Qiyou Li

Neuroprotective effect of memantine on the retinal ganglion cells of APPswe/PS1ΔE9 mice and its immunomodulatory mechanisms.

Besides the cognitive impairment and degeneration in the brain, vision dysfunction and retina damage are always prevalent in patients with Alzheimers disease (AD). The uncompetitive antagonist of the N-methyl-d-aspartate receptor, memantine (MEM), has been proven to improve the cognition of patients with AD. However, limited information exists regarding the mechanism of neurodegeneration and the possible neuroprotective mechanisms of MEM on the retinas of patients with AD. In the present study, by using APPswe/PS1ΔE9 double transgenic (dtg) mice, we found that MEM rescued the loss of retinal ganglion cells (RGCs), as well as improved visual impairments, including improving the P50 component in pattern electroretinograms and the latency delay of the P2 component in flash visual evoked potentials of APPswe/PS1ΔE9 dtg mice. The activated microglia in the retinas of APPswe/PS1ΔE9 dtg mice were also inhibited by MEM. Additionally, the level of glutamine synthetase expressed by Müller cells within the RGC layer was upregulated in APPswe/PS1ΔE9 dtg mice, which was inhibited by MEM. Simultaneously, MEM also reduced the apoptosis of choline acetyl transferase-immunoreactive cholinergic amacrine cells within the RGC layer of AD mice. Moreover, the phosphorylation level of extracellular regulated protein kinases 1 and 2 was increased in APPswe/PS1ΔE9 dtg mice, which was blocked by MEM treatment. These findings suggest that MEM protects RGCs in the retinas of APPswe/PS1ΔE9 dtg mice by modulating the immune response of microglia and the adapted response of Müller cells, making MEM a potential ophthalmic treatment alternative in patients with AD.

Evaluation of the toxicity of graphene oxide exposure to the eye

Abstract Graphene and its derivatives are the new carbon nanomaterials with the prospect for great applications in electronics, energy storage, biosensors and medicine. However, little is known about the toxicity of graphene or its derivatives in the case of occasional or repeated ocular exposure. We performed in vitro and in vivo studies to evaluate the toxicity of graphene oxide (GO) exposure to the eye. Primary human corneal epithelium cells (hCorECs) and human conjunctiva epithelium cells (hConECs) were exposed to GO (12.5–100 μg/mL). Acute GO exposure (2 h) did not induce cytotoxicity to hCorECs. However, short-term GO exposure (24 h) exerted significant cytotoxicity to hCorECs and hConECs with increased intracellular reactive oxygen species (ROS). Glutathione (GSH) reduced the GO-induced cytotoxicity. We further performed acute eye irritation tests in albino rabbits according to the Organization for Economic Cooperation and Development (OECD) guidelines, and the rabbits did not exhibit corneal opacity, conjunctival redness, abnormality of the iris, or chemosis at any time point after the instillation of 100 μg/mL of GO. However, 5-day repeated GO exposure (50 and 100 μg/mL) caused reversible mild corneal opacity, conjunctival redness and corneal epithelium damage to Sprague-Dawley rats, which was also alleviated by GSH. Therefore, our study suggests that GO-induced time- and dose-dependent cytotoxicity to hCorECs and hConECs via oxidative stress. Occasional GO exposure did not cause acute eye irritation; short-term repeated GO exposure generally resulted in reversible damage to the eye via oxidative stress, which may be alleviated by the antioxidant GSH.

Neural stem cells transplanted to the subretinal space of rd1 mice delay retinal degeneration by suppressing microglia activation

BACKGROUND AIMS Retinal degeneration (RD) is an inherited eye disease characterized by irreversible photoreceptor loss. Conventionally, the activation of the resident microglia is secondary to the disease. Stem cell-based therapy has recently made rapid progress in treating RD. Although it has been demonstrated that the effect of stem cell therapy may include immunomodulation, the specific mechanisms have not been clarified. METHODS Immunocytochemistry, terminal deoxynucleotidyl transferase UTP nick end labelling (TUNEL) assay and Western blot were used to analyze the microglia activation and photoreceptor apoptosis in the retina of rd1 mice. GFP-C17.2 neural stem cells (NSCs) were transplanted into the subretinal space to study the immunomodulatory and neuroprotective effects. The transwell co-culture of BV2 cells with GFP-C17.2 was performed to study the proliferation, apoptosis and secretion levels of inflammatory factors. Real time-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were performed to explore the gene and protein level of factors secreted by NSCs and microglia. RESULTS TUNEL-positive cells were primarily distributed in the inner nuclear layer (INL) of rd1 mice on P8d, appeared in the outer nuclear layer (ONL) on P10d and peaked on P14d. Meanwhile, microglia migrated to the ONL and reached the maximum level, accompanied by the changes in the levels of fractalkine and its unique receptor CX3CR1 protein. After transplantation of NSCs on P7d into the subretinal space of rd1 mice, the activated microglia were inhibited and the degeneration of ONL was delayed. In addition, microglia activation was suppressed by co-cultured NSCs in vitro. The gene and protein level of tissue inhibitor of metalloproteinase (TIMP1) in NSCs was elevated, whereas that of matrix metalloproteinase (MMP9) in BV2 microglia was markedly suppressed in this co-culture system. CONCLUSIONS Transplanted NSCs in the retina exerted immunomodulatory effects on microglia, thus delaying the degeneration of photoreceptors.

Sodium Iodate Influences the Apoptosis, Proliferation and Differentiation Potential of Radial Glial Cells In Vitro

Background/Aims: Sodium iodate (NaIO3)-induced acute retinal injury is typically used as an animal model for degenerative retinal disease; however, how NaIO3 influences the apoptosis, proliferation and differentiation of endogenous retinal stem cells is unknown. Methods: We exposed a radial glial cells (RGCs) line (L2.3) to different NaIO3 concentrations and determined the influence of NaIO3 on apoptosis, proliferation, and differentiation using flow cytometry and immunofluorescence assays. We used a real-time polymerase chain reaction assay to analyze the levels of mRNAs encoding GSK-3β, AXIN2, β-catenin, TGF-β1, SMAD2, SMAD3, NOG (Noggin), and BMP4. Results: Cell density decreased dramatically as a function of the NaIO3 dose. NaIO3 increased apoptosis, inhibited mitosis, proliferation, and the Wnt/β-catenin pathway. CHIR99021 (Wnt agonist) treatment efficiently reversed the effects of NaIO3 on the apoptosis and proliferation of RGCs. The number of neuronal class III β-tubulin-positive cells decreased markedly, whereas that of glial fibrillary acidic protein-positive cells increased significantly when RGCs were exposed to NaIO3. During differentiation, the Nog mRNA level decreased and transforming growth factor-β1 (Tgf-β1) and Smad2/3 mRNA levels increased significantly when RGCs were exposed to NaIO3. Conclusion: NaIO3 increased apoptosis, influenced the proliferation of RGCs and drove them toward astrocytic differentiation, likely through inhibition of the Wnt/β-catenin and noggin pathways and activation of the TGF-β1/SMAD2/3 pathway.

Intermittent high oxygen influences the formation of neural retinal tissue from human embryonic stem cells.

The vertebrate retina is a highly multilayered nervous tissue with a large diversity of cellular components. With the development of stem cell technologies, human retinas can be generated in three-dimensional (3-D) culture in vitro. However, understanding the factors modulating key productive processes and the way that they influence development are far from clear. Oxygen, as the most essential element participating in metabolism, is a critical factor regulating organic development. In this study, using 3-D culture of human stem cells, we examined the effect of intermittent high oxygen treatment (40% O2) on the formation and cellular behavior of neural retinas (NR) in the embryonic body (EB). The volume of EB and number of proliferating cells increased significantly under 40% O2 on day 38, 50, and 62. Additionally, the ratio of PAX6+ cells within NR was significantly increased. The neural rosettes could only develop with correct apical-basal polarity under 40% O2. In addition, the generation, migration and maturation of retinal ganglion cells were enhanced under 40% O2. All of these results illustrated that 40% O2 strengthened the formation of NR in EB with characteristics similar to the in vivo state, suggesting that the hyperoxic state facilitated the retinal development in vitro.

Features specific to retinal pigment epithelium cells derived from three-dimensional human embryonic stem cell cultures — a new donor for cell therapy

Retinal pigment epithelium (RPE) transplantation is a particularly promising treatment of retinal degenerative diseases affecting RPE-photoreceptor complex. Embryonic stem cells (ESCs) provide an abundant donor source for RPE transplantation. Herein, we studied the time-course characteristics of RPE cells derived from three-dimensional human ESCs cultures (3D-RPE). We showed that 3D-RPE cells possessed morphology, ultrastructure, gene expression profile, and functions of authentic RPE. As differentiation proceeded, 3D-RPE cells could mature gradually with decreasing proliferation but increasing functions. Besides, 3D-RPE cells could form polarized monolayer with functional tight junction and gap junction. When grafted into the subretinal space of Royal College of Surgeons rats, 3D-RPE cells were safe and efficient to rescue retinal degeneration. This study showed that 3D-RPE cells were a new donor for cell therapy of retinal degenerative diseases.

Evaluating the toxicity of silicon dioxide nanoparticles on neural stem cells using RNA-Seq

Neural stem cells are characterized by self-renewal and multipotency, and a capacity to regenerate in response to brain injury or neurodegenerative disease. Silicon dioxide nanoparticles (SiO2 NPs) are novel materials, which enable the delivery of specific payloads to stem cells; for example, genes or proteins, to enable cell-fate manipulation, or tracer materials, to enable in vivo tracing. However, little is known about the dose-dependent cytotoxicity of SiO2 NPs, and how exposure to SiO2 NPs changes mRNA expression profiles in neural stem cells. In this study, a mouse C17.2 neural stem cell line was treated with 90 nm monodisperse fluorescein isothiocyanate-SiO2 NPs at 0, 100, 200 and 400 μg mL−1 for 48 hours. Internalization of SiO2 NPs was observed in C17.2 cells in a dose-dependent manner. SiO2 NP exposure induced apoptosis and inhibited cell proliferation in the C17.2 cell line at dosage levels of 200 μg mL−1 and above. Microscopically, mitochondrial swelling and cristae fracture were observed. Furthermore, next generation RNA sequencing (RNA-Seq) indicated that high-dose SiO2 NP exposure specifically inhibited transcription of glutathione-S-transferase (GST) genes, including GSTM1, GSTM7 and GSTT1. These results suggest that application of high-dose SiO2 NPs to the nervous system may cause neurotoxicity, induce apoptosis and reduce neural stem cell proliferation by inhibiting GST gene expression.

Bone Marrow CD133 Stem Cells Ameliorate Visual Dysfunction in Streptozotocin-induced Diabetic Mice with Early Diabetic Retinopathy.

Diabetic retinopathy (DR), one of the leading causes of vision loss worldwide, is characterized by neurovascular disorders. Emerging evidence has demonstrated retinal neurodegeneration in the early pathogenesis of DR, and no treatment has been developed to prevent the early neurodegenerative changes that precede detectable microvascular disorders. Bone marrow CD133+ stem cells with revascularization properties exhibit neuroregenerative potential. However, whether CD133+ cells can ameliorate the neurodegeneration at the early stage of DR remains unclear. In this study, mouse bone marrow CD133+ stem cells were immunomagnetically isolated and analyzed for the phenotypic characteristics, capacity for neural differentiation, and gene expression of neurotrophic factors. After being labeled with enhanced green fluorescent protein, CD133+ cells were intravitreally transplanted into streptozotocin (STZ)-induced diabetic mice to assess the outcomes of visual function and retina structure and the mechanism underlying the therapeutic effect. We found that CD133+ cells co-expressed typical hematopoietic/endothelial stem/progenitor phenotypes, could differentiate to neural lineage cells, and expressed genes of robust neurotrophic factors in vitro. Functional analysis demonstrated that the transplantation of CD133+ cells prevented visual dysfunction for 56 days. Histological analysis confirmed such a functional improvement and showed that transplanted CD133+ cells survived, migrated into the inner retina (IR) over time and preserved IR degeneration, including retina ganglion cells (RGCs) and rod-on bipolar cells. In addition, a subset of transplanted CD133+ cells in the ganglion cell layer differentiated to express RGC markers in STZ-induced diabetic retina. Moreover, transplanted CD133+ cells expressed brain-derived neurotrophic factors (BDNFs) in vivo and increased the BDNF level in STZ-induced diabetic retina to support the survival of retinal cells. Based on these findings, we suggest that transplantation of bone marrow CD133+ stem cells represents a novel approach to ameliorate visual dysfunction and the underlying IR neurodegeneration at the early stage of DR.

Transplanted olfactory ensheathing cells restore retinal function in a rat model of light-induced retinal damage by inhibiting oxidative stress

There is still not an effective treatment for continuous retinal light exposure and subsequent photoreceptor degeneration. Olfactory ensheathing cell (OEC) transplantation has been shown to be neuroprotective in spinal cord, and optic nerve injury and retinitis pigmentosa. However, whether OECs protect rat photoreceptors against light-induced damage and how this may work is unclear. Thus, to elucidate this mechanism, purified rat OECs were grafted into the subretinal space of a Long-Evans rat model with light-induced photoreceptor damage. Light exposure decreased a- and b- wave amplitudes and outer nuclear layer (ONL) thickness, whereas the ONL of rats exposed to light for 24 h after having received OEC transplants in their subretinal space was thicker than the PBS control and untreated groups. A- and b- wave amplitudes from electroretinogram of OEC-transplanted rats were maintained until 8 weeks post OEC transplantation. Also, transplanted OECs inhibited formation of reactive oxygen species in retinas exposed to light. In vitro experiments showed that OECs had more total antioxidant capacity in a co-cultured 661W photoreceptor cell line, and cells were protected from damage induced by hydrogen-peroxide. Thus, transplanted OECs preserved retinal structure and function in a rat model of light-induced degeneration by suppressing retinal oxidative stress reactions.

Investigating oxidation state-induced toxicity of PEGylated graphene oxide in ocular tissue using gene expression profiles

Abstract Graphene and its derivatives are widely used for a variety of industrial, biomedical, and environmental applications. However, the potential harm caused by exposure of the eyes to graphene-based nanomaterials is scarce. Given the potential for these materials to be used in multiple applications, there is a pressing need to evaluate their ocular toxicity, and understand the relationships between their physico-chemical properties and the resulting toxicity. In this study, the toxicity of PEGylated graphene oxide (PEG-GO) with differing oxidation levels and/or surface charges (positive, negative and neutral charge) was evaluated using two in-vitro models of the eye: primary human corneal epithelial cells and human retinal capillary endothelial cells. The results showed that oxidation level, but not surface charge, had a pivotal effect on the toxicity of graphene-based nanomaterials. Typically, PEG-GO sample with a higher oxidation level caused more serious cytotoxicity than those with a lower oxidation level. Furthermore, by analysis of global gene expression profiles, we found that the foremost cellular response to PEG-GO sample with a high oxidation level was the oxidative stress response. Next, via exploring the underlying molecular mechanism of oxidative stress-induced cytotoxicity, we showed that PEG-GO sample with a high degree of oxidation induced reactive oxygen species (ROS) via NDUFB9-mediated biological pathway. This work has significant implications for design of safe graphene-based nanomaterials for biomedical applications.