of radial optic neurotomy in a rat model of glaucoma

Radial optic neurotomy (RON) has been proposed as a surgical treatment to alleviate the neurovascular compression and to improve the venous outflow in patients with central retinal vein occlusion. Glaucoma is characterized by specific visual field defects due to the loss of retinal ganglion cells and damage to the optic nerve head (ONH). One of the clinical hallmarks of glaucomatous neuropathy is the excavation of the ONH. The aim of this work was to analyze the effect of RON in an experimental model of glaucoma in rats induced by intracameral injections of chondroitin sulfate (CS). For this purpose, Wistar rats were bilaterally injected with vehicle or CS in the eye anterior chamber, once a week, for 10 weeks. At 3 or 6 weeks of a treatment with vehicle or CS, RON was performed by a single incision in the edge of the neuro-retinal ring at the nasal hemisphere of the optic disk in one eye, while the contralateral eye was submitted to a sham procedure. Electroretinograms (ERGs) were registered under scotopic conditions and visual evoked potentials (VEPs) were registered with skull-implanted electrodes. Retinal and optic nerve morphology was examined by optical microscopy. RON did not affect the ocular hypertension induced by CS. In eyes injected with CS, a significant decrease of retinal (ERG a- and b-wave amplitude) and visual pathway (VEP N2-P2 component amplitude) function was observed, whereas RON reduced these functional alterations in hypertensive eyes. Moreover, a significant loss of cells in the ganglion cell layer, and Thy-1-, NeuN- and Brn3a- positive cells was observed in eyes injected with CS, whereas RON significantly preserved these parameters. In addition, RON preserved the optic nerve structure in eyes with chronic ocular hypertension. These results indicate that RON reduces functional and histological alterations induced by experimental chronic ocular hypertension.     

Effects of bone marrow stromal cell injection in an experimental glaucoma model

We investigated if bone marrow stromal cells (BMSCs) transplanted into the vitreous body of a glaucoma model eye could be integrated in the host retina and also whether they could rescue the retinal ganglion cells (RGCs) from death induced by the elevated intraocular pressure. Glaucoma was induced in the right eye of adult Wistar rats by ligating the episcleral veins. The GFP-expressing BMSCs (GFP-BMSCs) were injected into the vitreous body of both the control and the glaucomatous eyes. After transplantation, GFP-BMSCs were mostly present along with the inner limiting membrane and only a few cells were integrated into the ganglion cell layer. At 2 or 4 weeks after transplantation, GFP-BMSCs were observed to express various trophic factors. The BMSCs injected glaucoma model eyes showed less reduction in the number of RGCs compared to the glaucomatous eyes with PBS injection. This study suggests that BMSC transplantation may be worthy as a neuroprotective tool to treat glaucoma.     

Pax-6 expression during retinal regeneration in the adult newt

The present study examined the expression of Pax-6 during retinal regeneration in adult newts using in situ hybridization. In a normal retina, Pax-6 is expressed in the ciliary marginal zone, the inner part of the inner nuclear layer, and the ganglion cell layer. After surgical removal of the neural retina, retinal pigment epithelial cells proliferate into retinal precursor cells and regenerate a fully functional retina. At the beginning of retinal regeneration, Pax-6 was expressed in all retinal precursor cells. As regeneration proceeded, differentiating cells appeared at the scleral and vitreal margins of the regenerating retina, which had no distinct plexiform layers. In this stage, the expression of Pax-6 was localized in a strip of cells along the vitreal margin of the regenerating retina. In the late stage of regeneration, when the layer structure was completed, the expression pattern of Pax-6 became similar to that of a normal retina. It was found that Pax-6 is expressed in the retinal precursor cells in the early regenerating retina and that the expression pattern of Pax-6 changed as cell differentiation proceeded during retinal regeneration.     

Imaging of retinal ganglion cells in glaucoma: pitfalls and challenges

Imaging has gained a key role in modern glaucoma management. Traditionally, interest was directed toward the appearance of the optic nerve head and the retinal nerve fiber layer. With the improvement of the resolution of optical coherence tomography, the ganglion cell complex has also become routinely accessible in the clinic. Further advances have been made in understanding the structure-function relationship in glaucoma. Nevertheless, direct imaging of the retinal ganglion cells in glaucoma would be advantageous. With the currently used techniques, this goal cannot be achieved, because the transversal resolution is limited by aberrations of the eye. The use of adaptive optics has significantly improved transversal resolution, and the imaging of several cell types including cones and astrocytes has become possible. Imaging of retinal ganglion cells, however, still remains a problem, because of the transparency of these cells. However, the visualization of retinal ganglion cells and their dendrites has been achieved in animal models. Furthermore, attempts have been made to visualize the apoptosis of retinal ganglion cells in vivo. Implementation of these techniques in clinical practice will probably improve glaucoma care and facilitate the development of neuroprotective strategies.     

Pigmented epithelium to retinal transdifferentiation and Pax6 expression in larval Xenopus laevis

This study examines the retinal transdifferentiation (TD) of retinal pigmented epithelium (RPE) fragments dissected from Xenopus laevis larvae and implanted into the vitreous chamber of non-lentectomized host eyes. In these experimental conditions, most RPE implants transformed into polarized vesicles in which the side adjacent to the lens maintained the RPE phenotype, while the side adjacent to the host retina transformed into a laminar retina with the photoreceptor layer facing the cavity of the vesicle and with the ganglionar cell layer facing the host retina. The formation of a new retina with a laminar organization is the result of depigmentation, proliferation and differentiation of progenitor cells under the influence of inductive factors from the host retina. The phases of the TD process were followed using BrdU labelling as a marker of the proliferation phase and using a monoclonal antibody (mAbHP1) as a definitive indicator of retina formation. Pigmented RPE cells do not express Pax6. In the early phase of RPE to retinal TD, all depigmented and proliferating progenitor cells expressed Pax6. Changes in the Pax6 expression pattern became apparent in the early phase of differentiation, when Pax6 expression decreased in the presumptive outer nuclear layer (ONL) of the new-forming retina. Finally, during the late differentiation phase, the ONL, which contains photoreceptors, no longer expressed Pax6, Pax6 expression being confined to the ganglion cell layer and the inner nuclear layer. These results indicate that Pax6 may have different roles during the different phases of RPE to retinal TD, acting as an early retinal determinant and later directing progenitor cell fate.     

转人APP基因小鼠视网膜视神经细胞超微结构改变

目的观察转人APP基因AD模型小鼠视网膜各层细胞超微结构的改变。方法实验采用C57BL/6J转人APP基因小鼠6只,采用同背景10个月龄的C57 BL/6J正常小鼠6只做为对照。灌注处死小鼠,完整取出右眼球分离视网膜,制作电镜标本观察视网膜各层细胞超微结构改变。结果与对照组相比,模型组视网膜各层细胞异常明显:膜盘排列结构模糊,局部间隙溶解、甚至消失;外核层细胞体干枯变形,染色质聚集浓缩;内核层可见固缩细胞;神经节细胞胞膜不完整,线粒体水肿,并可见染色质聚集。结论转人APP基因AD模型小鼠视网膜各层神经细胞超微结构存在明显病理改变。     

Temporary disruption of the retinal basal lamina and its effect on retinal histogenesis.

An experimental paradigm was devised to remove the retinal basal lamina for defined periods of development: the basal lamina was dissolved by injecting collagenase into the vitreous of embryonic chick eyes, and its regeneration was induced by a chase with mouse laminin-1 and alpha2-macroglobulin. The laminin-1 was essential in reconstituting a new basal lamina and could not be replaced by laminin-2 or collagen IV, whereas the macroglobulin served as a collagenase inhibitor that did not directly contribute to basal lamina regeneration. The regeneration occurred within 6 h after the laminin-1 chase by forming a morphologically complete basal lamina that included all known basal lamina proteins from chick embryos, such as laminin-1, nidogen-1, collagens IV and XVIII, perlecan, and agrin. The temporary absence of the basal lamina had dramatic effects on retinal histogenesis, such as an irreversible retraction of the endfeet of the neuroepithelial cells from the vitreal surface of the retina, the formation of a disorganized ganglion cell layer with an increase in ganglion cells by 30%, and the appearance of multiple retinal ectopias. Finally, basal lamina regeneration was associated with aberrant axons failing to correctly enter the optic nerve. The present data demonstrate that a transient disruption of the basal lamina leads to dramatic and probably irreversible aberrations in the histogenesis in the developing central nervous system.     

The development of retinal ganglion cells deprived of their targets

The influence of central targets on the morphological differentiation of retinal ganglion cells was investigated in Xenopus laevis. Since the ganglion cells mature into distinct morphological subtypes after their axons have reached their central targets, it is possible that the target tissues may influence or specify this aspect of neuronal cell development. To test this idea, Xenopus eyebuds were target-deprived by transplantation to the flank region of host embryos where they developed ectopically. The grafted eyes grew at normal rates, but could not make any projections into the central nervous system. To examine the morphological differentiation of the retinal ganglion cells their structures were revealed using an in vitro retinal preparation and intracellular injections of the dye Lucifer yellow. The elaboration and maturation of ganglion cell dendrites were found to be indistinguishable between control and transplanted eyes throughout development. Thus, the development of retinal ganglion cells into distinct morphological classes can occur even when their axons do not interact with the appropriate central targets.     

ADAM10 mediates N‐cadherin ectodomain shedding during retinal ganglion cell differentiation in primary cultured retinal cells from the developing chick retina

Here, we examined the role of ADAM10 during retinal cell differentiation in retinal sections and in vitro cultures of developing chick retinal cells from embryonic day 6 (ED6). Immunohistochemistry showed that ADAM10 is abundantly expressed in the inner zone of neuroblastic layer at ED5, and it becomes more highly expressed in the ganglion cell layer at ED7 and ED9. Western blotting confirmed that ADAM10 was expressed as an inactive pro‐form that was processed to a shorter, active form in control cultured cells, but in cultures treated with an ADAM10 inhibitor (GI254023X) and ADAM10‐specific siRNA, the level of mature ADAM10 decreased. Phase‐contrast microscopy showed that long neurite extensions were present in untreated cultures 24 h after plating, whereas cultures treated with GI254023X showed significant decreases in neurite extension. Immunofluorescence staining revealed that there were far fewer differentiated ganglion cells in ADAM10 siRNA and GI254023X‐treated cultures compared to controls, whereas the photoreceptor cells were unaltered. The Pax6 protein was more strongly detected in the differentiated ganglion cells of control cultures compared to ADAM10 siRNA and GI254023X‐treated cultures. N‐cadherin ectodomain shedding was apparent in control cultures after 24 h, when ganglion cell differentiation was observed, but ADAM10 siRNA and GI254023X treatment inhibited these processes. In contrast, N‐cadherin staining was strongly detected in photoreceptor cells regardless of ADAM10 siRNA and GI254023X treatment. Taken together, these data indicate that the inhibition of ADAM10 can inhibit Pax6 expression and N‐cadherin ectodomain shedding in retinal cells, possibly affecting neurite outgrowth and ganglion cell differentiation. J. Cell. Biochem. 114: 942–954, 2013. © 2013 Wiley Periodicals, Inc.     

Epac2-deficiency leads to more severe retinal swelling,glial reactivity and oxidative stress in transient middle cerebral artery occlusion induced ischemic retinopathy

Ischemia occurs in diabetic retinopathy with neuronal loss, edema, glial cell reactivity and oxidative stress. Epacs, consisting of Epac1 and Epac2, are c AMP mediators playing important roles in maintenance of endothelial barrier and neuronal functions. To investigate the roles of Epacs in the pathogenesis of ischemic retinopathy, transient middle cerebral artery occlusion(t MCAO) was performed on Epac1-deficient(Epac1-/-) mice, Epac2-deficient(Epac2-/-) mice, and their wild type counterparts(Epac1+/+ and Epac2+/+). Two-hour occlusion and 22-hour reperfusion were conducted to induce ischemia/reperfusion injury to the retina. After t MCAO, the contralateral retinae displayed similar morphology between different genotypes. Neuronal loss, retinal edema and increase in immunoreactivity for aquaporin 4(AQP4), glial fibrillary acidic protein(GFAP), peroxiredoxin 6(Prx6) were observed in ipsilateral retinae. Epac2-/- ipsilateral retinae showed more neuronal loss in retinal ganglion cell layer, increased retinal thickness and stronger immunostaining of AQP4, GFAP, and Prx6 than those of Epac2+/+. However, Epac1-/- ipsilateral retinae displayed similar pathology as those in Epac1+/+ mice. Our observations suggest that Epac2-deficiency led to more severe ischemic retinopathy after retinal ischemia/reperfusion injury.