Adaptive optics two-photon microscopy enables near-diffraction-limited and functional retinal imaging in vivo.

In vivo fundus imaging offers non-invasive access to neuron structures and biochemical processes in the retina. However, optical aberrations of the eye degrade the imaging resolution and prevent visualization of subcellular retinal structures. We developed an adaptive optics two-photon excitation fluorescence microscopy (AO-TPEFM) system to correct ocular aberrations based on a nonlinear fluorescent guide star and achieved subcellular resolution for in vivo fluorescence imaging of the mouse retina. With accurate wavefront sensing and rapid aberration correction, AO-TPEFM permits structural and functional imaging of the mouse retina with submicron resolution. Specifically, simultaneous functional calcium imaging of neuronal somas and dendrites was demonstrated. Moreover, the time-lapse morphological alteration and dynamics of microglia were characterized in a mouse model of retinal disorder. In addition, precise laser axotomy was achieved, and degeneration of retinal nerve fibres was studied. This high-resolution AO-TPEFM is a promising tool for non-invasive retinal imaging and can facilitate the understanding of a variety of eye diseases as well as neurodegenerative disorders in the central nervous system.

Disruption of retinal pigment epithelial cell properties under the exposure of cotinine.

Cigarette smoking is a major risk factor for age-related macular degeneration (AMD), in which progressive retinal pigment epithelial (RPE) cell degeneration is a major pathological change. Nicotine is a major biologically active component in cigarette smoke. It is continuously catabolized into cotinine, which has longer half-life and higher concentration in tissue cells and fluids. Here we hypothesized that continuous exposure of cotinine has more potent effects on human RPE cell properties than nicotine. Human RPE cell line (ARPE-19) was treated continuously with 1-2 µM of nicotine and/or cotinine for 7 days. RPE cells treated with 2 µM cotinine and nicotine-cotinine mixture has lower MTT signals without significant changes in cell apoptosis or integrity. Moreover, RPE cell migration was retarded under cotinine treatments, but not nicotine. Both nicotine and cotinine treatments attenuated the phagocytotic activity of RPE cells. In addition, cotinine and nicotine-cotinine mixture suppressed VEGF and IL-8 expression and upregulated TIMP-2 expression. Expressions of autophagy genes were upregulated by the cotinine treatment, whereas expressions of epithelial-to-mesenchymal transition markers were downregulated. In conclusion, our study, for the first time, demonstrated that cotinine, rather than nicotine, affects the properties of RPE cells in vitro, which could explain the smoking-induced RPE pathology.

Continuous exposure to non-lethal doses of sodium iodate induces retinal pigment epithelial cell dysfunction.

Age-related macular degeneration (AMD), characterized by progressive degeneration of retinal pigment epithelium (RPE), is the major cause of irreversible blindness and visual impairment in elderly population. We previously established a RPE degeneration model using an acute high dose sodium iodate to induce oxidative stress. Here we report findings on a prolonged treatment of low doses of sodium iodate on human RPE cells (ARPE-19). RPE cells were treated continuously with low doses (2-10 mM) of sodium iodate for 5 days. Low doses (2-5 mM) of sodium iodate did not reduce RPE cell viability, which is contrasting to cell apoptosis in 10 mM treatment. These low doses are sufficient to retard RPE cell migration and reduced expression of cell junction protein ZO-1. Phagocytotic activity of RPE cells was attenuated by sodium iodate dose-dependently. Sodium iodate also increased expression of FGF-2, but suppressed expression of IL-8, PDGF, TIMP-2 and VEGF. Furthermore, HTRA1 and epithelial-to-mesenchymal transition marker proteins were downregulated, whereas PERK and LC3B-II proteins were upregulated after sodium iodate treatment. These results suggested that prolonged exposure to non-lethal doses of oxidative stress induces RPE cell dysfunctions that resemble conditions in AMD. This model can be used for future drug/treatment investigation on AMD.

Pluripotent stem cell for modeling neurological diseases.

The availability of human pluriopotent stem cells, embryonic (ESC) and induced pluriopotent (iPSC) stem cells, not only can be a renewable source for investigating the early human development, etiology and progression of different diseases but also recapitulating the disease with the same genomic materials of the patient. In particular, specific neuronal subtypes generated from the patient ESC/iPSCs has become a source for studying disease mechanisms underlying different neurological disorders and allowed drug discovery. In this review, we summarize the recent advances in establishing patient ESC/iPSC to model various neurological diseases. We will also discuss the challenges and limitations of the current disease models and their potential future applications for untangling the unknowns in neurological disorders.