Cells In Inner Nuclear Layer Research Articles

Retinal regeneration after injury induced by gamma-ray irradiation during early embryogenesis in medaka, Oryzias latipes

Purpose Zebrafish, a small fish model, exhibits a multipotent ability for retinal regeneration after damage throughout its lifetime. Compared with zebrafish, birds and mammals exhibit such a regenerative capacity only during the embryonic period, and this capacity decreases with age. In medaka, another small fish model that has also been used extensively in biological research, the retina’s inner nuclear layer (INL) failed to regenerate after injury in the hatchling at eight days postfertilization (dpf). We characterized the regenerative process of the embryonic retina when the retinal injury occurred during the early embryonic period in medaka. Methods We employed a 10 Gy dose of gamma-ray irradiation to initiate retinal injury in medaka embryos at 3 dpf and performed histopathological analyses up to 21 dpf. Results One day after irradiation, numerous apoptotic neurons were observed in the INL; however, these neurons were rarely observed in the ciliary marginal zone and the photoreceptor layer. Numerous pyknotic cells were clustered in the irradiated retina until two days after irradiation. These disappeared four days after irradiation, but the abnormal bridging structures between the INL and ganglion cell layer (GCL) were present until 11 days after irradiation, and the neural layers were completely regenerated 18 days after irradiation. After gamma-ray irradiation, the spindle-like Müller glial cells in the INL became rounder but did not lose their ability to express SOX2. Conclusions Irradiated retina at 3 dpf of medaka embryos could be completely regenerated at 18 days after irradiation (21 dpf), although the abnormal layer structures bridging the INL and GCL were transiently formed in the retinas of all the irradiated embryos. Four days after irradiation, embryonic medaka Müller glia were reduced in number but maintained SOX2 expression as in nonirradiated embryos. This finding contrasts with previous reports that 8 dpf medaka larvae could not fully regenerate damaged retinas because of loss of SOX2 expression.

Vitreomacular Interface Abnormalities in Multiple Sclerosis; A Novel Signature of Disability (P10-3.013)

Vitreomacular Interface Abnormalities in Multiple Sclerosis; A Novel Signature of Disability (P10-3.013)

A Novel Artificial Visual System for Motion Direction Detection in Grayscale Images

How specific features of the environment are represented in the mammalian brain is an important unexplained mystery in neuroscience. Visual information is considered to be captured most preferentially by the brain. As one of the visual information elements, motion direction in the receptive field is thought to be collected already at the retinal direction-selective ganglion cell (DSGC) layer. However, knowledge of direction-selective (DS) mechanisms in the retina has remained only at a cellular level, and there is a lack of complete direction-sensitivity understanding in the visual system. Previous studies of DS models have been limited to the stage of one-dimensional black-and-white (binary) images or still lack biological rationality. In this paper, we innovatively propose a two-dimensional, eight-directional motion direction detection mechanism for grayscale images called the artificial visual system (AVS). The structure and neuronal functions of this mechanism are highly faithful to neuroscientific perceptions of the mammalian retinal DS pathway, and thus highly biologically reasonable. In particular, by introducing the horizontal contact pathway provided by horizontal cells (HCs) in the retinal inner nuclear layer and forming a functional collaboration with bipolar cells (BCs), the limitation that previous DS models can only recognize object motion directions in binary images is overcome; the proposed model can solve the recognizing problem of object motion directions in grayscale images. Through computer simulation experiments, we verified that AVS is effective and has high detection accuracy, and it is not affected by the shape, size, and location of objects in the receptive field. Its excellent noise immunity was also verified by adding multiple types of noise to the experimental data set. Compared to a classical convolutional neural network (CNN), it was verified that AVS is completely significantly better in terms of effectiveness and noise immunity, and has various advantages such as high interpretability, no need for learning, and easy hardware implementation. In addition, activation characteristics of neurons in AVS are highly consistent with those real in the retinal DS pathway, with strong neurofunctional similarity and brain-like superiority. Moreover, AVS will also provide a novel perspective and approach to understanding and analyzing mechanisms as well as principles of mammalian retinal direction-sensitivity in face of a cognitive bottleneck on the DS pathway that has persisted for nearly 60 years.

Monosodium Glutamate-Induced Mouse Model With Unique Diabetic Retinal Neuropathy Features and Artificial Intelligence Techniques for Quantitative Evaluation.

ObjectiveTo establish an artificial intelligence-based method to quantitatively evaluate subtle pathological changes in retinal nerve cells and synapses in monosodium glutamate (MSG) mice and provide an effective animal model and technique for quantitative evaluation of retinal neurocytopathies.MethodsICR mice were subcutaneously injected with MSG to establish a model of metabolic syndrome. We then established a mouse model of type 1 diabetes, type 2 diabetes, and KKAy mouse model as control. The HE sections of the retina were visualized using an optical microscope. AI technology was used for quantitative evaluation of the retinal lesions in each group of rats. The surface area custom parameters of the retinal nerve fiber layer (RNFL), inner plexiform layer (IPL), inner nuclear layer (INL), and outer plexiform layer (OPL) were defined as SR, SIPL, SINL, and SOPL, respectively. Their heights were defined as HR, HIPL, HINL, and HOPL, and the number of ganglion cells was defined as A. Then, the attention-augmented fully convolutional Unet network was used to segment the retinal HE images, and AI technology to identify retinal neurocytopathies quantitatively.ResultsThe attention-augmented fully convolutional Unet network increased PA and IOU parameters for INL, OPL, RNFL, and ganglion cells and was superior in recognizing fine structures. A quantitative AI identification of the height of each layer of the retina showed that the heights of the IPL and INL of the MSG model were significantly less than those of the control groups; the retinas of the other diabetic models did not exhibit this pathological feature. The RNFLs of type 2 diabetes were thinner, and the characteristics of retinopathy were not obvious in the other animal models. The pathological changes seen on HE images were consistent with the results of the quantitative AI evaluation. Immunohistochemistry results showed that NMDAR2A, GluR2, and NRG1 were significantly downregulated in the retina of MSG mice.ConclusionsThe MSG retinopathy model is closely associated with neurotransmitter abnormalities and exhibits important characteristics of retinal neurodegeneration, making it suitable for studying retinal neurocytopathies. The AI recognition technology for retinal images established in the present study can be used for the quantitative and objective evaluation of drug efficacy.

Expression of SCO1 and SCO2 after form-deprivation myopia in Guinea pigs.

The retina is a highly energy-consuming tissue associated with visual development, and the reduced quality of retinal imaging can be related to myopia. Synthesis of cytochrome c oxidase 1 (SCO1) and synthesis of cytochrome c oxidase 2 (SCO2) are involved in ATP (adenosine triphosphate) synthesis and energy metabolism. This study aimed to observe the morphologic changes and investigate the expression of SCO1 and SCO2 induced by form-deprivation myopia (FDM) in the retina and sclera of guinea pigs. Thirty-six 3-week-old male guinea pigs were randomly assigned to one of two groups: (1) the model group (n = 18), in which the right eyes were covered by a thin opaque balloon as FDM group, and the left eyes were uncovered and served as the contralateral control group; (2) the blank control group (n = 18), in which bilateral eye received no manipulation. Eyeballs were enucleated for histological analysis. The retina and sclera of the guinea pigs were separated to determine the protein and mRNA expression levels of SCO1 and SCO2, respectively. After four weeks of form deprivation (FD), the refractive degree and axial length increased significantly (P < 0.001). The retinal and scleral tissues were moderately thinner, and the ganglion cells and the cells of inner and outer nuclear layers in the retina became fewer. Compared with the contralateral control group (P < 0.001) and the blank control group (P < 0.001), the collagen content of the sclera became less in the FDM group. The protein and mRNA expression levels of SCO1 and SCO2 in the FDM group were significantly lower than those in the contralateral control group and the blank control group (P < 0.05). The morphologies of the retina and sclera were changed, and the expression of SCO1 and SCO2 at the protein and transcription levels was significantly reduced in the FDM group. Given these changes, SCO1 and SCO2 genes may be involved in myopic progression.

A multi-scale/multi-physics model for the theoretical study of the vascular configuration of retinal capillary plexuses based on OCTA data.

The retinal tissue is highly metabolically active and is responsible for translating the visual stimuli into electrical signals to be delivered to the brain. A complex vascular structure ensures an adequate supply of blood and oxygen, which is essential for the function and survival of the retinal tissue. To date, a complete understanding of the configuration of the retinal vascular structures is still lacking. Optical coherence tomography angiography has made available a huge amount of imaging data regarding the main retinal capillary plexuses, namely the superficial capillary plexuses (SCP), intermediate capillary plexuses (ICP) and deep capillary plexuses (DCP). However, the interpretation of these data is still controversial. In particular, the question of whether the three capillary plexuses are connected in series or in parallel remains a matter of debate. In this work, we address this question by utilizing a multi-scale/multi-physics mathematical model to quantify the impact of the two hypothesized vascular configurations on retinal hemodynamics and oxygenation. The response to central retinal vein occlusion (CRVO) and intraocular pressure (IOP) elevation is also simulated depending on whether the capillary plexuses are connected in series or in parallel. The simulation results show the following: (i) in the in series configuration, the plexuses exhibit a differential response, with DCP and ICP experiencing larger pressure drops than SCP; and (ii) in the in parallel configuration, the blood flow redistributes uniformly in the three plexuses. The different vascular configurations show different responses also in terms of oxygen profiles: (i) in the in series configuration, the outer nuclear layer, outer plexiform layer and inner nuclear layer (INL) are those most affected by CRVO and IOP elevation; and (ii) in the in parallel configuration the INL and ganglion cell layer are those most affected. The in series results are consistent with studies on paracentral acute middle maculopathy, secondary to CRVO and with studies on IOP elevation, in which DCP and ICP and the retinal tissues surrounding them are those most affected by ischemia. These findings seem to suggest that the in series configuration better describes the physiology of the vascular retinal capillary network in health and disease.

Synaptic repair and vision restoration in advanced degenerating eyes by transplantation of retinal progenitor cells.

SummaryStem cell transplantation shows enormous potential for treatment of incurable retinal degeneration (RD). To determine if and how grafts connect with the neural circuits of the advanced degenerative retina (ADR) and improve vision, we perform calcium imaging of GCaMP5-positive grafts in retinal slices. The organoid-derived C-Kit+/SSEA1− (C-Kit+) retinal progenitor cells (RPCs) become synaptically organized and build spontaneously active synaptic networks in three major layers of ADR. Light stimulation of the host photoreceptors elicits distinct neuronal responses throughout the graft RPCs. The graft RPCs and their differentiated offspring cells in inner nuclear layer synchronize their activities with the host cells and exhibit presynaptic calcium flux patterns that resemble intact retinal neurons. Once graft-to-host network is established, progressive vision loss is stabilized while control eyes continually lose vision. Therefore, transplantation of organoid-derived C-Kit+ RPCs can form functional synaptic networks within ADR and it holds promising avenue for advanced RD treatment.

Effect of constant light on the morphology of the retina in domestic chick (Gallus g. domesticus): A scanning electron microscope study

Light induces variable levels of damage to the retina and often, death of the photoreceptor cells. It is reported that chick retina, havingabundant melanin in the retinal pigment epithelium, undergoes no or minimal damage from exposure to light. However, the morphology of cone and rod photoreceptor cells after continuous exposure of light has not been studied in detail. The present study investigated the detailed changes of photoreceptor cells, by scanning electron microscopy (SEM).Domestic chicks (Gallus g. domesticus) from post-hatch day (PH) 1 to 7 were initially reared at 700 lux at 12 h light (L): 12 h dark (D). From PH 7 to 18, they were exposed to 12L: 12D (control) and 24L: 0D (constant light) cycles at 700 lux. At PH18, chicks were sacrificed, their eyes enucleated and preserved in Karnovsky’s fixative. Theretina was then separated from the adherent choroid, mounted on photoreceptor side up and processed for SEM. There was significant damage to rod and cone outer segmentsfrom both dorsal and ventral part of the retina and decrease in the abundance of cells of inner nuclear layer in constant light, compared to that found in 12L: 12D cycles. It is likely that constant light affects the retinal cells in the vertical pathway, which could alter the photo transduction process.

Ginsenoside Rg1 prevents early diabetic retinopathy via reducing retinal ganglion cell layer and inner nuclear layer cell apoptosis in db/db mice.

BackgroundDiabetic retinopathy (DR), a diabetic vascular complication, is prone to developing into blindness. Ginsenoside Rg1 (GRg1), a major saponin in ginseng, exerts high anti-apoptotic activity.MethodsThis study aimed to explore the protective effects of GRg1 against diabetes-induced retinal damage. Measurements of blood glucose, blood lipids and vascular permeability were performed, as well as assessments of pathological changes, and the retinal thickness of each layer. Retinal cell apoptosis related protein expression levels were measured by immunofluorescence and western blot assays.ResultsOur data demonstrated that GRg1 effectively reduced blood glucose and triglyceride levels and maintained normal retinal permeability and physiological structure. GRg1 maintained the thickness of the ganglion cell layer (GCL) and the inner nuclear layer (INL) by reducing cell apoptosis.ConclusionsThese data strongly indicate that GRg1 prevents diabetic retinal changes by decreasing GCL and INL cell apoptosis. GRg1 may be a promising drug for early DR treatment.

Neuronal apoptosis, axon damage and synapse loss occur synchronously in acute ocular hypertension

Retinal ganglion cells (RGCs) apoptosis and their axon degeneration are pivotal features in glaucoma. Previous studies suggest that the process of RGCs soma degeneration is distinct from axon degeneration and that both of them lead to vision loss but separately. However, since a normal visual function relies on the integrity of axon, synapse and soma in the retina, a comprehensive understanding of the changes of these neuron components in glaucoma is desired. Therefore, in an acute ocular hypertension (AOH) model in mice, we systematically evaluated retinal neuron soma, axon and synapse alteration at certain time points. We found that ocular hypertension led to a progressive apoptosis of retinal neural cells which proceeded from peripheral to central retina in the wholemount, meanwhile, started in the ganglion cell layer (GCL) and spread to the inner nuclear layer (INL) and then the outer nuclear layer (ONL) as time went on. The type of apoptotic cells was identified as RGCs in GCL, amacrine cells in INL and cone photoreceptor cells in ONL. Axon degeneration was observed at the same time as soma degenerated and also progressed from peripheral to central retina. More interestingly, accumulation of neurofilament in the soma caused by axon transport failure was detected synchronously. We also found that presynaptic and postsynaptic vesicle proteins were downregulated. Taken together, these data support a view that retinal neuronal apoptosis happens not only in RGCs, but also other neurons in laminar layers. Axon damage and synapse loss occur synchronously with soma loss in AOH. The combination of these three parameters might facilitate a systematic evaluation of the disease progression and treatment strategies in glaucoma.

Localization of cholecystokinin in the zebrafish retina from larval to adult stage

The peptide hormone cholecistokinin (CCK) plays a key role in the central and peripheral nervous system. It is known to be involved in the digestive physiology and in the regulation of food intake. Moreover, the CCK expression has also been detected in the retina of different vertebrates, including fish, although its biological activity in this tissue remains to be elucidated. In literature no data are yet available about the CCK-immunoreactivity in the zebrafish retina during development. Therefore, the aim of the study was to investigate the distribution of sulfated cholecystokinin octapeptide (CCK8-S) as a well preserved form during evolution in the zebrafish retina from 3days post hatching (dph) until adult stage, using immunohistochemistry in order to elucidate the potential role of this protein in the development and maintenance of normal retinal homeostasis. The cellular distribution of CCK in the retina was similar from 3 dph to 40days post fertilization (dpf) when immunoreactivity was found in the photoreceptors layer, in the outer plexiform layer, in the inner plexiform layer and, to a lesser extent, in the ganglion cell layer (GCL). Immunohistochemical localization at 50 dpf as well as in the adult stage was observed in a subpopulation of amacrine cells in the proximal inner nuclear layer, in the inner plexiform layer, in displaced amacrine cells and in retinal ganglion cells in the GCL. Our results demonstrate for the first time the occurrence of CCK in the zebrafish retina from larval to adult stage with a different pattern of distribution, suggesting different roles of CCK during retinal cells maturation.

Transduction Patterns of Adeno-associated Viral Vectors in a Laser-Induced Choroidal Neovascularization Mouse Model

Adeno-associated virus (AAV) vector is a promising platform technology for ocular gene therapy. Recently clinical successes to treat choroidal neovascularization (CNV) in wet type age-related macular degeneration have been reported. However, because pathologic conditions of the retina may alter the tropism of viral vectors, it is necessary to evaluate the transduction efficiency of different serotypes of AAV vectors in the retinas with CNVs. Here, we show the patterns and efficacy of transduction of AAV2, -5, and -8 vectors in a laser-induced CNV mouse model. C57BL/6J mice were subjected to unilateral laser photocoagulation on the right eye to induce CNV 5 days prior to intravitreal injection of AAV2, -5, and -8 capsids expressing EGFP. Transduction was increased around CNV lesions for all AAV capsid types, and AAV2 resulted in the highest transduction efficiency. In the absence of CNV, the AAV2 vector transduced ganglion and inner nuclear layer (INL) cells, and AAV5 and AAV8 transduced only a small proportion of cells in the retinal ganglion cell layer. CNV increased AAV2 vector expression throughout the retina and in and around CNVs; the transduced cells included retinal ganglion cells, Müller cells, cells from the INL and outer nuclear layer (ONL), photoreceptors, and retinal pigment epithelium (RPE) cells. Inflammatory cells and endothelial cells in CNVs were also transduced by AAV2. AAV5 and AAV8 were transduced in retinal ganglion, Müller, INL, ONL, and RPE cells in a localized pattern, and only endothelial cells at the surface of CNV lesions showed EGFP expression. Taken together, CNV formation resulted in enhanced transduction of AAV2, -5, and -8, and AAV2 exhibited the highest transduction efficiency in cells in CNV lesions.

Resveratrol Inhibits Diabetic-Induced Müller Cells Apoptosis through MicroRNA-29b/Specificity Protein 1 Pathway.

The aim of this study was to evaluate the anti-apoptosis effects of resveratrol (RSV) on diabetic rats retinal Müller cells in vivo and in vitro and to further investigate the roles of microRNA-29b (miR-29b)/specificity protein 1 (SP1) in the anti-apoptosis mechanism of RSV. Retina was obtained from normal and diabetic rats with or without RSV (5 and 10mg/kg/day) treatments at 1-7months. TdT-mediated dUTP-biotin nick end labeling (TUNEL) and Annexin V/PI staining were used to detect apoptosis. Immunofluorescence was used to assess distribution of SP1 in retina. MiR-29b and SP1 messenger RNA (mRNA) expression was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). SP1, Bax, and bcl-2 protein expression was evaluated by western blotting. Caspase-3 activity was detected by assay kit. Our study showed that the TUNEL-positive cells were mainly localized in the inner nuclear layer (INL) of retina and RSV administration effectively suppressed streptozotocin (STZ)-induced apoptosis of retinal cells in INL in vivo (P < 0.001). Our study also showed that RSV administration effectively suppressed high glucose (HG)-induced retinal Müller cells' apoptosis in vitro (P < 0.001). Furthermore, our study revealed that the diabetes-induced downregulated expression of miR-29b and upregulated expression of SP1 could be rescued by RSV in vivo and in vitro (P < 0.05). The anti-apoptosis effect and downregulated SP1 expression effect of RSV was prevented by miR-29b inhibitor (P < 0.05). MiR-29b mimic increased the above-mentioned effects of RSV (P < 0.001). These findings indicate that RSV is a potential therapeutic option for diabetic retinopathy (DR) and that miR-29b/SP1 pathway play roles in the anti-apoptosis mechanism of RSV.

Modulation of MEK/ERK and PI3K/Akt pathways to the expression of DDR2 and MMP-13 in choroidal neovascularization

Background It is estimated that discoidin domain receptor 2 (DDR2) and matrix metalloproteinase-13 (MMP-13) play an important role in the development of tumor angiogenesis. However, their effects on choroidal neovascularizaiton (CNV) have not been clarified yet. Objective This study was to observe the expression pattern of DDR2 and MMP-13 in CNV and to further investigate the regulation role of MEK/ERK and PI3K/Akt pathways to the expression of DDR2 and MMP-13 in CNV. Methods CNV models were established in 78 Brown Norway (BN) rats by retinal photocoagulation with 532 nm laser. Then the animals were randomly divided into the normal control group (n=7), the model control group (n=39), PD98059 (MEK1 inhibitor) group (n=16) and LY294002 (PI3K inhibitor) group (n=16), and 5 mmol/L PD98059 or 5 mmol/L LY294002 3 μl was intravitreally injected 1 day and 7 days after photocoagulation in the PD98059 group or LY294002 group. The expression of DDR2 and MMP-13 mRNA and proteins in the CNV area were detected by using reverse transcription PCR (RT-PCR), and the expression levels of p-ERK/ERK and p-Akt/Akt protein were detected by Western blot assay. CNV thickness was measured by pathological examination 14 days after photocoagulation, and the changes of CNV thickness, the expression levels of DDR2 and MMP-13 in CNV were compared among the model control group, PD98059 group and LY294002 group. Results Three days after photocoagulation, the cells within the lasered lesions proliferated, then CNV formed 7 days after photocoagulation and became stable 14 days after photocoagulation. Immunohistochemistry staining indicated that DDR2 was weakly expressed in the cells of ganglion cell layer, inner nuclear layer and vascular endothelial cells; while MMP-13 was strongly expressed in the cells of the inner limiting membrane layer, photoreceptor layer and sclera layer. Both DDR2 and MMP-13 were strongly expressed in CNV area. Double immunofluorescence staining revealed that MMP-13 and DDR2 co-expression in CNV area. RT-PCR revealed that the relative DDR2 mRNA levels at 1 day, 3 days, 7 days and 14 days after photocoagulation were 55. 22±4. 03, 47. 74±2. 23, 14. 82±4. 56 and 5. 59±2. 47 respectively, while the relative MMP-13 mRNA levels were 25. 54±3. 83, 43. 51±4. 36, 10. 90±4. 00 and 5. 23±3. 23 respectively. Compared with the normal control group, the expression of DDR2 and MMP-13 were significantly increased (all at P 0. 05). Both PD98059 and LY294002 treatment were able to attenuate the thickness of CNV to 57. 21% and 50. 34% at day 14 after photocoagulation and further decrease the expression levels of DDR2 and MMP-13 in CNV (all at P<0. 05). Conclusions The expressions of DDR2 and MMP-13 up-regulate in laser-induced CNV. MEK/ERK and PI3K/Akt pathways suppress the development of CNV by regulating the expression of DDR2 and MMP-13. Key words: Discoindin domain receptor 2; Choroid/pathology; Neovascularization; Matrix metalloproteinase-13; Disease model; Rat, Brown Norway

Neuropeptide Research in the Eye

The neuropeptide research in the eye is the main topic of our scientific group in Innsbruck. Most of the neuropeptides have been discovered more than 30 years ago and the presence and distribution of some of them has been explored in the eye in the 80´s mainly by Richard Stone. This concerns particularly substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY). Whereas SP and CGRP have been found to be constituents of the sensory innervation of the eye, VIP is present in the parasympathetic and NPY in the sympathetic innervation of the eye. These results have been reviewed in 1987 in “Experientia”. In the retina, the typical neuropeptide localization are amacrine cells in the proximal inner nuclear layer and displaced amacrine cells in the ganglion cell layer but some of them are also present in ganglion cells.

The effects of x-ray radiation on the eye development of zebrafish

The toxic effects of x-ray radiation on eye development was measured using zebrafish as a model organism. Zebrafish embryos at 8 h post-fertilization (hpf) were irradiated using X-rays at doses of 1, 2, 4, and 8 Gy. At 24 and 48 hpf, x-ray radiation induced a significant increase in reactive oxygen species (ROS) content and cell apoptotic signals. Both of these increases were dose dependent and there were significant positive relationships between them at 24 hpf. At 48 and 72 hpf, the increase of ROS concentration can be eliminated by increasing activities of superoxide dismutase and catalase. Although the ROS generated by x-ray radiation caused a significant increase in cell apoptosis at 24 and 48 hpf, the cellular layers of the retina and lens formation in the irradiated groups were not significantly disrupted at 144 hpf compared with the control group, with the exception of a heterogeneous distribution of the cells in inner nuclear cell layer and a significant decrease in the diameters of whole eyes after 8 Gy irradiation. X-Ray radiation at later stages of gastrulation may not cause distinct optic complications; however, there is still a risk of microophthalmia at high doses of irradiation.

Electrical Stimulation of Inner Retinal Neurons in Wild-Type and Retinally Degenerate (rd/rd) Mice

Electrical stimulation of the retina following photoreceptor degeneration in diseases such as retinitis pigmentosa and age-related macular degeneration has become a promising therapeutic strategy for the restoration of vision. Many retinal neurons remain functional following photoreceptor degeneration; however, the responses of the different classes of cells to electrical stimuli have not been fully investigated. Using whole-cell patch clamp electrophysiology in retinal slices we investigated the response to electrical stimulation of cells of the inner nuclear layer (INL), pre-synaptic to retinal ganglion cells, in wild-type and retinally degenerate (rd/rd) mice. The responses of these cells to electrical stimulation were extremely varied, with both extrinsic and intrinsic evoked responses observed. Further examination of the intrinsically evoked responses revealed direct activation of both voltage-gated Na+ channels and K+ channels. The expression of these channels, which is particularly varied between INL cells, and the stimulus intensity, appears to dictate the polarity of the eventual response. Retinally degenerate animals showed similar responses to electrical stimulation of the retina to those of the wild-type, but the relative representation of each response type differed. The most striking difference between genotypes was the existence of a large amplitude oscillation in the majority of INL cells in rd/rd mice (as previously reported) that impacted on the signal to noise ratio following electrical stimulation. This confounding oscillation may significantly reduce the efficacy of electrical stimulation of the degenerate retina, and a greater understanding of its origin will potentially enable it to be dampened or eliminated.

Differential expression of claudin-23 in retina of oxygen-induced retinopathy mice

Background Researches showed that some claudin subtypes are closely related to the integrity of blood retina barrier and participate in pathological angiogenesis.Ciaudin-23 is a novel discovered claudin subtype.Understanding its expression in the retina and its relationship with retinal vascular lesion is very important for the targeting treatment of retinal vascular diseases.Objective The aim of this study was to investigate the differential expression of claudin-23 in the retina of mice with oxygen-induced retinopathy(OIR).Methods OIR mice models were established by putting postnatal day 7 (P7)SPF C57BL/6J mice in the enclosed container with oxygen concentration of 75％ ±3％ together with mother mice for 5 days.The age-matched normal mice were used as controls.Fluorescein isothiocyanate dextran (FITC-dextran) was retro-orbitally injected to mice to confirm the models by whole-mount retina.Expressions of claudin-23 mRNA and protein in P12,P17 and P25 mice retinas were examined using real-time PCR and Western blot.lmmunofluorescent staining of frozen section was used to identify the expression and location of claudin-23 in the normal and OIR mice.Results Tortuous and engorged retinal vessels were found in P17 mice retina of the OIR group.Nonperfusion zones,abnormal capillary network and lots of pathological neovascular tufts were observed on the whole-mount retinas.The level of claudin-23 mRNA was slowly increased with the aging,reaching 2.3 fold in P25 normal mice more than P7 mice.However,that in OIR mice demonstrated 12.5-fold elevation from P7 to P25.There were no significant change in the mRNA and protein levels of claudin-23 in P7 mice retina between the normal group and OIR group(P＞0.05).But the mRNA level of claudin-23 in P12,P17 and P25 mice retinas was significantly higher in the OIR group than that in normal group(P＜0.05).The protein level of claudin-23 in P25 mice retina was significantly higher in the OIR group than that in normal group(P＜0.05).Claudin23 expressed mainly in inner nuclear cell layer and retinal ganglion cell layer of the normal mice,but claudin-23 expression was obviously increased in OIR mice.No colocalization of claudin-23 and Isolectin B4 was found in the retina in both normal and OIR mice by double immunofluorescent staining.Conclusions Claudin-23 expression upregulates in the retina of OIR mice.Claudin-23 locates in retinal neurons but not vascular endothelial cells. Key words: Claudin-23; Retina; Oxygen-induced retinopathy

Influence of 460-480 nm wavelength light at three different irradiance on retina tissue of SD rats

To investigate the survival of SD rat retinal cells after irradiated with 460-480 nm wavelength blue light at three different irradiance. Experimental study. Seventy-six SD rats six weeks old were divided into control group (4 animals) and experimental groups (18 different subgroups) with 4 animals for each group. Healthy SD rats were exposed to the 460-480 nm blue light for 3 or 12 hours on three different irradiance of 0.6, 1.5 and 10.0 W/m(2) respectively. After recovery in darkness for 4 hours, 24 hours or 3 days, retinal tissue was collected after the eyes were enucleated. Hematoxylin-eosin staining was performed to evaluate the general anatomical changes of the rat retinal layers. TdT-mediated dUTP nick-end labeling was performed to detect apoptotic cells and immunofluorescence staining was conducted to illustrate the expression and location of related proteins in Müller cells. The number of ganglion cells was analyzed by one-way ANOVA. No injury was observed in SD rat retina at the irradiance of 0.6 W/m(2). Retinal cells were damaged with irradiance at 10.0 W/m(2), which mainly affected photoreceptors and retinal pigment epithelial cells, while Müller cells of retinal inner nuclear layer were also involved. Apoptosis began after 4 hours of exposure and peaked at 24 hours. Moderate damage of retina was observed at the irradiance of 1.5 W/m(2) after 12 hours rather than 3 hours exposure. The number of ganglion cells was significantly different among the different groups 3 days after exposure. When the exposure time was 3 hours, the number of retinal ganglion cells under different irradiance at 0.0, 0.6, 1.5 and 10.0 W/m(2) was respectively 41.42 ± 0.17, 40.58 ± 0.50, 40.92 ± 0.32 and 22.83 ± 0.79 (F = 1305.86, P = 0.000). When the exposure time was 12 hours, the number of retinal ganglion cells under different irradiance at 0.0, 0.6, 1.5 and 10.0 W/m(2) was respectively 41.42 ± 0.17, 40.83 ± 0.69, 41.08 ± 0.17 and 22.75 ± 0.83 (F = 1095.78, P = 0.000). The number of retinal ganglion cells showed significant decrease 3 days after exposure at the irradiance 10.0 W/m(2) (exposure time 3 h:t = 52.32, P = 0.000; exposure time 12 h:t = 47.58, P = 0.000). The safe exposure time to SD rat retina layers is 12 hours for the blue light with wavelength 460-480 nm at the irradiance 0.6 W/m(2). And the safe exposure time descends to 3 hours at the irradiance 1.5 W/m(2).

Retinal ganglion cell loss on APP/PS1 transgenic mice with Alzheimer's disease

To investigate the retinal nerve cell changes in number by tests on APP/PS1 double transgenic mice. Experimental study. Comparison was made between 10 female 11-month-old APP/PS1 double transgenic mice with Alzheimer's disease (model group) and 10 non-transgenic female mice of the same background and age (control group). Retinal ganglion cell (RGC) was retrograde labeled by injecting horse radish peroxidase (HRP) into the bilateral optic nerve layer of the superior colliculus of two groups. After 48 hours, all mice were perfused sacrificed. The brains were frozen, mode slices, stained with methyl-alcohol-Congo. The retina peeled from right eye was conducted HRP histochemistry reaction to count RGC by computerized image analyzer. The left eyes were treated with paraffin-embedded, mode slices, stained with hematoxylin and eosin (HE) to count the neuron numbers of each retina layers. The neuron numbers of each retina layer for control group and model group were statistically analyzed using t test for independent samples. The brains showed positive in the model group and the brains showed negative in the control group after stained with methyl-alcohol-Congo. By counting retinal ganglion cells traced with HRP, the number of RGC was statistical different between two groups at 112.78 ± 7.70 within per field for the control group against 78.72 ± 11.35 within per field for the model group. The RGC of the model group was comparatively much less than the control group (t = 27.28, P < 0.01). By counting cells of retina stained with HE, the number of inner and outer nuclear layer cells were not statistical different between two groups of each layer (P > 0.05), the number of RGC in the control group and the model group were respectively 8.17 ± 1.17 within per field, 5.17 ± 0.75 within per field. The cells in retinal ganglion layer in the model group were significantly reduced (t = 52.88, P < 0.01). The results imply that the number of RGC reduced in 11-month-old APP/PS1 double transgenic mice with Alzheimer's disease.