Summary Stem cell-based transplantation therapies offer hope for currently untreatable retinal degenerations; however, preclinical progress has been largely confined to rodent models. Here, we describe an experimental platform for accelerating photoreceptor replacement therapy in the nonhuman primate, which has a visual system much more similar to the human. We deployed fluorescence adaptive optics scanning light ophthalmoscopy (FAOSLO) to noninvasively track transplanted photoreceptor precursors over time at cellular resolution in the living macaque. Fluorescently labeled photoreceptors generated from a CRX +/tdTomato human embryonic stem cell (hESC) reporter line were delivered subretinally to macaques with normal retinas and following selective ablation of host photoreceptors using an ultrafast laser. The fluorescent reporter together with FAOSLO allowed transplanted photoreceptor precursor survival, migration, and neurite formation to be monitored over time in vivo . Histological examination suggested migration of photoreceptor precursors to the outer plexiform layer and potential synapse formation in ablated areas in the macaque eye.

Diabetic retinopathy is a microvascular complication of diabetes that can lead to blindness. In the present study, we aimed to determine the nature of diabetes-induced, highly localized biochemical changes in the neuroretina at the onset of diabetes. High-resolution synchrotron Fourier transform infrared (s-FTIR) wide field microscopy coupled with multivariate analysis (PCA-LDA) was employed to identify biomarkers of diabetic retinopathy with spatial resolution at the cellular level. We compared retinal tissue prepared from 6-week-old Ins2Akita/+ heterozygous (Akita/+, N=6; a model of diabetes) male mice compared with the wild-type (control, N=6) mice. Male Akita/+ mice become diabetic at 4-weeks of age. Significant differences (P<0.001) in the presence of biomarkers associated with diabetes and segregation of spectra was achieved. Differentiating IR bands attributed to nucleic acids (964, 1051, 1087, 1226 and 1710 cm−1), proteins (1662, 1608 cm−1) and fatty acids (2854, 2923, 2956 and 3012 cm−1) were observed between the Akita/+ and the WT samples. Comparison between distinctive layers of the retina, namely the photoreceptor retinal layer (PRL), outer plexiform layer (OPL), inner nuclear layer (INL) and inner plexiform layer (IPL) suggested that the photoreceptor layer is the most susceptible layer to the oxidative stress in short-term diabetes. Spatially-resolved chemical images indicated heterogeneities and oxidative-stress induced alterations in the diabetic retina tissue morphology compared with WT retina. In this study, the spectral biomarkers and the spatial biochemical alterations in the diabetic retina and in specific layers were identified for the first time. We believe that the conclusions drawn from these studies will help to bridge the gap in our understanding of the molecular and cellular mechanism that contribute to pathobiology of diabetic retinopathy.

Background: Roles of lipids and carbohydrates in sensory transduction are understudied. Results: Localization of lipids and carbohydrates in neuronal subsets is identified and associated with enrichment of endoplasmic reticulum; lipids increased in nociceptors during inflammation but not in TRPA1-deficient neurons. Conclusion: Chemical morphology varies in sensory subpopulations; TRPA1-mediated lipid increases may underlie mechanical sensitization with inflammation. Significance: Lipids and carbohydrates may mediate inflammatory pain.

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Infrared (IR) spectromicroscopy, or chemical imaging, is an evolving technique that is poised to make significant contributions in the fields of biology and medicine. Recent developments in sources, detectors, measurement techniques and speciman holders have now made diffraction-limited Fourier transform infrared (FTIR) imaging of cellular chemistry in living cells a reality. The availability of bright, broadband IR sources and large area, pixelated detectors facilitate live cell imaging, which requires rapid measurements using non-destructive probes. In this work, we review advances in the field of FTIR spectromicroscopy that have contributed to live-cell two and three-dimensional IR imaging, and discuss several key examples that highlight the utility of this technique for studying the structure and chemistry of living cells.