Retinal Ganglion Cell Neurite Research Articles

The influence of polyacrylamide gel substrate elasticity on primary cultures of rat retinal ganglion cells

In vitro primary cell culture models of retinal ganglion cells (RGC) are widely used to study pathomechanisms of diseases such as glaucoma. The biomechanic interaction with the culture substrate is known to influence core cellular functions. RGC cultures, however, are usually grown on rigid plastic or glass substrates. We hypothesized that soft polyacrylamide gel substrates may alter survival and neurite outgrowth of primary cultured RGC.Primary retinal cultures from postnatal (day 1–6) Wistar rats were grown on glass coverslips or polyacrylamide (PA) gel substrate with different Young's elastic moduli (0.75, 10 or 30 kPa). Substrates were coated with Poly-l-lysine and/or laminin. RGC were immunostained with anti-beta-III-tubulin. Total neurite length, growth cone morphology, RGC density, mitochondrial morphology and transport as well as pro-survival pathways (Erk1/2, Akt, CREB) were assessed.PA gel substrates of E = 10 kPa significantly increased the total neurite length by factor 1.5 compared to glass (p = 0.02). The growth cone area was significantly larger by factor 5.3 on 30 kPa gels (p = 0.01). The presence of a substrate coating was more important for neurite outgrowth and RGC survival on PA gels (poly-l-lysine > laminin) than on glass. Neither mitochondrial morphology and motility nor the activation of pro-survival pathways significantly differed between the four substrates.PA gel substrates significantly enhanced RGC neurite outgrowth. The signaling cascades mediating this effect remain to be determined.

Shootin1 Regulates Retinal Ganglion Cell Neurite Development: Insights From an RGC Direct Somatic Cell Reprogramming Model.

Retinal ganglion cells (RGCs) connect the retina to the brain. Proper development of the axons and dendrites of RGCs is the basis for these cells to function as projection neurons to deliver visual information to the brain. The purpose of this study was to investigate the function of Shtn1 (which encodes shootin1) in RGC neurite development. Immunofluorescence (IF) was used to characterize the expression pattern of marker genes. An in vitro direct somatic cell reprogramming system was used to generate RGC-like neurons (iRGCs), which was subsequently used to study the function of Shtn1. Short-hairpin RNAs (shRNAs) were used to knock down Shtn1, and the coding sequence (CDS) of Shtn1 was used to overexpress the gene. Lentiviruses were used to deliver shRNAs or CDSs into iRGCs. The patch clamp technique was used to measure the electrophysiological properties of the iRGCs. RNA sequencing (RNA-seq) was used to examine transcriptome expression. Using IF, we demonstrated that shootin1 is distinctively expressed in RGCs during the period in which RGCs actively develop and adjust the connections of their neurites with upstream and downstream neurons. Using the iRGC system, we demonstrated that Shtn1 promotes the growth and complexity of neurites and thus the electrophysiological maturation, of iRGCs. RNA-seq analyses showed that Shtn1 may also regulate gene expression and neurogenesis in iRGCs. Shtn1 promotes RGC neurite development. These findings improve our understanding of the molecular machinery governing RGC neurite development and may help to optimize future RGC regeneration methods.

Combined treatment of human mesenchymal stem cells and green tea extract on retinal ganglion cell regeneration in rats after optic nerve injury

Retinal ganglion cell (RGC) death and axonal loss cause irreversible vision loss upon optic nerve (ON) injury. We have independently demonstrated that mesenchymal stem cells (MSCs) and green tea extract (GTE) promote RGC survival and axonal regeneration in rats with ON injury. Here we aimed to evaluate the combined treatment effect of human bone marrow-derived MSCs (hBM-MSCs) and GTE on RGC survival and axonal regeneration after ON injury. Combined treatment of hBM-MSCs and GTE promoted RGC survival and neurite outgrowth/axonal regeneration in ex vivo retinal explant culture and in rats after ON injury. GTE increased Stat3 activation in the retina after combined treatment, and enhanced brain-derived neurotrophic factor secretion from hBM-MSCs. Treatment of 10 μg/mL GTE would not induce hBM-MSC apoptosis, but inhibited their proliferation, migration, and adipogenic and osteogenic differentiation in vitro with reducing matrix metalloproteinase secretions. In summary, this study revealed that GTE can enhance RGC protective effect of hBM-MSCs, suggesting that stem cell priming could be a prospective strategy enhancing the properties of stem cells for ON injury treatment.

Design Parameters and Human Biocompatibility Assessment Protocols for Organic Semiconducting Neural Interfaces: Toward a Printed Artificial Retina with Color Vision

AbstractOrganic semiconductors have emerged as promising neural interfacing materials due to their innate biocompatibility, soft mechanical properties, and mixed electron/ion conduction. One exciting application is their use as artificial photosensors for retinal prostheses via optically induced neuromodulation. In this study, the optoelectronic and neural interfacing properties of six organic semiconductor polymers and small molecules, split into donor/acceptor pairs that form promising candidates for a trichromatic artificial retina that closely mimics the native response of the human eye are presented. The biocompatibility of these materials using primary human retinal cell cultures by systematic measurement of both cell viability and morphological analysis of retinal ganglion cell neurite elongation over time is investigated. Comparable cell viability between human retinal cell cultures established on all the organic semiconductors and a glass control, which is a standard measurement for biocompatibility in materials science is observed. In contrast, differences in the morphological biocompatibility between the organic semiconductor materials and glass control are detected by analyzing neurite elongation with specific immunomarkers. The difference in the two results has implications for the future assessment of material biocompatibility for bioelectronics, and optimal methodology for assessing morphological biocompatibility in neural interface devices is discussed.

Ginsenoside Rg1 promotes neurite growth of retinal ganglion cells through cAMP/PKA/CREB pathways

BackgroundMechanisms of synaptic plasticity in retinal ganglion cells (RGCs) are complex and the current knowledge cannot explain. Growth and regeneration of dendrites together with synaptic formation are the most important parameters for evaluating the cellular protective effects of various molecules. The effect of ginsenoside Rg1 (Rg1) on the growth of retinal ganglion cell processes has been poorly understood. Therefore, we investigated the effect of ginsenoside Rg1 on the neurite growth of RGCs. MethodsExpression of proteins and mRNA were detected by Western blot and qPCR. cAMP levels were determined by ELISA. In vivo effects of Rg1 on RGCs were evaluated by hematoxylin and eosin, and immunohistochemistry staining. ResultsThis study found that Rg1 promoted the growth and synaptic plasticity of RGCs neurite by activating the cAMP/PKA/CREB pathways. Meanwhile, Rg1 upregulated the expression of GAP43, Rac1 and PAX6, which are closely related to the growth of neurons. Meantime, H89, an antagonist of PKA, could block this effect of Rg1. In addition, we preliminarily explored the effect of Rg1 on enhancing the glycolysis of RGCs, which could be one of the mechanisms for its neuroprotective effects. ConclusionRg1 promoted neurite growth of RGCs through cAMP/PKA/CREB pathways. This study may lay a foundation for its clinical use of optic nerve diseases in the future.

Sigma 1 Receptor Contributes to Astrocyte-Mediated Retinal Ganglion Cell Protection.

PurposeSigma 1 receptor (S1R) is expressed in retinal ganglion cells (RGCs) and astrocytes, and its activation is neuroprotective. We evaluated the contribution of S1R within optic nerve head astrocytes (ONHAs) to growth and survival of RGCs in vitro.MethodsWild-type (WT) RGCs and WT or S1R knockout (S1R KO) ONHAs were cocultured for 2, 4, or 7 days. Total and maximal neurite length, neurite root, and extremity counts were measured. Cell death was measured using a TUNEL assay. Signal transducer and activator of transcription 3 phosphorylation levels were evaluated in ONHA-derived lysates by immunoblotting.ResultsThe coculture of WT RGCs with WT or S1R KO ONHAs increased the total and maximal neurite length. Neurite root and extremity counts increased at 4 and 7 days when WT RGCs were cocultured with WT or S1R KO ONHAs. At all timepoints, the total and maximal neurite length decreased for WT RGCs in coculture with S1R KO ONHAs compared with WT ONHAs. Root and extremity counts decreased for WT RGCs in coculture with S1R KO ONHAs compared with WT ONHAs at 2 and 7, but not 4 days. RGC apoptosis increased in S1R KO ONHA coculture and S1R KO-conditioned medium, compared with WT ONHA coculture or WT-conditioned medium. S1R KO ONHA-derived lysates showed decreased phosphorylated signal transducer and activator of transcription 3 levels compared with WT ONHA-derived lysates.ConclusionsThe absence of S1R within ONHAs has a deleterious effect on RGC neurite growth and RGC survival, reflected in analysis of WT RGC + S1R KO ONHA indirect cocultures. The data suggest that S1R may enhance ganglion cell survival via glia-mediated mechanisms.

Optic Nerve Engraftment of Neural Stem Cells.

PurposeTo evaluate the integrative potential of neural stem cells (NSCs) with the visual system and characterize effects on the survival and axonal regeneration of axotomized retinal ganglion cells (RGCs).MethodsFor in vitro studies, primary, postnatal rat RGCs were directly cocultured with human NSCs or cultured in NSC-conditioned media before their survival and neurite outgrowth were assessed. For in vivo studies, human NSCs were transplanted into the transected rat optic nerve, and immunohistology of the retina and optic nerve was performed to evaluate RGC survival, RGC axon regeneration, and NSC integration with the injured visual system.ResultsIncreased neurite outgrowth was observed in RGCs directly cocultured with NSCs. NSC-conditioned media demonstrated a dose-dependent effect on RGC survival and neurite outgrowth in culture. NSCs grafted into the lesioned optic nerve modestly improved RGC survival following an optic nerve transection (593 ± 164 RGCs/mm2 vs. 199 ± 58 RGCs/mm2; P < 0.01). Additionally, RGC axonal regeneration following an optic nerve transection was modestly enhanced by NSCs transplanted at the lesion site (61.6 ± 8.5 axons vs. 40.3 ± 9.1 axons, P < 0.05). Transplanted NSCs also differentiated into neurons, received synaptic inputs from regenerating RGC axons, and extended axons along the transected optic nerve to incorporate with the visual system.ConclusionsHuman NSCs promote the modest survival and axonal regeneration of axotomized RGCs that is partially mediated by diffusible NSC-derived factors. Additionally, NSCs integrate with the injured optic nerve and have the potential to form neuronal relays to restore retinofugal connections.

CXCL5/CXCR2 modulates inflammation-mediated neural repair after optic nerve injury

BackgroundPrevious studies reported that mild inflammation promotes retinal ganglion cell (RGC) survival and axonal regeneration after optic nerve (ON) injury with involvement of infiltrating macrophages and neutrophils. Here we aimed to evaluate the involvement and regulation of the main inflammatory chemokine pathway CXCL5/CXCR2 in the inflammation-mediated RGC survival and axonal regeneration in mice after ON injury. MethodsThe expressions and cellular locations of CXCL5 and CXCR2 were confirmed in mouse retina. Treatment effects of recombinant CXCL5 and CXCR2 antagonist SB225002 were studied in the explant culture and the ON injury model with or without lens injury. The number of RGCs, regenerating axons, and inflammatory cells were determined, and the activation of Akt andSTAT3 signaling pathways were evaluated. ResultsCxcr2 and Cxcl5 expressions were increased after ON and lens injury. Addition of recombinant CXCL5 promoted RGC survival and neurite outgrowth in retinal explant culture with increase in the number of activated microglia, which was inhibited by SB225002 or clodronate liposomes. Recombinant CXCL5 also alleviated RGC death and promoted axonal regeneration in mice after ON injury, and promoted the lens injury-induced RGC protection with increase in the number of activated CD68+ cells. SB225002 inhibited lens injury-induced cell infiltration and activation, and attenuated the promotion effect on RGC survival and axonal regeneration through reduction of lens injury-induced Akt activation. ConclusionsCXCL5 promotes RGC survival and axonal regeneration after ON injury and further enhances RGC protection induced by lens injury with CD68+ cell activation, which is attenuated by CXCR2 antagonist. CXCL5/CXCR2 could be a potential therapeutic target for RGC survival promotion after ON injury.

Role of the Internal Limiting Membrane in Structural Engraftment and Topographic Spacing of Transplanted Human Stem Cell-Derived Retinal Ganglion Cells.

SummaryRetinal ganglion cell (RGC) replacement holds potential for restoring vision lost to optic neuropathy. Transplanted RGCs must undergo neuroretinal integration to receive afferent visual signals for processing and efferent transmission. To date, retinal integration following RGC transplantation has been limited. We sought to overcome key barriers to transplanted human stem cell-derived RGC integration. Following co-culture ex vivo on organotypic mouse retinal explants, human RGCs cluster and extend bundled neurites that remain superficial to the neuroretina, hindering afferent synaptogenesis. To enhance integration, we increased the cellular permeability of the internal limiting membrane (ILM). Extracellular matrix digestion using proteolytic enzymes achieved ILM disruption while minimizing retinal toxicity and preserving glial reactivity. ILM disruption is associated with dispersion rather than clustering of co-cultured RGC bodies and neurites, and increased parenchymal neurite ingrowth. The ILM represents a significant obstacle to transplanted RGC connectivity and its circumvention may be necessary for functional RGC replacement.

Peritoneal macrophages attenuate retinal ganglion cell survival and neurite outgrowth.

Inflammation is a critical pathophysiological process that modulates neuronal survival in the central nervous system after disease or injury. However, the effects and mechanisms of macrophage activation on neuronal survival remain unclear. In the present study, we co-cultured adult Fischer rat retinas with primary peritoneal macrophages or zymosan-treated peritoneal macrophages for 7 days. Immunofluorescence analysis revealed that peritoneal macrophages reduced retinal ganglion cell survival and neurite outgrowth in the retinal explant compared with the control group. The addition of zymosan to peritoneal macrophages attenuated the survival and neurite outgrowth of retinal ganglion cells. Conditioned media from peritoneal macrophages also reduced retinal ganglion cell survival and neurite outgrowth. This result suggests that secretions from peritoneal macrophages mediate the inhibitory effects of these macrophages. In addition, increased inflammation- and oxidation-related gene expression may be related to the enhanced retinal ganglion cell degeneration caused by zymosan activation. In summary, this study revealed that primary rat peritoneal macrophages attenuated retinal ganglion cell survival and neurite outgrowth, and that macrophage activation further aggravated retinal ganglion cell degeneration. This study was approved by the Animal Ethics Committee of the Joint Shantou International Eye Center of Shantou University and the Chinese University of Hong Kong, Shantou, Guangdong Province, China, on March 11, 2014 (approval no. EC20140311(2)-P01).

Mild stress culture conditions promote neurite outgrowth of retinal explants from postnatal mice

The axons of retinal ganglion cells (RGCs) in adult mammals fail to regenerate after injury. It has been suggested that some extrinsic factors, such as neural activity, may promote the regeneration process. The present study tested the hypothesis that environmental stress such as slightly elevated osmolarity and temperature can enhance neural activity and thus promote axon regeneration of RGCs in postnatal mice. Retinal explants from P9-11 mice were cultured for 5 days to study the capacity of RGC neurite outgrowth. The neural activity of retinal explants in these two stress conditions was examined using the multi-electrode array. We found that RGC neurite outgrowth from P9-P11 mouse explants was significantly enhanced when the concentration of the culture medium was increased by 1.25 fold, but not when increased by 1.5 fold. Similarly, retinal explants from P9-P11 mice grew longer neurites when the overall temperature was increased from 35 °C to 38 °C, 40 °C or 42 °C for one hour each day, but not when they were kept at 40 °C or 42 °C constantly for five days. We further showed that there was increased neural activity during these two mild stress conditions. It was found that short-term 42 °C heat stress increased the expression of heat shock proteins 27 and 70 in postnatal retinas and they were RGC neural activity dependent. The present study thus provides insights into the cellular mechanism of retinal axon regeneration under the mild stress conditions.

Paired Immunoglobulin-like Receptor B Inhibition in Müller Cells Promotes Neurite Regeneration After Retinal Ganglion Cell Injury in vitro.

In the central nervous system (CNS), three types of myelin-associated inhibitors (MAIs) have major inhibitory effects on nerve regeneration. They include Nogo-A, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein. MAIs possess two co-receptors, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB). Previous studies have confirmed that the inhibition of NgR only results in a modest increase in regeneration in the CNS; however, the inhibitory effects of PirB with regard to nerve regeneration after binding to MAIs remain controversial. In this study, we demonstrated that PirB is expressed in primary cultures of retinal ganglion cells (RGCs), and the inhibitory effects of the three MAIs on the growth of RGC neurites are not significantly decreased after direct PirB knockdown using adenovirus PirB shRNA. Interestingly, we found that retinal Müller cells expressed PirB and that its knockdown enhanced the regeneration of co-cultured RGC neurites. PirB knockdown also activated the JAK/Stat3 signaling pathway in Müller cells and upregulated ciliary neurotrophic factor levels. These findings indicate that PirB plays a novel role in retinal Müller cells and that its action in these cells may indirectly affect the growth of RGC neurites. The results also reveal that PirB in Müller cells affects RGC neurite regeneration. Our findings provide a novel basis for the use of PirB as a target molecule to promote nerve regeneration.

The effect of extrinsic Wnt/β-catenin signaling in Muller glia on retinal ganglion cell neurite growth.

Muller glia are the predominant glial cell type in the retina, and they structurally and metabolically support retinal neurons. Wnt/β-catenin signaling pathways play essential roles in the central nervous system, including glial and neuronal differentiation, axonal growth, and neuronal regeneration. We previously demonstrated that Wnt signaling activation in retinal ganglion cells (RGC) induces axonal regeneration after injury. However, whether Wnt signaling within the adjacent Muller glia plays an axongenic role is not known. In this study, we characterized the effect of Wnt signaling in Muller glia on RGC neurite growth. Primary Muller glia and RGC cells were grown in transwell co-cultures and adenoviral constructs driving Wnt regulatory genes were used to activate and inhibit Wnt signaling specifically in primary Muller glia. Our results demonstrated that activation of Wnt signaling in Muller glia significantly increased RGC average neurite length and branch site number. In addition, the secretome of Muller glia after induction or inhibition of Wnt signaling was characterized using protein profiling of conditioned media by Q Exactive mass spectrometry. The Muller glia secretome after activation of Wnt signaling had distinct and more numerous proteins involved in regulation of axon extension, axon projection and cell adhesion. Furthermore, we showed highly redundant expression of Wnt signaling ligands in Muller glia and Frizzled receptors in RGCs and Muller glia. Therefore, this study provides new information about potential neurite growth promoting molecules in the Muller glia secretome, and identified Wnt-dependent target proteins that may mediate the axonal growth.

JIP1 Deficiency Protects Retinal Ganglion Cells From Apoptosis in a Rotenone-Induced Injury Model.

Retinal ganglion cells (RGCs) undergo apoptosis after injury. c-Jun N-terminal kinase (JNK)-interacting protein 1 (JIP1) is a scaffold protein that is relevant to JNK activation and a key molecule known to regulate neuronal apoptosis. However, the specific role of JIP1 in the apoptosis of RGCs is currently undefined. Here, we used JIP1 gene knockout (KO) mice to investigate the importance of JIP1-JNK signaling in the apoptosis of RGCs in a rotenone-induced injury model. In adult JIP1 KO mice, the number and electrophysiological functions of RGCs were not different from those of wild-type (WT) mice. Ablation of JIP1 attenuated the activation of JNK and the cleavage of caspase-3 in the retina after rotenone injury and contributed to a lower number of TUNEL-positive RGCs, a greater percentage of surviving RGCs, and a significant reduction in the electrophysiological functional loss of RGCs when compared to those in WT controls. We also found that JIP1 was located in the neurites of primary RGCs, but accumulated in soma in response to rotenone treatment. Moreover, the number of TUNEL-positive RGCs, the level of activation of JNK and the rate of cleavage of caspase-3 were reduced in primary JIP1-deficient RGCs after rotenone injury than in WT controls. Together, our results demonstrate that the JIP1-mediated activation of JNK contributes to the apoptosis of RGCs in a rotenone-induced injury model in vitro and in vivo, suggesting that JIP1 may be a potential therapeutic target for RGC degeneration.

Effects of siRNA-Mediated Knockdown of GSK3β on Retinal Ganglion Cell Survival and Neurite/Axon Growth.

There are contradictory reports on the role of the serine/threonine kinase isoform glycogen synthase kinase-3β (GSK3β) after injury to the central nervous system (CNS). Some report that GSK3 activity promotes axonal growth or myelin disinhibition, whilst others report that GSK3 activity prevents axon regeneration. In this study, we sought to clarify if suppression of GSK3β alone and in combination with the cellular-stress-induced factor RTP801 (also known as REDD1: regulated in development and DNA damage response protein), using translationally relevant siRNAs, promotes retinal ganglion cell (RGC) survival and neurite outgrowth/axon regeneration. Adult mixed retinal cell cultures, prepared from rats at five days after optic nerve crush (ONC) to activate retinal glia, were treated with siRNA to GSK3β (siGSK3β) alone or in combination with siRTP801 and RGC survival and neurite outgrowth were quantified in the presence and absence of Rapamycin or inhibitory Nogo-A peptides. In in vivo experiments, either siGSK3β alone or in combination with siRTP801 were intravitreally injected every eight days after ONC and RGC survival and axon regeneration was assessed at 24 days. Optimal doses of siGSK3β alone promoted significant RGC survival, increasing the number of RGC with neurites without affecting neurite length, an effect that was sensitive to Rapamycin. In addition, knockdown of GSK3β overcame Nogo-A-mediated neurite growth inhibition. Knockdown of GSK3β after ONC in vivo enhanced RGC survival but not axon number or length, without potentiating glial activation. Knockdown of RTP801 increased both RGC survival and axon regeneration, whilst the combined knockdown of GSK3β and RTP801 significantly increased RGC survival, neurite outgrowth, and axon regeneration over and above that observed for siGSK3β or siRTP801 alone. These results suggest that GSK3β suppression promotes RGC survival and axon initiation whilst, when in combination with RTP801, it also enhanced disinhibited axon elongation.

Matrix-bound nanovesicles prevent ischemia-induced retinal ganglion cell axon degeneration and death and preserve visual function

Injury to retinal ganglion cells (RGC), central nervous system neurons that relay visual information to the brain, often leads to RGC axon degeneration and permanently lost visual function. Herein this study shows matrix-bound nanovesicles (MBV), a distinct class of extracellular nanovesicle localized specifically to the extracellular matrix (ECM) of healthy tissues, can neuroprotect RGCs and preserve visual function after severe, intraocular pressure (IOP) induced ischemia in rat. Intravitreal MBV injections attenuated IOP-induced RGC axon degeneration and death, protected RGC axon connectivity to visual nuclei in the brain, and prevented loss in retinal function as shown by histology, anterograde axon tracing, manganese-enhanced magnetic resonance imaging, and electroretinography. In the optic nerve, MBV also prevented IOP-induced decreases in growth associated protein-43 and IOP-induced increases in glial fibrillary acidic protein. In vitro studies showed MBV suppressed pro-inflammatory signaling by activated microglia and astrocytes, stimulated RGC neurite growth, and neuroprotected RGCs from neurotoxic media conditioned by pro-inflammatory astrocytes. Thus, MBV can positively modulate distinct signaling pathways (e.g., inflammation, cell death, and axon growth) in diverse cell types. Since MBV are naturally derived, bioactive factors present in numerous FDA approved devices, MBV may be readily useful, not only experimentally, but also clinically as immunomodulatory, neuroprotective factors for treating trauma or disease in the retina as well as other CNS tissues.

Three-Dimensional Retinal Organoids Facilitate the Investigation of Retinal Ganglion Cell Development, Organization and Neurite Outgrowth from Human Pluripotent Stem Cells

Retinal organoids are three-dimensional structures derived from human pluripotent stem cells (hPSCs) which recapitulate the spatial and temporal differentiation of the retina, serving as effective in vitro models of retinal development. However, a lack of emphasis has been placed upon the development and organization of retinal ganglion cells (RGCs) within retinal organoids. Thus, initial efforts were made to characterize RGC differentiation throughout early stages of organoid development, with a clearly defined RGC layer developing in a temporally-appropriate manner expressing a complement of RGC-associated markers. Beyond studies of RGC development, retinal organoids may also prove useful for cellular replacement in which extensive axonal outgrowth is necessary to reach post-synaptic targets. Organoid-derived RGCs could help to elucidate factors promoting axonal outgrowth, thereby identifying approaches to circumvent a formidable obstacle to RGC replacement. As such, additional efforts demonstrated significant enhancement of neurite outgrowth through modulation of both substrate composition and growth factor signaling. Additionally, organoid-derived RGCs exhibited diverse phenotypes, extending elaborate growth cones and expressing numerous guidance receptors. Collectively, these results establish retinal organoids as a valuable tool for studies of RGC development, and demonstrate the utility of organoid-derived RGCs as an effective platform to study factors influencing neurite outgrowth from organoid-derived RGCs.

7,8,3′-Trihydroxyflavone ameliorate oxidative stress in vivo and promotes neurite regeneration in vitro in rat retinal ganglion cells

In this study, we investigate the mechanism of 7,8,3′-Trihydroxyflavone (THF) on retinal oxidative stress and retinal ganglion cell (RGC) growth. Oxidative stress was induced by ocular hypertension (O.H.) in rat retina in vivo. Rats were then systemically injected with THF. We found THF ameliorated O.H.-induced oxidative stress in retina by reducing caspase-3 activity, downregulating MDA level and augmenting glutathione peroxidase expression. In addition, neonatal RGCs were cultured in vitro, and treated with THF. It was demonstrated that THF promoted neurite regeneration in RGC explant, and activated receptor kinase B (TrkB) signaling pathway through phosphorylation. In THF-treated RGC explant, application of TrkB antagonist, ANA12, effectively reversed the pro-neuronal effect of THF on promoting RGC neurite outgrowth, and inhibited TrkB signaling pathway through dephosphorylation. Thus, we concluded that THF could inhibit retinal oxidative stress and promote RGC growth through TrkB-dependent signaling pathway, thus providing a novel pharmaceutical candidate for glaucoma.

Induced Pluripotent Stem Cells Promote Retinal Ganglion Cell Survival After Transplant.

PurposeThe purpose of this study was to characterize whether induced pluripotent stem cells (iPSCs) affect survival of grafted retinal ganglion cells (RGCs) after transplantation.MethodsFor in vitro studies, human iPSCs were either directly cocultured with mouse RGCs or plated in hanging inserts in RGC cultures for 1 week. For ex vivo studies, RGCs and iPSCs were seeded onto the inner surface of an adult rat retina explant and cultured for 1 week. For in vivo studies, RGCs and iPSCs were intravitreally coinjected into an adult rat eye 1 week before examining retinas by explant and immunostaining.ResultsA dose-dependent increase in RGC survival was observed in RGC-iPSC direct cocultures, and RGC-iPSC indirect cocultures showed a similar RGC protective effect, but to a lesser extent than in direct coculture. Enhanced RGC survival was also identified in RGC-iPSC cotransplantations to adult retinas ex vivo and in vivo. In addition, RGCs with iPSC cotransplantation extended significantly longer neurites than RGC-only transplants.ConclusionsHuman iPSCs promote transplanted RGC survival and neurite extension. This effect may be mediated at least partially through secretion of diffusible neuroprotective factors.

Human Periodontal Ligament-Derived Stem Cells Promote Retinal Ganglion Cell Survival and Axon Regeneration After Optic Nerve Injury.

Optic neuropathies are the leading cause of irreversible blindness and visual impairment in the developed countries, affecting more than 80 million people worldwide. While most optic neuropathies have no effective treatment, there is intensive research on retinal ganglion cell (RGC) protection and axon regeneration. We previously demonstrated potential of human periodontal ligament-derived stem cells (PDLSCs) for retinal cell replacement. Here, we report the neuroprotective effect of human PDLSCs to ameliorate RGC degeneration and promote axonal regeneration after optic nerve crush (ONC) injury. Human PDLSCs were intravitreally injected into the vitreous chamber of adult Fischer rats after ONC in vivo as well as cocultured with retinal explants in vitro. Human PDLSCs survived in the vitreous chamber and were maintained on the RGC layer even at 3 weeks after ONC. Immunofluorescence analysis of βIII-tubulin and Gap43 showed that the numbers of surviving RGCs and regenerating axons were significantly increased in the rats with human PDLSC transplantation. In vitro coculture experiments confirmed that PDLSCs enhanced RGC survival and neurite regeneration in retinal explants without inducing inflammatory responses. Direct cell-cell interaction and elevated brain-derived neurotrophic factor secretion, but not promoting endogenous progenitor cell regeneration, were the RGC protective mechanisms of human PDLSCs. In summary, our results revealed the neuroprotective role of human PDLSCs by strongly promoting RGC survival and axonal regeneration both in vivo and in vitro, indicating a therapeutic potential for RGC protection against optic neuropathies. Stem Cells 2018;36:844-855.