Neuroprotective Effects of Erinacine A on an Experimental Model of Traumatic Optic Neuropathy.

Erinacine A (EA), a natural neuroprotectant, is isolated from a Chinese herbal medicine, Hericium erinaceus. The aim of this study was to investigate the neuroprotective effects of EA in a rat model of traumatic optic neuropathy. The optic nerves (ONs) of adult male Wistar rats were crushed using a standardized method and divided into three experimental groups: phosphate-buffered saline (PBS control)-treated group, standard EA dose-treated group (2.64 mg/kg in 0.5 mL of PBS), and double EA dose-treated group (5.28 mg/kg in 0.5 mL of PBS). After ON crush, each group was fed orally every day for 14 days before being euthanized. The visual function, retinal ganglion cell (RGC) density, and RGC apoptosis were determined using flash visual-evoked potentials (fVEP) analysis, retrograde Fluoro-Gold labelling, and TdT-dUTP nick end-labelling (TUNEL) assay, respectively. Macrophage infiltration of ON was detected by immunostaining (immunohistochemistry) for ED1. The protein levels of phosphor-receptor-interacting serine/threonine-protein kinase1 (pRIP1), caspase 8 (Cas8), cleaved caspase 3 (cCas3), tumour necrosis factor (TNF)-α, tumour necrosis factor receptor1 (TNFR1), interleukin (IL)-1ß, inducible nitric oxide synthase (iNOS), nuclear factor erythroid 2-related factor 2 (Nrf2), haem oxygenase-1 (HO-1), and superoxide dismutase 1 (SOD1) were evaluated by Western blotting. When comparing the standard EA dose-treated group and the double EA dose-treated group with the PBS-treated group, fVEP analysis showed that the amplitudes of P1−N2 in the standard EA dose group and the double EA dose-treated group were 1.8 and 2.4-fold, respectively, higher than that in the PBS-treated group (p < 0.05). The density of RGC in the standard EA dose-treated group and the double EA dose-treated group were 2.3 and 3.7-fold, respectively, higher than that in the PBS-treated group (p < 0.05). The TUNEL assay showed that the standard EA dose-treated group and the double EA dose-treated group had significantly reduced numbers of apoptotic RGC by 10.0 and 15.6-fold, respectively, compared with the PBS-treated group (p < 0.05). The numbers of macrophages on ON were reduced by 1.8 and 2.2-fold in the standard EA dose-treated group and the double EA dose-treated group, respectively (p < 0.01). On the retinal samples, the levels of pRIP, Cas8, cCas3, TNF-α, TNFR1, IL-1ß, and iNOS were decreased, whereas those of Nrf2, HO-1, and SOD1 were increased in both EA-treated groups compared to those in the PBS-treated group (p < 0.05). EA treatment has neuroprotective effects on an experimental model of traumatic optic neuropathy by suppressing apoptosis, neuroinflammation, and oxidative stress to protect the RGCs from death as well as preserving the visual function.

Transcriptomic Analysis Reveals That Granulocyte Colony-Stimulating Factor Trigger a Novel Signaling Pathway (TAF9-P53-TRIAP1-CASP3) to Protect Retinal Ganglion Cells after Ischemic Optic Neuropathy.

Optic nerve head (ONH) infarct can result in progressive retinal ganglion cell (RGC) death. The granulocyte colony-stimulating factor (GCSF) protects the RGC after ON infarct. However, protective mechanisms of the GCSF after ONH infarct are complex and remain unclear. To investigate the complex mechanisms involved, the transcriptome profiles of the GCSF-treated retinas were examined using microarray technology. The retinal mRNA samples on days 3 and 7 post rat anterior ischemic optic neuropathy (rAION) were analyzed by microarray and bioinformatics analyses. GCSF treatment influenced 3101 genes and 3332 genes on days 3 and 7 post rAION, respectively. ONH infarct led to changes in 702 and 179 genes on days 3 and 7 post rAION, respectively. After cluster analysis, the levels of TATA box-binding protein (TBP)-associated factor were significantly reduced after ONH infarct, but these significantly increased after GCSF treatment. The network analysis revealed that TBP associated factor 9 (TAF9) can bind to P53 to induce TP53-regulated inhibitor of apoptosis 1 (TRIAP1) expression. To evaluate the function of TAF9 in RGC apoptosis, GCSF plus TAF9 siRNA-treated rats were evaluated using retrograde labeling with FluoroGold assay, TUNEL assay, and Western blotting in an rAION model. The RGC densities in the GCSF plus TAF9 siRNA-treated rAION group were 1.95-fold (central retina) and 1.75-fold (midperipheral retina) lower than that in the GCSF-treated rAION group (p < 0.05). The number of apoptotic RGC in the GCSF plus TAF9 siRNA-treated group was threefold higher than that in the GCSF-treated group (p < 0.05). Treatment with TAF9 siRNA significantly reduced GCSF-induced TP53 and TRIAP1 expression by 2.4-fold and 4.7-fold, respectively, in the rAION model. Overexpression of TAF9 significantly reduced apoptotic RGC and CASP3 levels, and induced TP53 and TRIAP1 expression in the rAION model. Therefore, we have demonstrated that GCSF modulated a new pathway, TAF9-P53-TRIAP1-CASP3, to control RGC death and survival after ON infarct.

The Benefits and Hazards of Intravitreal Mesenchymal Stem Cell (MSC) Based-Therapies in the Experimental Ischemic Optic Neuropathy.

Mesenchymal stem cell (MSC) therapy has been investigated intensively for many years. However, there is a potential risk related to MSC applications in various cell niches. METHODS: The safety of intravitreal MSC application and the efficacy of MSC-derived conditioned medium (MDCM) were evaluated in the normal eye and the diseased eye, respectively. For safety evaluation, the fundus morphology, visual function, retinal function, and histological changes of the retina were examined. For efficacy evaluation, the MDCM was intravitreally administrated in a rodent model of anterior ischemic optic neuropathy (rAION). The visual function, retinal ganglion cell (RGC) density, and neuroinflammation were evaluated at day 28 post-optic nerve (ON) infarct. RESULTS: The fundus imaging showed that MSC transplantation induced retinal distortion and venous congestion. The visual function, retinal function, and RGC density were significantly decreased in MSC-treated eyes. MSC transplantation induced astrogliosis, microgliosis, and macrophage infiltration in the retina due to an increase in the HLA-DR-positive MSC proportion in vitreous. Treatment with the MDCM preserved the visual function and RGC density in rAION via inhibition of macrophage infiltration and RGC apoptosis. CONCLUSIONS: The vitreous induced the HLA-DR expression in the MSCs to cause retinal inflammation and retina injury. However, the MDCM provided the neuroprotective effects in rAION.

Algae Oil Treatment Protects Retinal Ganglion Cells (RGCs) via ERK Signaling Pathway in Experimental Optic Nerve Ischemia.

BACKGROUND: We investigated the therapeutic effects and related mechanisms of algae oil (ALG) to protect retinal ganglion cells (RGCs) in a rat model of anterior ischemic optic neuropathy (rAION). METHODS: Rats were daily gavaged with ALG after rAION induction for seven days. The therapeutic effects of ALG on rAION were evaluated using flash visual evoked potentials (FVEPs), retrograde labeling of RGCs, TUNEL assay of the retina, and ED1 staining of optic nerves (ONs). The levels of inducible nitric oxide synthase (iNOS), IL-1ß, TNF-α, Cl-caspase-3, ciliary neurotrophic factor (CNTF), and p-ERK were analyzed by using western blots. RESULTS: Protection of visual function in FVEPs amplitude was noted, with a better preservation of the P1-N2 amplitude in the ALG-treated group (p = 0.032) than in the rAION group. The density of RGCs was 2.4-fold higher in the ALG-treated group compared to that in the rAION group (p < 0.0001). The number of ED1-positive cells in ONs was significantly reduced 4.1-fold in the ALG-treated group compared to those in the rAION group (p = 0.029). The number of apoptotic RGCs was 3.2-fold lower in number in the ALG-treated group (p = 0.001) than that in the rAION group. The ALG treatment inhibited ERK activation to reduce the levels of iNOS, IL-1ß, TNF-α, and Cl-caspase-3 and to increase the level of CNTF in the rAION model. CONCLUSION: The treatment with ALG after rAION induction inhibits ERK activation to provide both anti-inflammatory and antiapoptotic effects in rAION.

Haematococcus pluvialis-Derived Astaxanthin Is a Potential Neuroprotective Agent against Optic Nerve Ischemia.

Astaxanthin, a xanthophyll belonging to the family of carotenoids, is a potent antioxidant. However, much less is known about its protective effects on the oxidative stress of ischemic optic nerve. We hypothesized that astaxanthin treatment could protect retinal ganglion cells (RGCs) from death via anti-oxidative and anti-apoptotic responses. Adult male Wistar rats were fed astaxanthin (100 mg/kg/day) by daily gavage for seven consecutive days, either before or after inducing oxidative stress in the retina by photodynamic treatment. The visual function, RGC apoptosis, macrophage infiltration in the optic nerve, expression of p-Akt, p-mTOR, SGK1, pS6K, Nrf2, p62, TNFα, Il1ß in retinas were investigated. The visual function and the RGC densities were significantly higher in both pre- and post-treatment groups. The numbers of apoptotic RGCs and extrinsic macrophage infiltration in the optic nerve were significantly decreased in both astaxanthin-treated groups. Furthermore, pre- and post-treatment of astaxanthin showed a higher expression of p-Akt, p-mTOR, Nrf2 and superoxide dismutase activity, and a lower expression of cleaved caspase-3, suggesting anti-apoptotic and anti-oxidative roles. Our findings indicate that astaxanthin can preserve visual function and reduce RGC apoptosis after ischemic insults. Including astaxanthin in daily diet as a supplement may be beneficiary for ischemic optic neuropathy.

Optic nerve head width and retinal nerve fiber layer changes are proper indexes for validating the successful induction of experimental anterior ischemic optic neuropathy.

Reproducible skills are essential for successful induction of a rat model of anterior ischemic optic neuropathy (rAION). We established an in vivo validation index by measuring the natural course of optic nerve head (ONH) width and retinal nerve fiber layer (RNFL) thickness in the rAION model using optical coherence tomography (OCT). The rAION model was induced by photodynamic operations. We measured the ONH width, RNFL, Inner Plexiform layer (IPL) and Ganglion cell complex (GCC) thickness in the acute stage (<3 days), subacute stage (day-7 to day-14) and later stage (day-14 to day-28) post-infarct by OCT. Retinal layers were measured by hematoxylin and eosin stain (HE) to confirm the OCT findings. The RGCs survival rate was determined by retrograde Fluoro-gold labeling, and the visual function was assessed with flash visual-evoked potentials (FVEPs) 4 weeks post-infarct. We observed significant thinning in GCC, IPL, and RNFL at day-14 and day-28 but only RNFL showed significant thinning between day-14 and day-28. The ONH showed significant swelling in the acute stage which correlated at a greater extent with RNFL than GCC and IPL. Further RNFL correlated at a greater extent at with GCC than IPL. HE-stained retina cross sections also showed IPL and RNFL thinning, which further confirmed our OCT findings. The RGC density and P1-N2 amplitude were significantly reduced in rAION. Our data suggest that Swelling, reduction of swelling, and atrophy of RNFL in acute, sub-acute, and later stage, respectively and ONH swelling in the acute stage are essential events for confirming the successful induction of rAION.

Autocrine protective mechanisms of human granulocyte colony-stimulating factor (G-CSF) on retinal ganglion cells after optic nerve crush.

This study investigated the role of autocrine mechanisms in the anti-apoptotic effects of human granulocyte colony-stimulating factor (G-CSF) on retinal ganglion cells (RGCs) after optic nerve (ON) crush. We observed that both G-CSF and G-CSF receptor (G-CSFR) are expressed in normal rat retina. Further dual immunofluorescence staining showed G-CSFR immunoreactive cells were colocalized with RGCs, Müller cells, horizontal and amacrine cells. These results confirm that G-CSF is an endogenous ligand in the retina. The semi-quantitative RT-PCR finding demonstrated the transcription levels of G-CSF and G-CSFR were up-regulated after ON crush injury. G-CSF treatment further increased and prolonged the expression level of G-CSFR in the retina. G-CSF has been shown to enhance transdifferentiation of the mobilized hematopoietic stem cells into tissue to repair central nervous system injury. We test the hypothesis that the hematopoietic stem cells recruited by G-CSF treatment can transdifferentiate into RGCs after ON crush by performing sublethal irradiation of the rats 5 days before ON crush. The flow cytometric analysis showed the number of CD34 positive cells in the peripheral blood is significantly lower in the irradiated, crushed and G-CSF-treated group than the sham control group or crush and G-CSF treated group. Nevertheless, the G-CSF treatment enhances the RGC survival after sublethal irradiation and ON crush injury. These data indicate that G-CSF seems unlikely to induce hematopoietic stem cell transdifferentiation into RGCs after ON crush injury. In conclusion, G-CSF may serve an endogenous protective signaling in the retina through direct activation of intrinsic G-CSF receptors and downstream signaling pathways to rescue RGCs after ON crush injury, exogenous G-CSF administration can enhance the anti-apoptotic effects on RGCs.

Differential secretomics of Streptococcus pyogenes reveals a novel peroxide regulator (PerR)-regulated extracellular virulence factor mitogen factor 3 (MF3).

Streptococcus pyogenes is a human pathogen that causes various diseases. Numerous virulence factors secreted by S. pyogenes are involved in pathogenesis. The peroxide regulator (PerR) is associated with the peroxide resistance response and pathogenesis, but little is known about the regulation of the secretome involved in virulence. To investigate how PerR regulates the expression of the S. pyogenes secretome involved in virulence, a perR deficient mutant was used for comparative secretomic analysis with a wild-type strain. The conditioned medium containing secreted proteins of a wild-type strain and a perR deficient mutant at the stationary phase were collected for two-dimensional gel electrophoresis analysis, where protease inhibitors were applied to avoid the degradation of extracellular proteins. Differentially expressed protein spots were identified by liquid chromatography electrospray ionization tandem MS. More than 330 protein spots were detected on each gel. We identified 25 unique up-regulated proteins and 13 unique down-regulated proteins that were directly or indirectly controlled by the PerR regulator. Among these identified proteins, mitogen factor 3 (MF3), was selected to verify virulence and the expression of gene products. The data showed that MF3 protein levels in conditioned medium, as measured by immunoblot analysis, correlated well with protein levels determined by two-dimensional gel electrophoresis analysis. We also demonstrated that PerR bound to the promoter region of the mf3 gene. The result of an infection model showed that virulence was attenuated in the mf3 deficient mutant. Additional growth data of the wild-type strain and the mf3 deficient mutant suggested that MF3 played a role in digestion of exogenous DNA for promoting growth. To summarize, we conclude that PerR can positively regulate the expression of the secreted protein MF3 that contributes to the virulence in S. pyogenes. The analysis of the PerR-regulated secretome provided key information for the elucidation of the host-pathogen interactions and might assist in the development of potential chemotherapeutic strategies to prevent or treat streptococcal diseases.

Investigation of the Protective Effect of Extracellular Vesicle miR-124 on Retinal Ganglion Cells Using a Photolabile Paper-Based Chip.

Purpose: Photolabile paper-based chips were developed to isolate extracellular vesicles (EVs) from small-volume samples (less than 30 µL), such as vitreous humor. Putative neuroprotective effects of EVs' microRNAs were investigated by using the paper chip and a rodent model with nonarteritic anterior ischemic optic neuropathy (rNAION). Methods: rNAION was established using laser-induced photoactivation of rose bengal administered intravenously. On days 0, 0.25, 1, 3, and 7 after rNAION induction, CD63-positive EV microRNAs (CD63+-EV miRNAs) in vitreous humor samples were enriched using the paper chip and assessed using microarray and quantitative RT-PCR analyses. The viability and visual function of retinal ganglion cells (RGCs) were further assessed by measuring photopic flash visual evoked potentials (FVEPs). Results: We identified 38 different variations of CD63+-EV miRNAs with more than twofold altered expressions. Among them, M1-related miRNA, mR-31a-5p, and M2-related miRNA, miR-125a-5p, miR-182, miR-181a-5p, and miR-124-3, were capable of coordinating anti-inflammatory reactions during rNAION because of their capacity to activate macrophages. In particular, miR-124, having the most dramatic alteration of gene expression, was synthesized and injected intravitreally. Compared to controls, rats that received miR-124 had shown increased RGC survivability and improved visual function. Conclusions: Our research team has developed a paper-based chip capable of capturing EVs that can be released after UV exposure. The quantity and quality of EV-miRNAs extracted are adequate for microarray and quantitative RT-PCR analyses. Animal studies suggest that miR-124 may play a neuroprotective role in the natural recovery of rNAION and holds the potential to be a novel treatment option.

Long term therapeutic effects of icariin-loaded PLGA microspheres in an experimental model of optic nerve ischemia via modulation of CEBP-ß/G-CSF/noncanonical NF-κB axis.

An ischemic insult at optic nerve (ON) is followed by detrimental neuroinflammation that results in progressive and long-lasting retinal ganglion cell (RGC) death and vision loss. Icariin was reported to be a safe and effective natural anti-inflammatory drug. Herein, we evaluated the long-term therapeutic effects of a single intravitreal injection of poly(lactide-co-glycolide) PLGA-icariin in a rat model of anterior ischemic optic neuropathy (rAION). Treatment with PLGA microspheres of icariin preserved the visual function and RGC density for 1 month in the rAION model. In addition, ON edema and macrophage infiltration were inhibited by treating PLGA microspheres of icariin. We found that the binding complex of icariin and CCAAT enhancer binding protein beta (CEBP-ß) significantly induced endogenous granulocyte colony-stimulating factor (G-CSF) expression to activate noncanonical nuclear factor kappa B (NF-κB) signaling pathway by promoting an alternative phosphorylation reaction of IKK-ß. Activation of noncanonical NF-κB signaling pathway promoted the M2 microglia/macrophage polarization and AKT1 activation, which prevented neuroinflammation and RGC apoptosis after ON infarct. This study concluded that protective mechanism of icariin is a CEBP-ß/G-CSF axis-induced noncanonical NF-κB activation, which provides the long-term neuroprotective effects via anti-inflammatory and antiapoptotic actions after ON ischemia.

Induction of Human Umbilical Mesenchymal Stem Cell Differentiation Into Retinal Pigment Epithelial Cells Using a Transwell-Based Co-culture System.

There is an increasing interest in generating retinal pigment epithelial (RPE) cells from stem cells for treating degenerative eye diseases. However, whether human umbilical cord mesenchymal stem cells (HUCMSCs) can differentiate into RPE-like cells in a co-culture system has not been fully understood. In this study, induction of HUCMSC differentiation into RPE-like cells was performed by co-culturing HUCMSCs and a human RPE-like cell line (ARPE19) in a transwell system and then analyzed for biomarkers using quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescence staining technique. Moreover, the functional characterization of induced cells was carried out by examining their phagocytic and neurotrophic factor-secreting activities. Our results showed that mRNA expressions of RPE-specific markers-MITF, OTX2, RPE65, PEDF, PME17, and CRALBP-and protein markers-RPE65, CRALBP, and ZO-1-were significantly increased in HUCMSC-derived RPE-like cells. Functional characteristic studies showed that these induced cells were capable of engulfing photoreceptor outer segments and secreting brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF), which are typical functions of RPE-like cells. Overall, the study findings indicate that the morphology and proliferation of HUCMSCs can be maintained in a serum-free medium, and differentiation into RPE-like cells can be induced by simply co-culturing HUCMSCs with ARPE19 cells. Thus, the study provides fundamental information regarding the clinical-scale generation of RPE-like cells from HUCMSCs.

Vitamin B3 Provides Neuroprotection via Antioxidative Stress in a Rat Model of Anterior Ischemic Optic Neuropathy.

Supplementing with vitamin B3 has been reported to protect against retinal ganglion cell (RGC) damage events and exhibit multiple neuroprotective properties in a mouse model of optic nerve injury. In this study, a rat model of anterior ischemic optic neuropathy was used to assess the neuroprotective benefits of vitamin B3 (rAION). Vitamin B3 (500 mg/kg/day) or phosphate-buffered saline (PBS) was administered to the rAION-induced rats every day for 28 days. The vitamin B3-treated group had significantly higher first positive and second negative peak (P1-N2) amplitudes of flash visual-evoked potentials and RGC densities than the PBS-treated group (p < 0.05). A terminal deoxynucleotidyl transferase dUTP nick end labeling assay conducted on vitamin B3-treated rats revealed a significant reduction in apoptotic cells (p < 0.05). Superoxide dismutase and thiobarbituric acid reactive substance activity showed that vitamin B3 treatment decreased reactive oxygen species (p < 0.05). Therefore, vitamin B3 supplementation preserves vision in rAION-induced rats by reducing oxidative stress, neuroinflammation, and mitochondrial apoptosis.

Neuroprotective Effects of Cordyceps cicadae (Ascomycetes) Mycelium Extract in the Rat Model of Optic Nerve Crush.

Cordyceps cicadae mycelium is an herbal medicine used to provide anti-inflammatory and antiapoptotic actions. However, little is known about the role of C. cicadae mycelium in neuroprotection. This study aimed to investigate the neuroprotective effects of C. cicadae mycelium extract (CCME) in the optic nerve crush (ONC) model. The optic nerves of adult male Wistar rats (aged 7-8 weeks) were crushed by a standardized method. Rats were divided equally into three groups: 1) a sham-operated group (sham), 2) a phosphate buffered saline-treated control group (crush), and 3) a CCME-treated group (CCME) that received CCME once daily for 7 consecutive days at doses of 100 mg/kg before ONC. Two weeks after ONC in rats, retinal ganglion cell (RGC) density and visual function were determined by using retrograde labeling with FluoroGold and flash visual evoked potentials. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunohistochemistry of ED1 (a marker of macrophage/microglia) were used to evaluate the antiapoptotic and anti-inflammatory effects of CCME in the optic nerve section. The P1-N2 amplitude and RGC density in the CCME-treated group were higher than those in the ONC control (crush) group by 5.15- and 3.13-fold, respectively. The numbers of TUNEL-positive cells and ED1-positive cells in the CCME-treated group were reduced by 4.38- and 6.63-fold, respectively, compared to those in the crush group. Oral administration of CCME provided neuroprotective effects in the ONC model via antiapoptotic and anti-inflammatory actions, which provides a potential treatment for patient with traumatic optic neuropathy.

Mitochondrial Transplantation Moderately Ameliorates Retinal Degeneration in Royal College of Surgeons Rats.

Retinal pigmented epithelial (RPE) cells possess high mitochondria content for energy production, which is required for phagocytosis and vision cycle metabolism. The mitochondrial integrity in RPE cells helps the homeostasis of photoreceptor turnover and prevents retina aging and degeneration. Mitochondrial transplantation benefits the recovery of several acute inflammatory diseases, leading us to investigate the effects of mitochondrial transplantation on retina degeneration. Allogeneic mitochondria were isolated and delivered into the vitreous chamber in the Royal College of Surgeons (RCS) rats, which exhibit inherited and early-onset retina degeneration. The progress of retina degeneration was examined with optical coherence tomography (OCT) and visual evoked potential (VEP) to determine the retina thickness and integrity of afferent electrical signals from affected eyes, respectively. We found that mitochondria engraftment moderately attenuated the degeneration of retinal layers in RCS rats by histological examination. This result was consistent with the OCT measurement of retina thickness around the optic disc. The VEP analysis revealed that the peak one (N1) latency, representing the arriving time of electrical impulse from the retina to cortex, was substantially maintained as the normal value after the mitochondrial transplantation. This result suggests that the intra-vitreous transplanted mitochondria ameliorate the degeneration of photoreceptors in RCS rats and might be potential for clinical application.

Intravitreal Injection of Long-Acting Pegylated Granulocyte Colony-Stimulating Factor Provides Neuroprotective Effects via Antioxidant Response in a Rat Model of Traumatic Optic Neuropathy.

Traumatic optic neuropathy (TON) may cause severe visual loss following direct or indirect head trauma which may result in optic nerve injuries and therefore contribute to the subsequent loss of retinal ganglion cells by inflammatory mediators and reactive oxygen species (ROS). Granulocyte colony-stimulating factor (G-CSF) provides the anti-inflammatory and anti-oxidative actions but has a short half-life and also induces leukocytosis upon typical systemic administration. The purpose of the present study was to investigate the relationship between the anti-oxidative response and neuroprotective effects of long-acting pegylated human G-CSF (PEG-G-CSF) in a rat model of optic nerve crush (ONC). Adult male Wistar rats (150-180 g) were chosen to have a sham operation in one eye and have ONC in the other. PEG-G-CSF or phosphate-buffered saline (PBS control) was immediately administered after ONC by intravitreal injection (IVI). We found the IVI of PEG-G-CSF does not induce systemic leukocytosis, but increases survival of RGCs and preserves the visual function after ONC. TUNEL assays showed fewer apoptotic cells in the retina in the PEG-G-CSF-treated eyes. The number of sorely ED1-positive cells was attenuated at the lesion site in the PEG-G-CSF-treated eyes. Immunoblotting showed up-regulation of p-Akt1, Nrf2, Sirt3, and HO-1 in the ON of the PEG-G-CSF-treated eyes. Our results demonstrated that one IVI of long-acting PEG-G-CSF is neuroprotective in the rONC. PEG-G-CSF activates the p-Akt1/Nrf2/Sirt3 and the p-Akt1/Nrf2/HO-1 axes to provide the antioxidative action and further attenuated RGC apoptosis and neuroinflammation. This provides crucial preclinical information for the development of alternative therapy with IVI of PEG-G-CSF in TON.

Role of Oxidative Stress in Ocular Diseases Associated with Retinal Ganglion Cells Degeneration.

Ocular diseases associated with retinal ganglion cell (RGC) degeneration is the most common neurodegenerative disorder that causes irreversible blindness worldwide. It is characterized by visual field defects and progressive optic nerve atrophy. The underlying pathophysiology and mechanisms of RGC degeneration in several ocular diseases remain largely unknown. RGCs are a population of central nervous system neurons, with their soma located in the retina and long axons that extend through the optic nerve to form distal terminals and connections in the brain. Because of this unique cytoarchitecture and highly compartmentalized energy demand, RGCs are highly mitochondrial-dependent for adenosine triphosphate (ATP) production. Recently, oxidative stress and mitochondrial dysfunction have been found to be the principal mechanisms in RGC degeneration as well as in other neurodegenerative disorders. Here, we review the role of oxidative stress in several ocular diseases associated with RGC degenerations, including glaucoma, hereditary optic atrophy, inflammatory optic neuritis, ischemic optic neuropathy, traumatic optic neuropathy, and drug toxicity. We also review experimental approaches using cell and animal models for research on the underlying mechanisms of RGC degeneration. Lastly, we discuss the application of antioxidants as a potential future therapy for the ocular diseases associated with RGC degenerations.

Inhibition of Retinal Ganglion Cell Loss By a Novel ROCK Inhibitor (E212) in Ischemic Optic Nerve Injury Via Antioxidative and Anti-Inflammatory Actions.

Purpose: This study investigated the neuroprotective effects of administration of ROCK inhibitor E212 on ischemic optic neuropathy. Methods: Rats received an intravitreal injection of either E212 or PBS immediately after optic nerve infarct. The oxidative stress in the retina was detected by performing superoxide dismutase activity and CellROX assays. The integrity of retinal pigment epithelium was determined by staining of zona occludens 1. The visual function, retinal ganglion cell (RGC) density, and RGC apoptosis were determined by using flash visual-evoked potential analysis, retrograde FluoroGold labeling, and TdT-dUTP nick end-labeling assay. Macrophage infiltration was detected by staining for ED1. The protein levels of TNF-α, p-CRMP, p-AKT1, p-STAT3, and CD206 were evaluated using Western blotting. Results: Administration of E212 resulted in a 1.23-fold increase in the superoxide dismutase activity of the retina and 2.28-fold decrease in RGC-produced reactive oxygen species as compared to the levels observed upon treatment with PBS (P < 0.05). Moreover, E212 prevented the disruption of the blood-retinal barrier (BRB) in contrast to PBS. The P1-N2 amplitude and RGC density in the E212-treated group were 1.75- and 2.05-fold higher, respectively, than those in the PBS-treated group (P < 0.05). The numbers of apoptotic RGCs and macrophages were reduced by 2.93- and 2.54-fold, respectively, in the E212-treated group compared with those in the PBS-treated group (P < 0.05). The levels of p-AKT1, p-STAT3, and CD206 were increased, whereas those of p-PTEN, p-CRMP2, and TNF-α were decreased after treatment with E212 (P < 0.05). Conclusions: Treatment with E212 suppresses oxidative stress, BRB disruption, and neuroinflammation to protect the visual function in ischemic optic neuropathy.

Induced pluripotent stem cells and derivative photoreceptor precursors as therapeutic cells for retinal degenerations.

The visual impairment associated with inherited retinal degeneration and age-related degeneration of photoreceptors is causing substantial challenges in finding effective therapies. However, induced pluripotent stem cell (iPSC)-derived therapeutic cells such as photoreceptor and retinal pigment epithelium (RPE) cells provide the ultimate options in the rescue of lost photoreceptors to improve the visual function in end-stage degeneration. Retinal cells derived from iPSC are therapeutic cells that could be promising in the field of cell replacement therapy and regenerative medicine. This review presents an overview of the photoreceptor degeneration, methods of iPSC generation, iPSC in retinal disease modeling, summarizes the photoreceptor differentiation protocols, and challenges remained with photoreceptor cell replacement for the treatment of retinal diseases. Thus, the burden and increased incidence of visual impairment emphasizes the need of novel therapy, where iPSC-derived photoreceptor and RPE cells proved to be promising for curing the retinal dysfunction and act as renovation in approach to improve visual function.

Effective Differentiation and Biological Characterization of Retinal Pigment Epithelium Derived from Human Induced Pluripotent Stem Cells.

PURPOSE: Human induced pluripotent stem cells (hiPSC)-derived retinal pigment epithelium (RPE) cells are therapeutic cells that have been shown to be promising in the rescue of lost photoreceptors. In this study, we generated hiPSC from human epidermal keratinocytes and subsequently differentiated them into RPE cells to investigate their ability to influence the retinal functions of the Royal College of Surgeon (RCS) rats. METHODS: Keratinocytes were reprogrammed to hiPSC using a non-integrating Sendai reprogramming system. Established hiPSCs were differentiated into RPE cells, and complete characterization was performed. Next, the suspension of hiPSC-RPE cells was transplanted into the subretinal space of 3-week-old RCS rats (n = 12). Posttransplantation evaluations were performed using optical coherence tomography (OCT), electroretinography, and immunohistochemical analysis. RESULTS: The hiPSC colonies were identical to embryonic stem-like cells that revealed the expression of pluripotency markers and retention of the normal genome. These cells exhibited the ability to differentiate into an amalgam of germ layers and produce RPE cells. The differentiated RPE cells exhibited an identical pigmented morphology that expressed RPE-specific markers, such as CRALBP, BESTROPHIN, RPE65, and MERTK. At 8 weeks of longitudinal culture, the RPE cells exhibited maximum pigmentation with in vitro phagocytotic activity. Furthermore, transplantation data showed improved retinal function till week 12 post-transplantation and a significantly higher number of rod/cone ratios in transplanted eyes compared to non-surgery control eyes. CONCLUSION: hiPSC-derived RPE cells exhibited naïve RPE cell properties and functionality that provided trophic support and the transient rescue of photoreceptor cells.

Neuroprotective effects of low-dose G-CSF plus meloxicam in a rat model of anterior ischemic optic neuropathy.

Non-arteritic anterior ischemic optic neuropathy (NAION) causes a sudden loss of vision and lacks effective treatment. Granulocyte colony-stimulating factor (G-CSF) provides neuroprotection against the experimental optic nerve injuries but also induce leukocytosis upon typical administration. We found synergetic neuroprotective effects of meloxicam and low dose G-CSF without leukocytosis in a rat model of anterior ischemic optic neuropathy (rAION). The WBC counts in the low-dose G-CSF-plus meloxicam-treated group were similar to the sham-operated group. Combination treatment of low-dose G-CSF plus meloxicam preserved RGCs survival and visual function, reduced RGC apoptosis and the macrophages infiltration, and promote more M2 phenotype of macrophage/microglial transition than the low-dose GCSF treatment or the meloxicam treatment. Moreover, the combination treatment induced higher serine/threonine kinase 1 (Akt1) expression. The combination treatment of low-dose G-CSF plus meloxicam lessened the leukocytotic side effect and provided neuroprotective effects via Akt1 activation in the rAION model. This approach provides crucial preclinical information for the development of alternative therapy in AION.