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Calcium is involved in vision processes in the retina and implicated in various pathologies, including glaucoma. Rod cells rely on store-operated calcium entry (SOCE) to safeguard against the prolonged lowering of intracellular calcium ion concentrations. Zebrafish that lacked the endoplasmic reticulum Ca 2+ sensor Stim2 (stim2 knockout [KO]) exhibited impaired vision and lower light perception-related gene expression. We sought to understand mechanisms that are responsible for vision impairment in stim2 KO zebrafish. The single-cell RNA (scRNA) sequencing of neuronal cells from brains of 5 days postfertilization larvae distinguished 27 cell clusters, 10 of which exhibited distinct gene expression patterns, including amacrine and γ-aminobutyric acid (GABA)ergic retinal interneurons and GABAergic optic tectum cells. Five clusters exhibited significant changes in cell proportions between stim2 KO and controls, including GABAergic diencephalon and optic tectum cells. Transmission electron microscopy of stim2 KO zebrafish revealed decreases in width of the inner plexiform layer, ganglion cells, and their dendrites numbers (a hallmark of glaucoma). GABAergic neuron densities in the inner nuclear layer, including amacrine cells, as well as photoreceptors significantly decreased in stim2 KO zebrafish. Our study suggests a novel role for Stim2 in the regulation of neuronal insulin expression and GABAergic-dependent vision causing glaucoma-like retinal pathology., (© 2024. The Author(s).)

Purpose: This study investigates alterations in intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells (DACs) in lid suture myopia (LSM) rats., Methods: LSM was induced in rats by suturing the right eyes for 4 weeks. Double immunofluorescence staining of ipRGCs and DACs in whole-mount retinas was performed to analyze changes in the density and morphology of control, LSM, and fellow eyes. Real-time quantitative PCR and Western blotting were used to detect related genes and protein expression levels., Results: Significant myopia was induced in the lid-sutured eye, but the fellow eye was not different to control. Decreased ipRGC density with paradoxically increased overall melanopsin expression and enlarged dendritic beads was observed in both the LSM and fellow eyes of the LSM rat retinas. In contrast, DAC changes occurred only in the LSM eyes, with reduced DAC density and tyrosine hydroxylase (TH) expression, sparser dendritic processes, and fewer varicosities. Interestingly, contacts between ipRGCs and DACs in the inner plexiform layer (IPL) and the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and vesicular monoamine transporter protein 2 (VMAT2) mRNA were decreased in the LSM eyes., Conclusions: The ipRGCs and DACs in LSM rat retinas undergo multiple alterations in density, morphology, and related molecule expressions. However, the ipRGC changes alone appear not to be required for the development of myopia, given that myopia is only induced in the lid-sutured eye, and they are unlikely alone to drive the DAC changes. Reduced contacts between ipRGCs and DACs in the LSM eyes may be the structural foundation for the impaired signaling between them. PACAP and VMAT2, strongly associated with ipRGCs and DACs, may play important roles in LSM through complex mechanisms.

One of the causes of nervous system degeneration is an excess of glutamate released upon several diseases. Glutamate analogs, like N-methyl-DL-aspartate (NMDA) and kainic acid (KA), have been shown to induce experimental retinal neurotoxicity. Previous results have shown that NMDA/KA neurotoxicity induces significant changes in the full field electroretinogram response, a thinning on the inner retinal layers, and retinal ganglion cell death. However, not all types of retinal neurons experience the same degree of injury in response to the excitotoxic stimulus. The goal of the present work is to address the effect of intraocular injection of different doses of NMDA/KA on the structure and function of several types of retinal cells and their functionality. To globally analyze the effect of glutamate receptor activation in the retina after the intraocular injection of excitotoxic agents, a combination of histological, electrophysiological, and functional tools has been employed to assess the changes in the retinal structure and function. Retinal excitotoxicity caused by the intraocular injection of a mixture of NMDA/KA causes a harmful effect characterized by a great loss of bipolar, amacrine, and retinal ganglion cells, as well as the degeneration of the inner retina. This process leads to a loss of retinal cell functionality characterized by an impairment of light sensitivity and visual acuity, with a strong effect on the retinal OFF pathway. The structural and functional injury suffered by the retina suggests the importance of the glutamate receptors expressed by different types of retinal cells. The effect of glutamate agonists on the OFF pathway represents one of the main findings of the study, as the evaluation of the retinal lesions caused by excitotoxicity could be specifically explored using tests that evaluate the OFF pathway.

Cytoplasmic dynein (dynein 1), a major retrograde motor of eukaryotic cells, is a 1.4 MDa protein complex consisting of a pair of heavy chains (DYNC1H1) and a set of heterodimeric noncatalytic accessory components termed intermediate, light intermediate and light chains. DYNC1H1 (4644 amino acids) is the dynein backbone encoded by a gene consisting of 77 exons. We generated a floxed Dync1h1 allele that excises exons 24 and 25 and truncates DYNC1H1 during Six3Cre-induced homologous recombination. Truncation results in loss of the motor and microtubule-binding domain. Dync1h1F/F;Six3Cre photoreceptors degenerated rapidly within two postnatal weeks. In the postnatal day 6 (P6) Dync1h1F/F;Six3Cre central retina, outer and inner nuclear layers were severely disorganized and lacked a recognizable outer plexiform layer (OPL). Although the gene was effectively silenced by P6, DYNC1H1 remnants persisted and aggregated together with rhodopsin, PDE6 and centrin-2-positive centrosomes in the outer nuclear layer. As photoreceptor degeneration is delayed in the Dync1h1F/F;Six3Cre retina periphery, retinal lamination and outer segment elongation are in part preserved. DYNC1H1 strongly persisted in the inner plexiform layer (IPL) beyond P16 suggesting lack of clearance of the DYNC1H1 polypeptide. This persistence of DYNC1H1 allows horizontal, rod bipolar, amacrine and ganglion cells to survive past P12. The results show that cytoplasmic dynein is essential for retina lamination, nuclear positioning, vesicular trafficking of photoreceptor membrane proteins and inner/outer segment elaboration., Competing Interests: The authors have declared that no competing interests exist.

Glaucoma is a chronic neurodegenerative disease of the optic nerve and a leading cause of irreversible blindness, worldwide. While the experimental research using animal models provides growing information about cellular and molecular processes, parallel analysis of the clinical presentation of glaucoma accelerates the translational progress towards improved understanding, treatment, and clinical testing of glaucoma. Optic nerve axon injury triggers early alterations of retinal ganglion cell (RGC) synapses with function deficits prior to manifest RGC loss in animal models of glaucoma. For testing the clinical relevance of experimental observations, this study analyzed the functional correlation of localized alterations in the inner plexiform layer (IPL), where RGCs establish synaptic connections with retinal bipolar and amacrine cells. Participants of the study included a retrospective cohort of 36 eyes with glaucoma and a control group of 18 non-glaucomatous subjects followed for two-years. The IPL was analyzed on consecutively collected macular SD-OCT scans, and functional correlations with corresponding 10-2 visual field scores were tested using generalized estimating equations (GEE) models. The GEE-estimated rate of decrease in IPL thickness (R = 0.36, P<0.001) and IPL density (R = 0.36, P<0.001), as opposed to unchanged or increased IPL thickness or density, was significantly associated with visual field worsening at corresponding analysis locations. Based on multivariate logistic regression analysis, this association was independent from the patients' age, the baseline visual field scores, or the baseline thickness or alterations of retinal nerve fiber or RGC layers (P>0.05). These findings support early localized IPL alterations in correlation with progressing visual field defects in glaucomatous eyes. Considering the experimental data, glaucoma-related increase in IPL thickness/density might reflect dendritic remodeling, mitochondrial redistribution, and glial responses for synapse maintenance, but decreased IPL thickness/density might correspond to dendrite atrophy. The bridging of experimental data with clinical findings encourages further research along the translational path., Competing Interests: The authors declare that they have no competing interests.

Objective: Considering the demonstrated implication of the retina in Parkinson disease (PD) pathology and the importance of dopaminergic cells in this tissue, we aimed to analyze the state of the dopaminergic amacrine cells and some of their main postsynaptic neurons in the retina of PD., Methods: Using immunohistochemistry and confocal microscopy, we evaluated morphology, number, and synaptic connections of dopaminergic cells and their postsynaptic cells, AII amacrine and melanopsin-containing retinal ganglion cells, in control and PD eyes from human donors., Results: In PD, dopaminergic amacrine cell number was reduced between 58% and 26% in different retinal regions, involving a decline in the number of synaptic contacts with AII amacrine cells (by 60%) and melanopsin cells (by 35%). Despite losing their main synaptic input, AII cells were not reduced in number, but they showed cellular alterations compromising their adequate function: (1) a loss of mitochondria inside their lobular appendages, which may indicate an energetic failure; and (2) a loss of connexin 36, suggesting alterations in the AII coupling and in visual signal transmission from the rod pathway., Interpretation: The dopaminergic system impairment and the affection of the rod pathway through the AII cells may explain and be partially responsible for the reduced contrast sensitivity or electroretinographic response described in PD. Also, dopamine reduction and the loss of synaptic contacts with melanopsin cells may contribute to the melanopsin retinal ganglion cell loss previously described and to the disturbances in circadian rhythm and sleep reported in PD patients. These data support the idea that the retina reproduces brain neurodegeneration and is highly involved in PD pathology. ANN NEUROL 2020;88:893-906., (© 2020 American Neurological Association.)

Connectomics has demonstrated that synaptic networks and their topologies are precise and directly correlate with physiology and behavior. The next extension of connectomics is pathoconnectomics: to map neural network synaptology and circuit topologies corrupted by neurological disease in order to identify robust targets for therapeutics. In this report, we characterize a pathoconnectome of early retinal degeneration. This pathoconnectome was generated using serial section transmission electron microscopy to achieve an ultrastructural connectome with 2.18nm/px resolution for accurate identification of all chemical and gap junctional synapses. We observe aberrant connectivity in the rod-network pathway and novel synaptic connections deriving from neurite sprouting. These observations reveal principles of neuron responses to the loss of network components and can be extended to other neurodegenerative diseases., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Retinal bipolar cells (BCs) connect with photoreceptors and relay visual information to retinal ganglion cells (RGCs). Retina-specific deletion of Lhx4 in mice results in a visual defect resembling human congenital stationary night blindness. This visual dysfunction results from the absence of rod bipolar cells (RBCs) and the loss of selective rod-connecting cone bipolar cell (CBC) subtypes and AII amacrine cells (ACs). Inactivation of Lhx4 causes the apoptosis of BCs and cell fate switch from some BCs to ACs, whereas Lhx4 overexpression promotes BC genesis. Moreover, Lhx4 positively regulates Lhx3 expression to drive the fate choice of type 2 BCs over the GABAergic ACs. Lhx4 inactivation ablates Bhlhe23 expression, whereas overexpression of Bhlhe23 partially rescues RBC development in the absence of Lhx4. Thus, by acting upstream of Bhlhe23, Prdm8, Fezf2, Lhx3, and other BC genes, Lhx4, together with Isl1, could play essential roles in regulating the subtype-specific development of RBCs and CBCs., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genome duplication leads to an emergence of gene paralogs that are essentially free to undergo the process of neofunctionalization, subfunctionalization or degeneration (gene loss). Onecut1 (Oc1) and Onecut2 (Oc2) transcription factors, encoded by paralogous genes in mammals, are expressed in precursors of horizontal cells (HCs), retinal ganglion cells and cone photoreceptors. Previous studies have shown that ablation of either Oc1 or Oc2 gene in the mouse retina results in a decreased number of HCs, while simultaneous deletion of Oc1 and Oc2 leads to a complete loss of HCs. Here we study the genetic redundancy between Oc1 and Oc2 paralogs and focus on how the dose of Onecut transcription factors influences abundance of individual retinal cell types and overall retina physiology. Our data show that reducing the number of functional Oc alleles in the developing retina leads to a gradual decrease in the number of HCs, progressive thinning of the outer plexiform layer and diminished electrophysiology responses. Taken together, these observations indicate that in the context of HC population, the alleles of Oc1/Oc2 paralogous genes are mutually interchangeable, function additively to support proper retinal function and their molecular evolution does not follow one of the typical routes after gene duplication., Competing Interests: The authors have declared that no competing interests exist.

Purpose: Disruption in blood supply to active retinal circuits is the earliest hallmark of diabetic retinopathy (DR) and has been primarily attributed to vascular deficiency. However, accumulating evidence supports an early role for a disrupted neuronal function in blood flow impairment. Here, we tested the hypothesis that selectively stimulating cholinergic neurons could restore neurovascular signaling to preserve the capillary circulation in DR., Methods: We used wild type (wt) and choline acetyltransferase promoter (ChAT)-channelrhodopsin-2 (ChR2) mice expressing ChR2 exclusively in cholinergic cells. Mice were made diabetic by streptozotocin (STZ) injections. Two to 3 months after the last STZ injection, the rate of capillary blood flow was measured in vivo within each retinal vascular layer using high speed two-photon imaging. Measurements were done at baseline and following ChR2-driven activation of retinal cholinergic interneurons, the sole source of the vasodilating neurotransmitter acetylcholine. After recordings, retinas were collected and assessed for physiological and structural features., Results: In retinal explants from ChAT-ChR2 mice, we found that channelrhodopsin2 was selectively expressed in all cholinergic amacrine cells. Its direct activation by blue light led to dilation of adjacent retinal capillaries. In living diabetic ChAT-ChR2 animals, basal capillary blood flow was significantly higher than in diabetic mice without channelrhodopsin. However, optogenetic stimulation with blue light did not result in flickering light-induced functional hyperemia, suggesting a necessity for a concerted neurovascular interaction., Conclusions: These findings provide direct support to the utility and efficacy of an optogenetic approach for targeting selective retinal circuits to treat DR and its complications.

The presence of α-synuclein aggregates in the retina of Parkinson's disease patients has been associated with vision impairment. In this study we sought to determine the effects of α-synuclein overexpression on the survival and function of dopaminergic amacrine cells (DACs) in the retina. Adult mice were intravitreally injected with an adeno-associated viral (AAV) vector to overexpress human wild-type α-synuclein in the inner retina. Before and after systemic injections of levodopa (L-DOPA), retinal responses and visual acuity-driven behavior were measured by electroretinography (ERG) and a water maze task, respectively. Amacrine cells and ganglion cells were counted at different time points after the injection. α-synuclein overexpression led to an early loss of DACs associated with a decrease of light-adapted ERG responses and visual acuity that could be rescued by systemic injections of L-DOPA. The data show that α-synuclein overexpression affects dopamine neurons in the retina. The approach provides a novel accessible method to model the underlying mechanisms implicated in the pathogenesis of synucleinopathies and for testing novel treatments.

Combined administration of N-Methyl-D-Aspartate (NMDA) and kainic acid (KA) on the inner retina was studied as a model of excitotoxicity. The right eye of C57BL6J mice was injected with 1 µL of PBS containing NMDA 30 mM and KA 10 mM. Only PBS was injected in the left eye. One week after intraocular injection, electroretinogram recordings and immunohistochemistry were performed on both eyes. Retinal ganglion cell (RGC) projections were studied by fluorescent-cholerotoxin anterograde labeling. A clear decrease of the retinal "b" wave amplitude, both in scotopic and photopic conditions, was observed in the eyes injected with NMDA/KA. No significant effect on the "a" wave amplitude was observed, indicating the preservation of photoreceptors. Immunocytochemical labeling showed no effects on the outer nuclear layer, but a significant thinning on the inner retinal layers, thus indicating that NMDA and KA induce a deleterious effect on bipolar, amacrine and ganglion cells. Anterograde tracing of the visual pathway after NMDA and KA injection showed the absence of RGC projections to the contralateral superior colliculus and lateral geniculate nucleus. We conclude that glutamate receptor agonists, NMDA and KA, induce a deleterious effect of the inner retina when injected together into the vitreous chamber., Competing Interests: The authors declare no financial conflicts of interest.

N-Methyl-D-aspartate (NMDA)-induced neuronal cell death is involved in a large spectrum of diseases affecting the brain and the retina such as Alzheimer's disease and diabetic retinopathy. Associated neurological impairments may result from the inhibition of neuronal plasticity by Nogo-A. The objective of the current study was to determine the contribution of Nogo-A to NMDA excitotoxicity in the mouse retina. We observed that Nogo-A is upregulated in the mouse vitreous during NMDA-induced inflammation. Intraocular injection of a function-blocking antibody specific to Nogo-A (11C7) was carried out 2 days after NMDA-induced injury. This treatment significantly enhanced visual function recovery in injured animals. Strikingly, the expression of potent pro-inflammatory molecules was downregulated by 11C7, among which TNFα was the most durably decreased cytokine in microglia/macrophages. Additional analyses suggest that TNFα downregulation may stem from cofilin inactivation in microglia/macrophages. 11C7 also limited gliosis presumably via P.Stat3 downregulation. Diabetic retinopathy was associated with increased levels of Nogo-A in the eyes of donors. In summary, our results reveal that Nogo-A-targeting antibody can stimulate visual recovery after retinal injury and that Nogo-A is a potent modulator of excitotoxicity-induced neuroinflammation. These data may be used to design treatments against inflammatory eye diseases.

Simulation of hypoxic processes in vitro can be achieved through cobalt chloride (CoCl 2 ), which induces strong neurodegeneration. Hypoxia plays an important role in the progression of several retinal diseases. Thus, we investigated whether hypoxia can be reduced by hypothermia. Porcine retinal explants were cultivated for four and eight days and hypoxia was mimicked by adding 300 µM CoCl 2 from day one to day three. Hypothermia treatment (30 °C) was applied simultaneously. Retinal ganglion, bipolar and amacrine cells, as well as microglia were evaluated via immunohistological and western blot analysis. Furthermore, quantitative real-time PCR was performed to analyze cellular stress and apoptosis. In addition, the expression of specific marker for the previously described cell types were investigated. A reduction of ROS and stress markers HSP70, iNOS, HIF-1α was achieved via hypothermia. In accordance, an inhibition of apoptotic proteins (caspase 3, caspase 8) and the cell cycle arrest gene p21 was found in hypothermia treated retinae. Furthermore, neurons of the inner retina were protected by hypothermia. In this study, we demonstrate that hypothermia lowers hypoxic processes and cellular stress. Additionally, hypothermia inhibits apoptosis and protects neurons. Hence, this seems to be a promising treatment for retinal neurodegeneration.

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., (Copyright © 2018 Sun Yat-sen University Zhongshan Ophthalmic Center. Published by Elsevier Ltd.. All rights reserved.)

We determined whether the structural and functional integrity of amacrine cells (ACs), the largest cohort of neurons in the mammalian retina, are affected in glaucoma. Intraocular injection of microbeads was made in mouse eyes to elevate intraocular pressure as a model of experimental glaucoma. Specific immunocytochemical markers were used to identify AC and displaced (d)ACs subpopulations in both the inner nuclear and ganglion cell layers, respectively, and to distinguish them from retinal ganglion cells (RGCs). Calretinin- and γ-aminobutyric acid (GABA)-immunoreactive (IR) cells were highly vulnerable to glaucomatous damage, whereas choline acetyltransferase (ChAT)-positive and glycinergic AC subtypes were unaffected. The AC loss began 4 weeks after initial microbead injection, corresponding to the time course of RGC loss. Recordings of electroretinogram (ERG) oscillatory potentials and scotopic threshold responses, which reflect AC and RGC activity, were significantly attenuated in glaucomatous eyes following a time course that matched that of the AC and RGC loss. Moreover, we found that it was the ACs coupled to RGCs via gap junctions that were lost in glaucoma, whereas uncoupled ACs were largely unaffected. Our results suggest that AC loss in glaucoma occurs secondary to RGC death through the gap junction-mediated bystander effect. J. Comp. Neurol. 527:159-173, 2019. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

BNN27, a C17-spiroepoxy derivative of DHEA, was shown to have antiapoptotic properties via mechanisms involving the nerve growth factor receptors (tropomyosin-related kinase A [TrkA]/neurotrophin receptor p75 [p75 NTR ]). In this study, we examined the effects of BNN27 on neural/glial cell function, apoptosis, and inflammation in the experimental rat streptozotocin (STZ) model of diabetic retinopathy (DR). The ability of BNN27 to activate the TrkA receptor and regulate p75 NTR expression was investigated. BNN27 (2,10, and 50 mg/kg i.p. for 7 days) administration 4 weeks post-STZ injection (paradigm A) reversed the diabetes-induced glial activation and loss of function of amacrine cells (brain nitric oxide synthetase/tyrosine hydroxylase expression) and ganglion cell axons via a TrkA receptor (TrkAR)-dependent mechanism. BNN27 activated/phosphorylated the TrkA Y490 residue in the absence but not the presence of TrkAR inhibitor and abolished the diabetes-induced increase in p75 NTR expression. However, it had no effect on retinal cell death (TUNEL + cells). A similar result was observed when BNN27 (10 mg/kg i.p.) was administered at the onset of diabetes, every other day for 4 weeks (paradigm B). However, BNN27 decreased the activation of caspase-3 in both paradigms. Finally, BNN27 reduced the proinflammatory (TNFα and IL-1β) and increased the anti-inflammatory (IL-10 and IL-4) cytokine levels. These findings suggest that BNN27 has the pharmacological profile of a therapeutic for DR, since it targets both the neurodegenerative and inflammatory components of the disease., (© 2017 by the American Diabetes Association.)

Previous studies have revealed a loss of retinal ganglion cells (RGCs) and optic nerve fibers after immunization with the S100B protein. Addition of heat shock protein 27 (HSP27) also leads to a decrease of RGCs. Our present aim has been to analyze various retinal cell types after immunization with S100B or S100B + HSP27 (S100 + HSP). After 28 days, retinas were processed for immunohistology and Western blot. RGCs, immunostained for NeuN, were significantly decreased in the S100 and the S100 + HSP groups. Significantly fewer ChAT + cells were noted in both groups, whereas parvalbumin + cells were only affected in the S100 + HSP group. Western blot results also revealed fewer ChAT signals in both immunized groups. No changes were noted with regard to PKCα + rod bipolar cells, whereas a significant loss of recoverin + cone bipolar cells was observed in both groups via immunohistology and Western blot. The presynaptic marker Bassoon and the postsynaptic marker PSD95 were significantly reduced in the S100 + HSP group. Opsin + and rhodopsin + photoreceptors revealed no changes in either group. Thus, the inner retinal layers are affected by immunization. However, the combination of S100 and HSP27 has a stronger additive effect on the retinal synapses and AII amacrine cells.

Major histocompatibility complex (MHC) class I molecules and their receptors play fundamental roles in neuronal death during diseases. T-cell receptors (TCR) function as MHCI receptor on T-cells and both MHCI and a key component of TCR, CD3ζ, are expressed by mouse retinal ganglion cells (RGCs) and displaced amacrine cells. Mutation of these molecules compromises the development of RGCs. We investigated whether CD3ζ regulates the development and degeneration of amacrine cells after RGC death. Surprisingly, mutation of CD3ζ not only impairs the proper development of amacrine cells expressing CD3ζ but also those not expressing CD3ζ. In contrast to effects of MHCI and its receptor, PirB, on other neurons, mutation of CD3ζ has no effect on RGC death and starburst amacrine cells degeneration after optic nerve crush. Thus, unlike MHCI and PirB, CD3ζ regulates the development of RGCs and amacrine cells but not their degeneration after optic nerve crush.

Inherited mitochondrial complex 1 deficiency causes Leber's hereditary Optic Neuropathy (LHON) and retinal ganglion cell (RGC) degeneration, and optic neuropathies are common in many inherited mitochondrial diseases. How mitochondrial defects pathomechanistically trigger optic neuropathy remains unclear. We observe that complex 1-deficient Ndufs4-/- mice present with acute vision loss around p30, and this vision loss is coincident with an 'inflammatory wave'. In order to understand what causes the inflammatory wave we explored retinal pathology that occurs from p20-p30. The results indicated that in the period p20-p30 in Ndufs4-/- retinas, there is: significant reduction in bipolar cells, RGC dendritic atrophy, reduced PSD95, increased oxidative stress as manifested by increased 4HNE, HO1 and Cuzn-SOD, increased mitochondrial biogenesis and increased apoptosis. These precede the major induction of 'inflammatory wave' at p30 shown previously, but occur earlier than frank RGC loss at p42. In general, complex 1 deficiency in retina triggers oxidative stress and mitochondrial respiratory dysfunction that causes death of the most sensitive cells, including bipolar cells and their synaptic contacts and amacrine cells in the early period, 20-24days. The early death of these cells is the likely precursor to the sharp rise in inflammatory molecules that occurs at day 30 and coincides with vision loss, and greatly precedes the death of RGCs that occurs at p42. These data suggest that metabolic antioxidant support of the most sensitive cells in the retina, or anti-inflammatory suppression of the consequences of their death, are both rational strategies for mitochondrial blinding disease., (Copyright © 2016. Published by Elsevier B.V.)

Diabetic retinopathy is a common complication of type 2 diabetes and the leading cause of blindness in adults of working age. The aim of this work was to study the repercussions of high fat diet (HFD) induced diabetes on the retina of Meriones shawi (M.sh). Two groups of six M.sh each was studied. Group I was a normal control, fed with standard laboratory granules. In Group II, rodents received a HFD of enriched laboratory granules, for a period of 3 months. Body weight and plasma glucose were determined in the two groups. Retinal sections of the two groups were stained with the Hematoxylin-Eosin. Photoreceptors were identified by immunolabeling for rhodopsin (rods) and PNA (cones). Gliosis and microglial activation were identified by immunolabeling for GFAP and Iba-1. Labeling of calretinin and parvalbumin were also carried out to study the AII amacrine cells. Retinal layers thicknesses, gliosis, and specific neural cell populations were quantified by microscopy. The body weight (+77%) and plasma glucose (+108%) were significantly greater in the HFD rodents. Three months of HFD induced a significant loss of 38.77% of cone photoreceptors, as well as gliosis and an increase of 70.67% of microglial cells. Calcium homeostatic enzymes were depleted. This work shows that HFD in Meriones shawi induces a type II diabetes-like condition that causes loss of retinal neurons and photoreceptors, as well as gliosis. Meriones shawi could be a useful experimental animal model for this physiopathology particularly in the study of retinal neuro-glial alterations in Type II diabetes., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

In retinal degenerative disease (RD), the diminished light signal from dying photoreceptors has been considered the sole cause of visual impairment. Recent studies show a 10-fold increase in spontaneous activity in the RD network, challenging this paradigm. This aberrant activity forms a new barrier for the light signal, and not only exacerbates the loss of vision, but also may stand in the way of visual restoration. This activity originates in AII amacrine cells and relies on excessive activation of gap junctions. However, it remains unclear whether aberrant activity affects central visual processing and what mechanisms lead to this excessive activation of gap junctions. By combining genetic manipulation with electrophysiological recordings of light-induced activity in both living mice and isolated wholemount retina, we demonstrate that aberrant activity extends along retinotectal projections to alter activity in higher brain centers. Next, to selectively eliminate Cx36-containing gap junctions, which are the primary type expressed by AII amacrine cells, we crossed rd10 mice, a slow-degenerating model of RD, with Cx36 knockout mice. We found that retinal aberrant activity was reduced in the rd10/Cx36KO mice compared to rd10 controls, a direct evidence for involvement of Cx36-containing gap junctions in generating aberrant activity in RD. These data provide an essential support for future experiments to determine if selectively targeting these gap junctions could be a valid strategy for reducing aberrant activity and restoring light responses in RD., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: Retinal dopamine deficiency is a potential cause of myopia and visual deficits in retinopathy of prematurity (ROP). We investigated the cellular mechanisms responsible for lowered levels of retinal dopamine in an oxygen-induced retinopathy (OIR) mouse model of ROP., Methods: Retinopathy was induced by exposing mice to 75% oxygen from postnatal day 7 (P7) to P12. Oxygen-induced retinopathy and age-matched control mice were euthanized at P12, P17, P25, or P42 to P50. Immunohistochemistry, electrophysiology, and biochemical approaches were used to determine the effect of OIR on the structure and function of dopaminergic amacrine cells (DACs)., Results: The total number of DACs was unchanged in OIR retinas at P12 despite significant capillary dropout in the central retina. However, a significant loss of DACs was observed in P17 OIR retinas (in which neovascularization was maximal), with the cell loss being more profound in the central (avascular) than in the peripheral (neovascular) regions. Cell loss was persistent in both regions at P25, at which time retinal neovascularization had regressed. At P42, the percentage of DACs lost (54%) was comparable to the percent decrease in total dopamine content (53%). Additionally, it was found that DACs recorded in OIR retinas at P42 to P50 had a complete dendritic field and exhibited relatively normal spontaneous and light-induced electrical activity., Conclusions: The results suggest that remaining DACs are structurally and functionally intact and that loss of DACs is primarily responsible for the decreased levels of retinal dopamine observed after OIR.

Dopaminergic amacrine cells (DACs) release dopamine in response to light-driven synaptic inputs, and are critical to retinal light adaptation. Retinal degeneration (RD) compromises the light responsiveness of the retina and, subsequently, dopamine metabolism is impaired. As RD progresses, retinal neurons exhibit aberrant activity, driven by AII amacrine cells, a primary target of the retinal dopaminergic network. Surprisingly, DACs are an exception to this physiological change; DACs exhibit rhythmic activity in healthy retina, but do not burst in RD. The underlying mechanism of this divergent behavior is not known. It is also unclear whether RD leads to structural changes in DACs, impairing functional regulation of AII amacrine cells. Here we examine the anatomical details of DACs in three mouse models of human RD to determine how changes to the dopaminergic network may underlie physiological changes in RD. By using rd10, rd1, and rd1/C57 mice we were able to dissect the impacts of genetic background and the degenerative process on DAC structure in RD retina. We found that DACs density, soma size, and primary dendrite length are all significantly reduced. Using a novel adeno-associated virus-mediated technique to label AII amacrine cells in mouse retina, we observed diminished dopaminergic contacts to AII amacrine cells in RD mice. This was accompanied by changes to the components responsible for dopamine synthesis and release. Together, these data suggest that structural alterations of the retinal dopaminergic network underlie physiological changes during RD., (© 2015 Wiley Periodicals, Inc.)

Amacrine cells were targeted for whole cell recording using two-photon fluorescence microscopy in a transgenic mouse line in which the promoter for dopamine receptor 2 drove expression of green fluorescent protein in a narrow field tristratified amacrine cell (TNAC) that had not been studied previously. Light evoked a multiphasic response that was the sum of hyperpolarizing and depolarization synaptic inputs consistent with distinct dendritic ramifications in the off and on sublamina of the inner plexiform layer. The amplitude and waveform of the response, which consisted of an initial brief hyperpolarization at light onset followed by recovery to a plateau potential close to dark resting potential and a hyperpolarizing response at the light offset varied little over an intensity range from 0.4 to ~10^6 Rh*/rod/s. This suggests that the cell functions as a differentiator that generates an output signal (a transient reduction in inhibitory input to downstream retina neurons) that is proportional to the derivative of light input independent of its intensity. The underlying circuitry appears to consist of rod and cone driven on and off bipolar cells that provide direct excitatory input to the cell as well as to GABAergic amacrine cells that are synaptically coupled to TNAC. Canonical reagents that blocked excitatory (glutamatergic) and inhibitory (GABA and glycine) synaptic transmission had effects on responses to scotopic stimuli consistent with the rod driven component of the proposed circuit. However, responses evoked by photopic stimuli were paradoxical and could not be interpreted on the basis of conventional thinking about the neuropharmacology of synaptic interactions in the retina.

Cannabinoids have been suggested to protect retinal ganglion cells in different models of toxicity, but their effects on other retinal neurons are poorly known. We investigated the neuroprotective actions of the endocannabinoid N-arachidonoyl ethanolamine (Anandamide/AEA) and the synthetic cannabinoids R1-Methanandamide (MethAEA) and HU-210, in an in vivo retinal model of AMPA excitotoxicity, and the mechanisms involved in the neuroprotection. Sprague-Dawley rats were intravitreally injected with PBS or AMPA in the absence or presence of the cannabinoid agonists. Brain nitric oxide synthase (bNOS) and choline acetyltransferase (ChAT) immunoreactivity (IR), as well as TUNEL staining, assessed the AMPA-induced retinal amacrine cell loss and the dose-dependent neuroprotection afforded by cannabinoids. The CB1 receptor selective antagonist AM251 and the PI3K/Akt inhibitor wortmannin reversed the cannabinoid-induced neuroprotection, suggesting the involvement of CB1 receptors and the PI3K/Akt pathway in cannabinoids' actions. Experiments with the CB2 agonist JWH015 and [(3)H]CP55940 radioligand binding suggested that the CB2 receptor is not involved in the neuroprotection. AEA and HU-210 induced phosphorylation of Akt but only AEA induced phosphorylation of ERK1/2 kinases, as revealed by western blot analysis. To investigate the role of caspase-3 in the AMPA-induced cell death, the caspase-3 inhibitor Z-DEVD-FMK was co-injected with AMPA. Z-DEVD-FMK had no effect on AMPA excitotoxicity. Moreover, no difference was observed in the phosphorylation of SAPK/JNK kinases between PBS- and AMPA-treated retinas. These results suggest that endogenous and synthetic cannabinoids protect retinal amacrine neurons from AMPA excitotoxicity in vivo via a mechanism involving the CB1 receptors, and the PI3K/Akt and/or MEK/ERK1/2 signaling pathways., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Retinal ischemia is a common risk factor for visual impairment and blindness. Two common changes after retinal ischemia are retinal ganglion cell (RGC) loss and Müller glial cell (MGC)-mediated endogenous repair. Matrix metalloproteinase 9 (MMP-9) has been shown to be responsible to RGC death. However, the effects of MMP-9 on the loss of other neurons and the reactivity of MGCs after retinal injury remain unclear. Ouabain, a Na/K-ATPase inhibitor, was injected into the vitreous body of rat eyes to induce cell death in the inner nuclear layer (INL). MMP-9 expression and activation in the retinas were examined by gelatin zymography and immunohistochemistry. The role of MMP-9 inhibitor (MMP-9i) in ouabain-treated retinas was assessed. After ouabain injection, there was an upregulation of MMP-9 activity in the inner retinas, and the activation of MMP-9 reached a maximum at 2 day. Unexpectedly, MMP-9i enhanced the thinning of the INL, the loss of Calbindin D-28k-positive cells and Syntaxin-positive amacrine cells (ACs) in the INL and decreased levels of Calbindin D-28k protein, while leaving the outer nuclear layer (ONL) unchanged. In addition, MMP-9i led to a minor increase in the number of BrdU positive cells that did not express GS in the INL. Collectively, these results revealed that the inhibition of MMP-9 activity facilitated AC loss and promoted the generation of MGC-derived cells in ouabain-treated retinas, which indicates that treating retinal diseases with drugs that inhibit MMP-9 activity should be considered with caution., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The intrinsic mechanisms that promote the degeneration of retinal ganglion cells (RGCs) following the activation of N-Methyl-D-aspartic acid-type glutamate receptors (NMDARs) are unclear. In this study, we have investigated the role of downstream regulatory element antagonist modulator (DREAM) in NMDA-mediated degeneration of the retina. NMDA, phosphate-buffered saline (PBS), and MK801 were injected into the vitreous humor of C57BL/6 mice. At 12, 24, and 48 hours after injection, expression of DREAM in the retina was determined by immunohistochemistry, western blot analysis, and electrophoretic mobility-shift assay (EMSA). Apoptotic death of cells in the retina was determined by terminal deoxynucleotidyl transferace dUTP nick end labeling (TUNEL) assays. Degeneration of RGCs in cross sections and in whole mount retinas was determined by using antibodies against Tuj1 and Brn3a respectively. Degeneration of amacrine cells and bipolar cells was determined by using antibodies against calretinin and protein kinase C (PKC)-alpha respectively. DREAM was expressed constitutively in RGCs, amacrine cells, bipolar cells, as well as in the inner plexiform layer (IPL). NMDA promoted a progressive decrease in DREAM levels in all three cell types over time, and at 48 h after NMDA-treatment very low DREAM levels were evident in the IPL only. DREAM expression in retinal nuclear proteins was decreased progressively after NMDA-treatment, and correlated with its decreased binding to the c-fos-DRE oligonucleotides. A decrease in DREAM expression correlated significantly with apoptotic death of RGCs, amacrine cells and bipolar cells. Treatment of eyes with NMDA antagonist MK801, restored DREAM expression to almost normal levels in the retina, and significantly decreased NMDA-mediated apoptotic death of RGCs, amacrine cells, and bipolar cells. Results presented in this study show for the first time that down-regulation of DREAM promotes the degeneration of RGCs, amacrine cells, and bipolar cells.

Glaucoma is the second leading cause of blindness in the United States and the world, characterized by progressive degeneration of the optic nerve and retinal ganglion cells (RGCs). Glaucoma patients exhibit an early diffuse loss of retinal sensitivity followed by focal loss of RGCs in sectored patterns. Recent evidence has suggested that this early sensitivity loss may be associated with dysfunctions in the inner retina, but detailed cellular and synaptic mechanisms underlying such sensitivity changes are largely unknown. In this study, we use whole-cell voltage-clamp techniques to analyze light responses of individual bipolar cells (BCs), AII amacrine cells (AIIACs), and ON and sustained OFF alpha-ganglion cells (ONαGCs and sOFFαGCs) in dark-adapted mouse retinas with elevated intraocular pressure (IOP). We present evidence showing that elevated IOP suppresses the rod ON BC inputs to AIIACs, resulting in less sensitive AIIACs, which alter AIIAC inputs to ONαGCs via the AIIAC→cone ON BC→ONαGC pathway, resulting in lower ONαGC sensitivity. The altered AIIAC response also reduces sOFFαGC sensitivity via the AIIAC→sOFFαGC chemical synapses. These sensitivity decreases in αGCs and AIIACs were found in mice with elevated IOP for 3-7 wk, a stage when little RGC or optic nerve degeneration was observed. Our finding that elevated IOP alters neuronal function in the inner retina before irreversible structural damage occurs provides useful information for developing new diagnostic tools and treatments for glaucoma in human patients.

Purpose: To evaluate the effect of optic nerve head drusen (ONHD) on the retinal nerve fiber layer (RNFL) and macular ganglion cell-inner plexiform layer (GCIPL) using Cirrus optical coherence tomography (OCT)., Methods: Fifty-seven eyes of thirty patients with ONHD and thirty-eight eyes of twenty age-matched and sex-matched control subjects underwent circumpapillary and macular scanning using Cirrus OCT. The percentages of eyes with abnormal GCIPL and RNFL values according to the Cirrus normative data were analysed and compared., Results: Overall, eyes with ONHD showed abnormally reduced values for average and minimum GCIPL thicknesses in 35 % and 45 % of cases compared to 2 % for both values in control eyes (P < 0.001). Average RNFL thickness comparison between eyes with ONHD and normal eyes revealed abnormal thinning in 33 % vs. 0 %, respectively (p = 0.002). The percentage of abnormal thinning increased with higher grades of ONHD for all the parameters evaluated, so that in grade III drusen, values were abnormally reduced in 80 % of eyes in all three analyses. Regarding buried ONHD, 30 % and 4 % of eyes had an abnormally reduced minimum GCIPL and average RNFL thickness, respectively. Furthermore, 26 % of these eyes had abnormal GCIPL exams with a normal or increased RNFL thickness., Conclusions: Both RNFL and GCIPL analysis reveal significant thinning in eyes with ONHD directly correlated with drusen severity. In buried ONHD, the abnormality rate was significantly higher with GCIPL compared to RNFL evaluation, suggesting that GCIPL analysis might be an early structural indicator of neuronal loss in the setting of thickened RNFL.

Age-related decreases in neural function result in part from alterations in synapses. To identify molecular defects that lead to such changes, we focused on the outer retina, in which synapses are markedly altered in old rodents and humans. We found that the serine/threonine kinase LKB1 and one of its substrates, AMPK, regulate this process. In old mice, synaptic remodeling was accompanied by specific decreases in the levels of total LKB1 and active (phosphorylated) AMPK. In the absence of either kinase, young adult mice developed retinal defects similar to those that occurred in old wild-type animals. LKB1 and AMPK function in rod photoreceptors where their loss leads to aberrant axonal retraction, the extension of postsynaptic dendrites and the formation of ectopic synapses. Conversely, increasing AMPK activity genetically or pharmacologically attenuates and may reverse age-related synaptic alterations. Together, these results identify molecular determinants of age-related synaptic remodeling and suggest strategies for attenuating these changes.

The N-methyl-d-aspartate (NMDA) receptor-induced apoptosis is implicated in the pathological mechanisms of neural tissues, increasing the release of reactive oxygen species (ROS), resulting in a type of apoptotic cell death called excitotoxicity. Although intrinsic mechanisms to remove ROS, such as antioxidant enzymes, are provided by the tissue, the association between NMDA-induced excitotoxicity and antioxidative enzymes is not well understood. In this study, we focused on superoxide dismutase 1 (SOD1), an antioxidant enzyme, and investigated the role of SOD1 in the NMDA-induced neuronal cell death in the retina. NMDA was intravitreally injected into wild-type (WT) and SOD1 total knock-out (SOD1-deficient) mice. The number of TUNEL-positive cells in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) counted in the retinal sections and flatmount retinas were significantly higher in the SOD1-deficient mice than the WT mice after NMDA injection. Visual function assessed by dark-adapted electroretinogram (ERG) showed that the amplitudes of a-wave, b-wave, and oscillatory potential 2 were significantly reduced in the NMDA-injected SOD1-deficient mice. The level of ROS in the GCL and INL, measured using dihydroethidium, and the number of positive cells for γ-H2AX, a marker for DNA double strand breaks, and 8-OHdG, a marker for DNA oxidation, in the GCL were significantly increased in the SOD1-deficient mice after NMDA injection. We also measured mRNA and protein levels of SOD1 and SOD2 in the retina of WT mice, to find that mRNA and protein levels of SOD1, but not SOD2, were significantly reduced after NMDA injection. SOD1 deficiency exacerbated NMDA-induced damage to the inner retinal neurons, and NMDA reduced SOD1 levels in the retina of WT mice. Therefore, SOD1 protected retinal neurons against NMDA-induced retinal neurotoxicity, and NMDA-induced SOD1 reduction may be involved in neuronal vulnerability to excitotoxicity., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Insulin-like growth factor I (IGF-I) exerts multiple effects on different retinal cell types in both physiological and pathological conditions. Despite the growth factor's extensively described neuroprotective actions, transgenic mice with increased intraocular levels of IGF-I showed progressive impairment of electroretinographic amplitudes up to complete loss of response, with loss of photoreceptors and bipolar, ganglion, and amacrine neurons. Neurodegeneration was preceded by the overexpression of genes related to retinal stress, acute-phase response, and gliosis, suggesting that IGF-I altered normal retinal homeostasis. Indeed, gliosis and microgliosis were present from an early age in transgenic mice, before other alterations occurred, and were accompanied by signs of oxidative stress and impaired glutamate recycling. Older mice also showed overproduction of pro-inflammatory cytokines. Our results suggest that, when chronically increased, intraocular IGF-I is responsible for the induction of deleterious cellular processes that can lead to neurodegeneration, and they highlight the importance that this growth factor may have in the pathogenesis of conditions such as ischemic or diabetic retinopathy.

Aim: Dietary deficiencies in ω-3 polyunsaturated fatty acids are known to effect retinal function including retinal ganglion cell (RGC) activity, which may have implications for glaucoma. In this study we consider retinal function after dietary manipulation and intraocular pressure (IOP) stress designed to compromise RGCs., Methods: Sprague-Dawley dams were fed either ω-3 sufficient (ω-3, n=15) or deficient (ω-3, n=16) diets 5 weeks before conception with pups subsequently weaned onto their mothers diets. At 20 weeks of age, acute IOP elevation was induced repeatedly through anterior chamber cannulation to 70 mm Hg for 1 hour on 3 separate occasions separated by 1 week. Electroretinograms were recorded 1 week after each IOP elevation to assay the photoreceptors (PIII), ON-bipolar cells (PII), and ganglion/amacrine cells (STR)., Results: Repeat IOP insult results in a specific RGC dysfunction (pSTR -14.5%, P<0.035) as does ω-3 deficiency (-26.4%, P<0.01). However, the combination of both causes an even larger RGC functional loss (-40.1%, P<0.001) than does either diet or IOP insult in isolation (P<0.001)., Conclusions: Both ω-3 deficiency and repeat acute IOP insult cause RGC dysfunction and the combination of these factors results in a cumulative effect. Our data indicate that sufficient dietary ω-3 improves RGC function making it less susceptible to IOP insult.

How synaptic neuropil is formed within the CNS is poorly understood. The retinal inner plexiform layer (IPL) is positioned between the cell bodies of amacrine cells (ACs) and retinal ganglion cells (RGCs). It consists of bipolar cell (BC) axon terminals that synapse on the dendrites of ACs and RGCs intermingled with projections from Müller glia (MG). We examined whether any of these cellular processes are specifically required for the formation of the IPL. Using genetic and pharmacological strategies, we eliminated RGCs, ACs, and MG individually or in combination. Even in the absence of all of these partner cells, an IPL-like neuropil consisting of only BC axon terminals still forms, complete with presynaptic specializations and sublaminar organization. Previous studies have shown that an IPL can form in the complete absence of BCs; therefore, we conclude that neither presynaptic nor postsynaptic processes are individually essential for the formation of this synaptic neuropil., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Sodium iodate (NaIO(3))-induced retina injury is one of models that is commonly used to study various retinal diseases caused by retinal pigment epithelium (RPE) injury such as AMD. Previous researches have revealed that RPE and photoreceptors are main impaired objects in this model. By comparison, intra-retinal layer has not been studied in detail after NaIO(3) administration. In this study, we present evidences that intra-retinal neurons can be directly injured by NaIO(3) at early stage and that the morphology had taken obvious changes, the decreased areas of dendritic fields of dopaminergic amacrine cells (DA-ACs), horizontal cells, and melanopsin-expressing retinal ganglion cells (mRGCs). Moreover, we found that miRNA 133b that was considered specifically to express in midbrain dopaminergic neurons was markedly upregulated in retinal DA-ACs after NaIO(3) administration. The overexpression of mir-133b negatively regulated the expression of pitx3, an important transcription factor, and led to a series of deficits of DA-ACs such as TH and D2 receptor expression and DA producing, which may play a causative role in pathological events of horizontal cells and mRGCs. After mir-133b was interfered with mir-133b/RNAi, not only those deficits were rescued, but also the amplitude of b-wave and summed OPs of ERG were improved significantly. In conclusion, our data demonstrate, for the first time, that intra-retinal neurons can be directly injured by NaIO(3) at early stage, and that mir-133b level effectively controls synaptic contacts or neural interactions among DA-ACs, horizontal cells, and mRGCs. Delivering mir-133b/RNAi intravitreally can rescue NaIO(3)-induced failure and improve visual function by restoring synaptic contacts.

Purpose: To investigate the longitudinal profile of N-methyl-D-aspartate (NMDA) injection-induced damage in retinal ganglion cells (RGCs) by imaging retinal Thy 1-cyan fluorescent protein (CFP) expression and inner retinal layers using a custom-made imaging device containing short-wavelength confocal scanning laser ophthalmoscope (scSLO) and speckle noise-reduced spectral-domain optical coherence tomography (SD-OCT)., Methods: Simultaneous scSLO and SD-OCT examinations were performed in Thy 1-CFP mice injected with NMDA (1-20 nanomoles). CFP-expressing RGCs were counted using scSLO images. Ganglion cell complex (GCC: retinal nerve fiber layer, ganglion cell layer, and inner plexiform layer) thickness around the optic disc was measured in SD-OCT images., Results: The RGCs rapidly decreased 1 day after NMDA injection in a dose-dependent manner (65.3%, 71.7%, 49.5%, and 27.1% of the preinjection level, 2, 5, 10, and 20 nanomoles, respectively) and continued to decrease slightly (to 53.7%, 44.1%, 28.3%, and 20.2% of the preinjection level on days 14, 2, 5, 10, and 20 nanomoles, respectively). In contrast, dose-dependent reduction of GCC thickness was first detected 4 days after injection. The thickness further decreased to 84.6%, 75.7%, 76.5%, and 71.4% of the preinjection level on day 14 (2, 5, 10, and 20 nanomoles, respectively)., Conclusions: NMDA-induced RGC damage is characterized by rapid RGCs loss followed by gradual reduction in GCC thickness. Simultaneous imaging of CFP expression in the RGCs and inner retinal layers provides a sensitive, reliable, and new method for longitudinal evaluation of progressive RGC damage in experimental models of glaucoma.

Gestational lead exposure (GLE) produces supernormal scotopic electroretinograms (ERG) in children, monkeys and rats, and a novel retinal phenotype characterized by an increased number of rod photoreceptors and bipolar cells in adult mice and rats. Since the loss of dopaminergic amacrine cells (DA ACs) in GLE monkeys and rats contributes to supernormal ERGs, the retinal DA system was analyzed in mice following GLE. C57BL/6 female mice were exposed to low (27 ppm), moderate (55 ppm) or high (109 ppm) lead throughout gestation and until postnatal day 10 (PN10). Blood [Pb] in control, low-, moderate- and high-dose GLE was ≤ 1, ≤ 10, ~25 and ~40 μg/dL, respectively, on PN10 and by PN30 all were ≤ 1 μg/dL. At PN60, confocal-stereology studies used vertical sections and wholemounts to characterize tyrosine hydroxylase (TH) expression and the number of DA and other ACs. GLE dose-dependently and selectively decreased the number of TH-immunoreactive (IR) DA ACs and their synaptic plexus without affecting GABAergic, glycinergic or cholinergic ACs. Immunoblots and confocal revealed dose-dependent decreases in retinal TH protein expression and content, although monoamine oxidase-A protein and gene expression were unchanged. High-pressure liquid chromatography showed that GLE dose-dependently decreased retinal DA content, its metabolites and DA utilization/release. The mechanism of DA selective vulnerability is unknown. However, a GLE-induced loss/dysfunction of DA ACs during development could increase the number of rods and bipolar cells since DA helps regulate neuronal proliferation, whereas during adulthood it could produce ERG supernormality as well as altered circadian rhythms, dark/light adaptation and spatial contrast sensitivity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: To investigate retinal cell population changes under chronic elevated intraocular pressure in an inducible mouse model of glaucoma., Methods: Chronic unilateral ocular hypertension was induced in 40 C57BL6/J mice by ablation of the limbal episcleral veins. After 5, 20, 40 and 60 days of elevated intraocular pressure, specific retinal cell types were identified and/or quantified by immunohistochemistry for protein kinase C α, glial fibrillary acidic protein, parvalbumin and calretinin. Apoptotic cells were identified by TUNEL and cleaved caspase-3 immunohistochemistry., Results: Elevations in intraocular pressure in the range 22-30 mmHg were developed and sustained in mice for up to 60 days. Protein kinase C α immunoreactivity localized to bipolar cells was unchanged. We observed a rapid increase in glial fibrillary acidic protein expression in Müller cells and a progressive loss of parvalbumin-labelled ganglion cells. After 60 days of elevated intraocular pressure, calretinin-immunoreactive cell counts declined by 55.4% and 46.4% in the inner nuclear and ganglion cell layers, respectively. However, at all time points examined, the markers of cell death were only observed in the ganglion cell layer, not in the inner nuclear layer., Conclusions: In addition to ganglion cell death and reactive Müller cell changes, chronic experimental elevation of intraocular pressure alters calcium-binding protein immunohistochemistry in amacrine cells. However, these changes are not indicative of amacrine cell loss but may represent early indicators of cellular distress that precede physiological dysfunction or cell death., (© 2011 The Authors. Clinical and Experimental Ophthalmology © 2011 Royal Australian and New Zealand College of Ophthalmologists.)

Purpose: Staurosporine (SS) causes retinal ganglion cell (RGC) death in vivo, but the underlying mechanisms have been unclear. Since previous studies on RGC-5 cells indicated that SS induces cell death by elevating proteases, this study was undertaken to investigate whether SS induces RGC loss by elevating proteases in the retina, and curcumin prevents SS-mediated death of RGCs., Methods: Transformed mouse retinal ganglion-like cells (RGC-5) were treated with 2.0 μM SS and various doses of curcumin. Two optimal doses of SS (12.5 and 100 nM) and curcumin (2.5 and 10 μM) were injected into the vitreous of C57BL/6 mice. Matrix metalloproteinase (MMP)-9, tissue plasminogen activator (tPA), and urokinase plasminogen activator (uPA) activities were assessed by zymography assays. Viability of RGC-5 cells was assessed by MTT assays. RGC and amacrine cell loss in vivo was assessed by immunostaining with Brn3a and ChAT antibodies, respectively. Frozen retinal cross sections were immunostained for nuclear factor-κB (NF-κB)., Results: Staurosporine induced uPA and tPA levels in RGC-5 cells, and MMP-9, uPA, and tPA levels in the retinas and promoted the death of RGC-5 cells in vitro and RGCs and amacrine cells in vivo. In contrast, curcumin attenuated RGC and amacrine cell loss, despite elevated levels of proteases. An NF-κB inhibitory peptide reversed curcumin-mediated protective effect on RGC-5 cells, but did not inhibit protease levels. Curcumin did not inhibit protease levels in vivo, but attenuated RGC and amacrine cell loss by restoring NF-κB expression., Conclusions: The results show that curcumin attenuates RGC and amacrine cell death despite elevated levels of proteases and raises the possibility that it may be used as a plausible adjuvant therapeutic agent to prevent the loss of these cells in retinal degenerative conditions.

Excitotoxicity, induced either by N-Methyl-d-aspartate (NMDA) or kainic acid (KA), promotes irreversible loss of retinal ganglion cells (RGCs). Although the intracellular signaling mechanisms underlying excitotoxic cell death are still unclear, recent studies on the retina indicate that NMDA promotes RGC death by increasing phosphorylation of cyclic AMP (cAMP) response element (CRE)-binding protein (CREBP), while studies on the central nervous system indicate that KA promotes neuronal cell death by decreasing phosphorylation of CREBP, suggesting that CREBP can elicit dual responses depending on the excitotoxic-agent. Interestingly, the role of CREBP in KA-mediated death of RGCs has not been investigated. Therefore, by using an animal model of excitotoxicity, the aim of this study was to investigate whether excitotoxicity induces RGC death by decreasing Ser(133)-CREBP in the retina. Death of RGCs was induced in CD-1 mice by an intravitreal injection of 20 nmoles of kainic acid (KA). Decrease in CREBP levels was determined by immunohistochemistry, western blot analysis, and electrophoretic mobility gel shift assays (EMSAs). Immunohistochemical analysis indicated that CREBP was constitutively expressed in the nuclei of cells both in the ganglion cell layer (GCL) and in the inner nuclear layer (INL) of CD-1 mice. At 6 h after KA injection, nuclear localization of Ser(133)-CREBP was decreased in the GCL. At 24 h after KA injection, Ser(133)-CREBP was decreased further in GCL and the INL, and a decrease in Ser(133)-CREBP correlated with apoptotic death of RGCs and amacrine cells. Western blot analysis indicated that KA decreased Ser(133)-CREBP levels in retinal protein extracts. EMSA assays indicated that KA also reduced the binding of Ser(133)-CREBP to CRE consensus oligonucleotides. In contrast, intravitreal injection of CNQX, a non-NMDA glutamate receptor antagonist, restored the KA-induced decrease in Ser(133)-CREBP both in the GCL and INL, and inhibited loss of RGCs and amacrine cells. These results, for the first time, suggest that KA promotes retinal degeneration by reducing phosphorylation of Ser(133)-CREBP in the retina., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: Intrauterine infection is a common antecedent of preterm birth. Infants born very preterm are at increased risk for neurologic dysfunction, including visual deficits. With increasing survival of very preterm infants, there is a need for therapies that prevent adverse neurologic outcomes. Using an ovine model, the authors investigated the neuroprotective potential of recombinant human erythropoietin (rhEPO) on retinal injury induced by intrauterine inflammation., Methods: At 107 ± 1 days of gestational age (DGA), chronically catheterized fetal sheep received either of the following on 3 consecutive days: intravenous (IV) bolus dose of lipopolysaccharide (LPS; ∼0.9 μg/kg; n = 8); IV bolus dose of LPS, followed at 1 hour by 5000 IU/kg rhEPO (LPS + rhEPO; n = 8); rhEPO alone (n = 5). Untreated fetuses (n = 8) were used for comparison with the three treatment groups. Fetal physiological parameters were monitored. At 116 ± 1 DGA, fetal retinas were assessed quantitatively for morphologic and neurochemical alterations., Results: Exposure to LPS alone, but not to rhEPO alone, resulted in fetal hypoxemia and hypotension (P < 0.05). Exposure to LPS alone caused retinal changes, including reductions in thickness of the inner nuclear layer (INL), somal areas of INL neurons, process growth in the plexiform layers, and numbers of ganglion and tyrosine hydroxylase immunoreactive (TH-IR) dopaminergic amacrine cells. Treatment of LPS-exposed fetuses with rhEPO did not alter the physiological effects of LPS but significantly reduced alterations in retinal layers and ganglion and TH-IR cell numbers., Conclusions: rhEPO treatment was beneficial in protecting the developing retina after LPS-induced inflammation. Retinal protection could occur by the antiapoptotic or anti-inflammatory actions of EPO.

To determine if the absence of c-Jun N-terminal kinase 3 (JNK3) in the mouse retina would reduce retinal ganglion cell (RGC) loss in mice with experimental glaucoma. C57BL/6 mice underwent experimental intraocular pressure (IOP) elevation with a bead/viscoelastic injection into one eye. One-half of the mice were Jnk3 homozygous knockouts (KO) and were compared to wild type (WT) mice. IOP was measured under anesthesia with the TonoLab, axial length was measured post-mortem with calipers after inflation to 15mmHg, and RGC layer counts were performed on retinal whole mount images stained with DAPI, imaged by confocal microscopy, and counted by masked observers in an image analysis system. Axon counts were performed in optic nerve cross-sections by semi-automated image analysis. Both WT and Jnk3(-/-) mice had mean elevations of IOP of more than 50% after bead injection. Both groups underwent the expected axial globe elongation due to chronic IOP elevation. The absence of JNK3 in KO retina was demonstrated by Western blots. RGC layer neuron counts showed modest loss in both WT and Jnk3(-/-) animals; local differences by retinal eccentricity were detected, in each case indicating greater loss in KO animals than in WT. The baseline number of RGC layer cells in KO animals was 10% higher than in WT, but the number of optic nerve axons was identical in KO and WT controls. A slightly greater loss of RGC in Jnk3(-/-) mice compared to controls was detected in experimental mouse glaucoma by RGC layer counting and there was no protective effect shown in axon counts. Counts of RGC layer cells and optic nerve axons indicate that Jnk3(-/-) mice have an increased number of amacrine cells compared to WT controls., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aim of this study was to examine synaptic connectivity changes in the retina and the location and rate of apoptosis in transgenic S334ter line-3 and line-5 rats with photoreceptor degeneration. Heterozygous S334ter-line-3 and line-5 at P11-13, P30, P60, P90 and several control non-dystrophic rats (Long Evans and Sprague-Dawley) at P60, were studied anatomically by immunohistochemistry for various cell and synaptic markers, and by PNA and TUNEL label.- S334ter line-3 exhibited the fastest rate of degeneration with an early loss of photoreceptors, with 1-2 layers remaining at P30, and only cones left at P60. Line-5 had 4-5 layers left at P30, and very few rods left at P60-90. In both lines, horizontal cell processes (including dendrites and axon) were diminished at P11-13, showing gaps in the outer plexiform layer (OPL) at P60, and at P90, almost no terminal tips could be seen. Bipolar cells showed a retraction of their dendrites forming clusters along the OPL. Synaptic terminals of A-II amacrine cells in the IPL lost most of their parvalbumin-immunoreactivity. The apoptosis rate was different in both lines. Line-3 rats showed many photoreceptors affected at P11, occupying the innermost part of the outer nuclear layer. Line-5 showed a lower number of apoptotic cells within the same location at P13. In summary, the S334ter line-3 rat has a faster progression of degeneration than line-5. The horizontal and bipolar terminals are already affected at P11-P13 in both models. Apoptosis is related to the mutated rhodopsin transgene; the first photoreceptor cells affected are those close to the OPL., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Purpose: Displaced cholinergic amacrine cell neurons comprise a significant fraction of the retinal ganglion cell (RGC) layer. Rodent anterior ischemic optic neuropathy (rAION) is an optic nerve infarct, which results in RGC loss in mice. The goal was to determine whether rAION produces changes in amacrine cell neurons., Methods: rAION was generated in transgenic mice carrying a cyan fluorescent reporter protein (CFP) gene linked to the Thy-1 promoter, which expresses CFP in RGCs. rAION was induced with standard parameters. Retinas were examined pre-and post-induction by retinal fundus microscopy. rAION induction severity was scored by changes in retinal transparency and RGC loss. Cholinergic amacrine cells were identified via choline acetyltransferase (ChAT) immunohistochemistry. ChAT and CFP expression was evaluated in flat-mounted retinas examined by confocal microscopy and western analysis., Results: Moderate rAION induction levels (defined as early retention of retinal transparency and <70% RGC loss) did not alter amacrine cell numbers in the RGC layer, but changed the relative levels of ChAT expression by immunohistochemistry. No changes in total ChAT protein were seen. Severe rAION induction (defined as loss of retinal transparency and >70% RGC loss) resulted in a trend toward amacrine cell loss and decreased ChAT protein levels., Conclusions: There is wide disparity in mouse rAION induction levels using standardized parameters. Moderate rAION induction levels without direct retinal compromise produces isolated RGC loss, with displaced amacrine cell changes likely due to changes in RGC-amacrine communication. Severe rAION induction results in both RGC and amacrine cell loss, possibly due to intra-retinal ischemic changes.

The S334ter-line-3 rat is a transgenic model of retinal degeneration developed to express a rhodopsin mutation similar to that found in human retinitis pigmentosa (RP) patients. Previous studies have focused on physiological changes in retinal cells and higher centers of the visual system with this model of retinal degeneration. However, little is known about the morphological changes in retinal cells during the development of the S334ter-line-3 rat. In order to understand and aid vision-rescue strategies, our aim has been to describe the retinal degeneration pattern in this model. We focus on changes in the morphologies of horizontal, bipolar, and amacrine cells in developing S334ter-line-3 rat retinas. Degeneration of photoreceptors begins in the central retina and progresses toward the periphery. In retinas at post-natal day 15 (P15), horizontal and rod bipolar cells show normal morphology. However, at P21, horizontal and rod bipolar cells exhibit abnormal processes at the outer plexiform layer, whereas the outer nuclear layer is significantly thinner. A glial reaction occurs concomitantly. In contrast, modifications in cone-bipolar and amacrine cells are much slower and do not occur until P90 and P180, respectively. The density of horizontal and rod-bipolar cells significantly drops after P60. Overall, the S334ter-line-3 model exhibits the hallmarks of cellular remodeling caused by photoreceptor degeneration. Its moderately fast time course makes the S334ter-line-3 a good model for studying vision-rescue strategies.

Retinopathy of prematurity (ROP) is characterized by deficits in the scotopic pathway, although the cellular locus for these deficits is not clear. Here we examined neurochemical and cellular changes that develop during oxygen-induced retinopathy, a model of ROP. In addition, we examined whether treatment with the angiotensin II type-1 receptor inhibitor, valsartan, prevented these changes. Newborn Sprague-Dawley rats were exposed from postnatal day (P) 0 to 11 to 80%:20% O(2) (22:2 hr/day) and then room air until P18. Valsartan (40 mg/kg/day) was administered from P12-P18. Pattern recognition analysis of overlapping amino acid profiles was used to provide a statistically robust and spatially complete classification of neural elements for each experimental condition. Classification yielded 12 neuronal theme classes in controls and nine classes following ROP. ROP was associated with a reduction in the number of amacrine and bipolar cell theme classes. The reduction in theme classes was confirmed as true neuronal loss by quantifying anatomical changes and using an apoptotic marker. ROP was associated with shifts in amino acid levels across all neuronal populations except for horizontal cells. A reduction in the density of glycine-immunoreactive amacrine cells, and particularly parvalbumin-immunoreactive AII amacrine cells, was observed following ROP. Valsartan treatment during ROP prevented loss of theme classes and loss of AII amacrine cells. This study suggests that deficits in scotopic vision during ROP may be associated with loss of AII amacrine cells. In addition, this study highlights the potential of AT(1)R blockade in preventing neuronal anomalies in this condition.

Purpose: To study alterations in different retinal cell types associated with retinal ganglion cell (RGC) death after elevation of intraocular pressure (IOP) in rats., Methods: IOP was elevated by episcleral vein cauterization of the rat left eye. The right unoperated eye was kept as the control. IOP was measured when rats were awake. The animals were euthanized after one week (n=4) and five weeks (n=4). Their eyes were enucleated, postfixed, cryoprotected, and embedded in optimal cutting temperature (OCT) medium. Cryosections of the retina were cut at 14 microm thickness and processed for immunocytochemistry with 15 antibodies that specifically stain different retinal cell types. The distribution and intensity of the label was analyzed by comparing sections of control and glaucomatous retinas obtained from identical locations., Results: The amount of amacrine cells identified by calcium binding proteins and choline acetyltransferase antibodies decreased after five weeks of elevated IOP. By using the anti-protein kinase C-alpha antibody, we were able to label a subpopulation of rod bipolar cells in control retinas but not in retinas that had elevated IOP. No changes were found in RGCs labeled with brain derived neurotrophic factor when comparing control and glaucomatous retinas. Glial fibrillary acidic protein and vimentin expression in glial cells increased after one week of elevated IOP., Conclusions: After one week of elevated IOP and before the onset of RGC death, it was evident that inner retinal cells showed remarkable changes in their molecular expression.

A DBA/2J (D2) transgenic mouse line with cyan fluorescent protein (CFP) reporter expression in ganglion cells was developed for the analysis of ganglion cells during progressive glaucoma. The Thy1-CFP D2 (CFP-D2) line was created by congenically breeding the D2 line, which develops pigmentary glaucoma, and the Thy1-CFP line, which expresses CFP in ganglion cells. Microsatellite marker analysis of CFP-D2 progeny verified the genetic inclusion of the D2 isa and ipd loci. Specific mutations within these loci lead to dysfunctional melanosomal proteins and glaucomatous phenotype in D2 mice. Polymerase chain reaction analysis confirmed the inclusion of the Thy1-CFP transgene. CFP-fluorescent ganglion cells, 6-20 microm in diameter, were distributed in all retinal regions, CFP processes were throughout the inner plexiform layer, and CFP-fluorescent axons were in the fiber layer and optic nerve head. Immunohistochemistry with antibodies to ganglion cell markers NF-L, NeuN, Brn3a, and SMI32 was used to confirm CFP expression in ganglion cells. Immunohistochemistry with antibodies to amacrine cell markers HPC-1 and ChAT was used to confirm weak CFP expression in cholinergic amacrine cells. CFP-D2 mice developed a glaucomatous phenotype, including iris disease, ganglion cell loss, attrition of the fiber layer, and elevated intraocular pressure. A CFP-D2 transgenic line with CFP-expressing ganglion cells was developed, which has (1) a predominantly D2 genetic background, (2) CFP-expressing ganglion cells, and (3) age-related progressive glaucoma. This line will be of value for experimental studies investigating ganglion cells and their axons in vivo and in vitro during the progressive development of glaucoma.

The purpose of this study was to establish, and characterize a porcine model of acute, controlled retinal ischemia. The controlled retinal ischemia was produced by clamping the ocular perfusion pressure (OPP) in the left eye to 5 mm Hg for 2 h. The OPP was defined as mean arterial blood pressure (MAP) minus the intraocular pressure (IOP). It was clamped to 0-30 mm Hg by continuous monitoring of MAP and adjustment of the IOP, which was controlled by cannulation of the anterior chamber. Inner retinal function was assessed by induced multifocal electroretinography (mfERG) with comparisons of the amplitudes obtained in the experimental, left eye, and the control, right eye. Quantitative histology was performed to measure the survival of ganglion cells, amacrine cells and horizontal cells 2-6 weeks after the ischemic insult. An OPP of 5 mm Hg for 2h induced significant reductions in the amplitudes of iN1 to 20% (CI: 13-30%), and iP2 to 14% (95% CI: 8-22%) of their baseline values. No signs of recovery were found within the 6-week observation period. Quantitative histology revealed a highly significant reduction in the number of ganglion cells, amacrine cells and horizontal cells after the ischemic insult. This model seems to be suitable for investigations of therapeutic initiatives in diseases involving acute retinal ischemia.