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.

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.

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.

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.

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.

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.

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.

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.

THE ASSOCIATION BETWEEN CENTRAL SEROUS CHORIORETINOPATHY AND SLEEP APNEA: A Nationwide Population-Based Study.

PURPOSE: To identify the association between sleep apnea (SA) and central serous chorioretinopathy (CSC). METHODS: In this nationwide population-based study using the Taiwan National Health Insurance Database, we enrolled adult patients with a diagnosis of SA and matched each patient to 30 age- and gender-matched control subjects without any SA diagnosis. Using Poisson regression analyses, the incidence rate of CSC was compared between SA patients and control subjects. RESULTS: A total of 10,753 SA patients and 322,590 control subjects were identified. After adjusting for age, gender, residency, income level, and comorbidities, the incidence rate of CSC was significantly higher in SA patients than in the control subjects (adjusted incident rate ratio for probable SA: 1.2 [95% CI: 1.1-1.4], P < 0.0001). Analyses of the propensity score-matched subpopulations also confirmed our findings. Risk factors for CSC in SA patients included male gender, age ≤50 years, higher income, presence of heart disease, absence of chronic pulmonary disease, and presence of liver disease. In SA patients, those who had received continuous positive airway pressure titration had a significantly lower incidence rate of CSC than the others. CONCLUSION: Our study revealed a significantly higher incidence rate of CSC in SA patients compared with the control subjects.

Therapeutic Effects of Puerarin Against Anterior Ischemic Optic Neuropathy Through Antiapoptotic and Anti-Inflammatory Actions.

Purpose: This study investigated the therapeutic effects of puerarin (PR) on a rat model of anterior ischemic optic neuropathy (rAION). Methods: The neuroprotective effects of PR on rAION were evaluated using flash visual-evoked potentials (FVEP), retrograde labeling of retinal ganglion cells (RGCs), TUNEL assay of the retina, optical coherence tomography (OCT) images of optic nerve width, and ED1 staining of the optic nerve (ON). The inflammatory response of ON and Akt signaling pathways were analyzed through Western blot. M2 polarization was determined by immunostaining and immunoblotting in ONs. Results: In FVEP analysis, the amplitude of P1-N2 and the RGC density in the PR-treated group were 2.3- and 1.6-fold higher than those in the PBS-treated group, respectively (P < 0.05). The number of apoptotic RGC in the PR-treated group was 2.8-fold lower than that in the PBS-treated group. OCT images demonstrated that PR treatment-reduced ON edema in the acute phase compared to PBS treatment (P < 0.05). Macrophage infiltration was reduced by 5.2-fold by PR treatment compared with the PBS treatment (P < 0.05). PR treatment inhibited the levels of iNOS, IL-1ß, and TNF-α, induced the levels of IL-10, Arg1, and Fizz1 in the rAION model. The levels of p-Akt1 and C/EBPß in the PR-treated group increased by 3.4-fold and 5.89-fold compared with those in the PBS-treated group (P < 0.05). Inhibition of Akt activation reduced the number of M2 macrophage in the PR-treated group (P < 0.05). Conclusions: PR treatment provided the neuroprotective effects in the rAION model, which may lead to new clinical applications.

mTORC2 activation protects retinal ganglion cells via Akt signaling after autophagy induction in traumatic optic nerve injury.

Traumatic optic neuropathy is an injury to the optic nerve that leads to vision loss. Autophagy is vital for cell survival and cell death in central nervous system injury, but the role of autophagy in traumatic optic nerve injury remains uncertain. Optic nerve crush is a robust model of traumatic optic nerve injury. p62 siRNA and rapamycin are autophagy inducers and have different neuroprotective effects in the central nervous system. In this study, p62 and rapamycin induced autophagy, but only p62 siRNA treatment provided a favorable protective effect in visual function and retinal ganglion cell (RGC) survival. Moreover, the number of macrophages at the optic nerve lesion site was lower in the p62-siRNA-treated group than in the other groups. p62 siRNA induced more M2 macrophage polarization than rapamycin did. Rapamycin inhibited both mTORC1 and mTORC2 activation, whereas p62 siRNA inhibited only mTORC1 activation and maintained mTORC2 and Akt activation. Inhibition of mTORC2-induced Akt activation resulted in blood-optic nerve barrier disruption. Combined treatment with rapamycin and the mTORC2 activator SC79 improved RGC survival. Overall, our findings suggest that mTORC2 activation after autophagy induction is necessary for the neuroprotection of RGCs in traumatic optic nerve injury and may lead to new clinical applications.

Pigment epithelium-derived factor from ARPE19 promotes proliferation and inhibits apoptosis of human umbilical mesenchymal stem cells in serum-free medium.

Clinical expansion of mesenchymal stem cells (MSCs) is hampered by the lack of knowledge regarding how to prevent MSC apoptosis and promote their proliferation in serum-free medium. Our in vitro studies demonstrated that human umbilical cord MSCs (HUCMSCs) underwent apoptosis in the serum-free medium. When HUCMSCs were co-cultured with retinal pigment epithelial cells (ARPE19), however, HUCMSCs exhibited normal growth and morphology in serum-free medium. Their colony formation was promoted by the conditioned medium (CM) of ARPE19 cells on Matrigel. Proteomics analysis showed that pigment epithelium-derived factor (PEDF) was one of the most abundant extracellular proteins in the ARPE19 CM, whereas enzyme-linked immunosorbent assay confirmed that large amounts of PEDF was secreted from ARPE19 cells. Adding anti-PEDF-blocking antibodies to the co-culture of HUCMSCs with ARPE19 cells increased apoptosis of HUCMSCs. Conversely, treatment with PEDF significantly reduced apoptosis and increased proliferation of HUCMSCs in serum-free medium. PEDF was further demonstrated to exert this anti-apoptotic effect by inhibiting P53 expression to suppress caspase activation. In vivo studies demonstrated that co-injection of HUCMSCs with ARPE19 cells in immunocompromised NOD-SCID mice also increased survival and decreased apoptosis of HUCMSCs. PEDF also showed no negative effect on the mesoderm differentiation capability of HUCMSCs. In conclusion, this study is the first to demonstrate that PEDF promotes HUCMSC proliferation and protects them from apoptosis by reducing p53 expression in the serum-free medium. This study provides crucial information for clinical-scale expansion of HUCMSCs.

Neuroprotective Effects of Omega-3 Polyunsaturated Fatty Acids in a Rat Model of Anterior Ischemic Optic Neuropathy.

Purpose: The purpose of this study was to investigate the therapeutic effect of omega-3 polyunsaturated fatty acid (ω-3 PUFA) administration in a rat model of anterior ischemic optic neuropathy (rAION). Methods: The level of blood arachidonic acid/eicosapentaenoic acid (AA/EPA) was measured to determine the suggested dosage. The rAION-induced rats were administered fish oil (1 g/day EPA) or phosphate-buffered saline (PBS) by daily gavage for 10 consecutive days to evaluate the neuroprotective effects. Results: Blood fatty acid analysis showed that the AA/EPA ratio was reduced from 17.6 to ≤1.5 after 10 days of fish oil treatment. The retinal ganglion cell (RGC) densities and the P1-N2 amplitude of flash visual-evoked potentials (FVEP) were significantly higher in the ω-3 PUFA-treated group, compared with the PBS-treated group (P < 0.05). The number of apoptotic cells in the RGC layer of the ω-3 PUFA-treated rats was significantly decreased (P < 0.05) compared with that of the PBS-treated rats. Treatment with ω-3 PUFAs reduced the macrophage recruitment at the optic nerve (ON) by 3.17-fold in the rAION model. The M2 macrophage markers, which decrease inflammation, were induced in the ω-3 PUFA-treated group in contrast to the PBS-treated group. In addition, the mRNA levels of tumor necrosis factor-alpha, interleukin-1 beta, and inducible nitric oxide synthase were significantly reduced in the ω-3 PUFA-treated group. Conclusions: The administration of ω-3 PUFAs has neuroprotective effects in rAION, possibly through dual actions of the antiapoptosis of RGCs and anti-inflammation via decreasing inflammatory cell infiltration, as well as the regulation of macrophage polarization to decrease the cytokine-induced injury of the ON.

Cisplatin-Impaired Adipogenic Differentiation of Adipose Mesenchymal Stem Cells1.

Adipose tissue-derived mesenchymal stem cells (ADSCs) are derived from adipose tissue and can be induced in vitro to differentiate into osteoblasts, chondroblasts, myocytes, neurons, and other cell types. Cisplatin is a commonly used chemotherapy drug for cancer patients. However, the effects of cisplatin on ADSCs remain elusive. This study found that a high concentration of cisplatin affects the viability of ADSCs. First, the IC50 concentration of cisplatin was evaluated. Proliferation of ADSCs, as assessed by the XTT method, decreased immediately after treatment with various concentrations of cisplatin. ADSCs maintained mesenchymal stem cell surface markers after cisplatin treatment, as determined by flow cytometry. Upon differentiation by adding specific reagents, a significant decrease in adipogenic differentiation (by Oil red O staining) and osteogenic differentiation (by Alizarin red staining), and significant chondrogenic differentiation (by Alcian blue staining) were found after cisplatin treatment. Quantitative RT-PCR was also used in evaluating expression of specific genes to confirm differentiation. Finally, ADSCs from one donor who had received cisplatin showed significantly decreased adipogenic differentiation but increased osteogenic differentiation compared with ADSCs derived from one healthy donor. In conclusion, cisplatin affects the viability, proliferation, and differentiation of ADSCs both in vitro and in vivo via certain signaling pathways, such as p53 and Fas/FasL. The differentiation abilities of ADSCs should be evaluated before their transplantation for repairing cisplatin-induced tissue damage.

Early applications of granulocyte colony-stimulating factor (G-CSF) can stabilize the blood-optic-nerve barrier and ameliorate inflammation in a rat model of anterior ischemic optic neuropathy (rAION).

Granulocyte colony-stimulating factor (G-CSF) was reported to have a neuroprotective effect in a rat model of anterior ischemic optic neuropathy (rAION model). However, the therapeutic window and anti-inflammatory effects of G-CSF in a rAION model have yet to be elucidated. Thus, this study aimed to determine the therapeutic window of G-CSF and investigate the mechanisms of G-CSF via regulation of optic nerve (ON) inflammation in a rAION model. Rats were treated with G-CSF on day 0, 1, 2 or 7 post-rAION induction for 5 consecutive days, and a control group were treated with phosphate-buffered saline (PBS). Visual function was assessed by flash visual evoked potentials at 4 weeks post-rAION induction. The survival rate and apoptosis of retinal ganglion cells were determined by FluoroGold labeling and TUNEL assay, respectively. ON inflammation was evaluated by staining of ED1 and Iba1, and ON vascular permeability was determined by Evans Blue extravasation. The type of macrophage polarization was evaluated using quantitative real-time PCR (qRT-PCR). The protein levels of TNF-α and IL-1ß were analyzed by western blotting. A therapeutic window during which G-CSF could rescue visual function and retinal ganglion cell survival was demonstrated at day 0 and day 1 post-infarct. Macrophage infiltration was reduced by 3.1- and 1.6-fold by G-CSF treatment starting on day 0 and 1 post-rAION induction, respectively, compared with the PBS-treated group (P<0.05). This was compatible with 3.3- and 1.7-fold reductions in ON vascular permeability after G-CSF treatment compared with PBS treatment (P<0.05). Microglial activation was increased by 3.8- and 3.2-fold in the early (beginning treatment at day 0 or 1) G-CSF-treated group compared with the PBS-treated group (P<0.05). Immediate (within 30 mins of infarct) treatment with G-CSF also induced M2 microglia/macrophage activation. The cytokine levels were lower in the group that received immediate G-CSF treatment compared to those in the later G-CSF treatment group (P<0.05). Early treatment with G-CSF stabilized the blood-ON barrier to reduce macrophage infiltration and induced M2 microglia/macrophage polarization to decrease the expressions of pro-inflammatory cytokines in this rAION model.

Cross-Regulation of Proinflammatory Cytokines by Interleukin-10 and miR-155 in Orientia tsutsugamushi-Infected Human Macrophages Prevents Cytokine Storm.

Scrub typhus is caused by the obligate intracellular bacterium Orientia tsutsugamushi. Macrophages are host cells for its replication and clearance. Severe complications in patients are mainly caused by a cytokine storm resulting from overproduction of proinflammatory cytokines; nevertheless, the molecular mechanism for the occurrence remains obscure. Herein, we investigate the interactive regulation of cytokines and micro-RNA (miR) in human macrophages infected with low and high doses of O. tsutsugamushi. During low dose infection, macrophages produce high levels of IL-10 through extracellular signal-regulated kinase activation, which inhibits proinflammatory cytokine production and facilitates pathogen replication. Increasing levels of pathogen results in reduced levels of IL-10, and macrophages begin to generate high levels of proinflammatory cytokines through NF-κB activation. However, during a high dose infection, macrophages produce high levels of miR-155 to slow the proinflammatory response. The extracellular signal-regulated kinase/IL-10 axis suppresses the NF-κB/tumor necrosis factor alpha axis via activation of signal transducer and activator of transcription 3. Both IL-10 and miR-155 inhibit the NF-κB signaling pathway. Furthermore, IL-10 is a potent inhibitor of miR-155. Patients susceptible to a cytokine storm, peripheral blood mononuclear cells showed significantly lower IL-10 and miR-155 responses to O. tsutsugamushi challenge. Thus, IL-10 and miR-155 operate inhibitory mechanisms to achieve a proper defense mechanism and prevent a cytokine storm.

Primary diffuse large B cell lymphoma of the lacrimal sac.

A 47-year-old man presented with epiphora and a mass around the lacrimal sac in his left eye. Imaging studies revealed a favorable diagnosis of sinusitis-related mucocele. However, the pathological study of the excised tumor demonstrated a diffuse large B cell lymphoma. The patient was consequently referred to the oncology department for further management. Malignant lymphomas of the lacrimal sac are rare and they can mimic mucoceles. As such, they should be included in the list of differential diagnoses for lacrimal sac mass.

Neuroprotective effect of 4-(Phenylsulfanyl)butan-2-one on optic nerve crush model in rats.

This study is to investigate the effect of coral-related compound, 4-(phenylsulfanyl)butan-2-one (4-PSB-2) on optic nerves (ON) and retinal ganglion cells (RGC) in a rat model subjected to ON crush. The ONs of adult male Wistar rat (150-180 g) were crushed by a standardized method. The control eyes received a sham operation. 4-PSB-2 (5 mg/kg in 0.2 mL phosphate-buffered saline) or phosphate-buffered saline (PBS control) was immediately administered after ON crush once by subcutaneous injection. Rats were euthanized at 2 weeks after the crush injury. RGC density was counted by retrograde labeling with FluoroGold (FG) application to the superior colliculus, and visual function was assessed by flash visual evoked potentials (FVEP). TUNEL assay, immunoblotting analysis of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) in the retinas, and immunohistochemistry of ED1 in the ON were evaluated. Two weeks after the insult, the RGC densities in the central and mid-peripheral retinas in ON-crushed, 4-PSB-2-treated rats were significantly higher than that of the corresponding ON-crushed, PBS-treated rats FVEP measurements showed a significantly better preserved latency of the P1 wave in the ON-crushed, 4-PSB-2-treated rats than the ON-crushed, PBS treated rats. TUNEL assays showed fewer TUNEL positive cells in the ON-crushed, 4-PSB-2-treated rats. The number of ED1 positive cells was reduced at the lesion site of the optic nerve in the ON-crushed, 4-PSB-2-treated group. Furthermore, administration of 4-PSB-2 significantly attenuated ON crush insult-stimulated iNOS and COX2 expression in the retinas. These results demonstrated that 4-PSB-2 protects RGCs and helps preserve the visual function in the rat model of optic nerve crush. 4-PSB-2 may work by being anti-apoptotic and by attenuation of the inflammatory responses involving less ED1 positive cells infiltration in ON as well as suppression of iNOS/COX-2 signaling pathway in the retinas to rescue RGCs after ON crush injury.

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.