ADIPOR1 is essential for vision and its RPE expression is lost in the Mfrp

The knockout (KO) of the adiponectin receptor 1 (AdipoR1) gene causes retinal degeneration. Here we report that ADIPOR1 protein is primarily found in the eye and brain with little expression in other tissues. Further analysis of AdipoR1 KO mice revealed that these animals exhibit early visual system abnormalities and are depleted of RHODOPSIN prior to pronounced photoreceptor death. A KO of AdipoR1 post-development either in photoreceptors or the retinal pigment epithelium (RPE) resulted in decreased expression of retinal proteins, establishing a role for ADIPOR1 in supporting vision in adulthood. Subsequent analysis of the Mfr

Tracking adaptive optics scanning laser ophthalmoscope

ABSTRACT Active image stabilization for an adaptive optics scanning laser ophthalmoscope (AOSLO) was developed and tested in human subjects. The tracking device, a high speed, closed-loop optical servo which uses retinal features as tracking target, is separate from AOSLO optical path. The tracking system and AOSLO beams are combined via a dichroic beam splitter in front of the eye. The primary tracking system galvanometer mirrors follow the motion of the eye. The AOSLO raster is stabilized by a secondary set of galvanometer mirrors in the AOSLO optical train which are "slaved" to the primary mirrors with fixed scaling factors to match the angular gains of the optical systems. The AO system (at 830 nm) uses a MEMS-based deformable mirror (Boston Micromachines Inc.) for wave-front correction. The third generation retinal tracking system achieves a bandwidth of greater than 1 kHz allowing acquisition of stabilized AO images with an accuracy of <10 µm. However, such high tracking bandwidth, required for tracking saccades, results in finite tracking position noise which is evident in AOSLO images. By means of filtering algorithms, the AOSLO raster is made to follow the eye accurately with reduced tracking noise artifacts. The system design includes simultaneous presentation of non-AO, wide-field (~40 deg) live reference image captured with a line scanning laser ophthalmoscope (LSLO) typically operating from 900 to 940 nm. High-magnification (1-2 deg) AOSLO retinal scans easily positioned on the retina in a drag-and-drop manner. Normal adult human volunteers were tested to optimize the tracking instrumentation and to characterize AOSLO imaging performance. Automatic blink detection and tracking re-lock, enabling reacquisition without operator intervention, were also tested. The tracking-enhanced AOSLO may become a useful tool for eye research and for early detection and treatment of retinal diseases

Community as an Institutional Learning Goal at the Unversity of Dayton

This working paper summarizes the work of the Habits of Inquiry and Reflection Community Fellows. It considers the meaning of community both in UD’s historic mission and in the ways it is practiced at UD now; identifies obstacles and failures; and offers recommendations for advancing community as a learning goal at UD

Biological Applications of Confocal Fluorescence Polarization Microscopy

Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of metatetrahydroxyphenylchlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proofof- principle studies are performed in a model system consisting of fluorescently labeled bovine serum albumin attached to sepharose beads. The action of trypsin and proteinase K on the albumin is monitored to demonstrate validity of the technique. Images of the processing of the albumin in J774 murine macrophages are also presented indicating large intercellular differences in enzyme activity. Future directions for the technique are also presented, including the design of enzyme probes specific for prostate specific antigen based on fluorescently-labeled dendrimers. A technique for enzyme imaging based on extracellular autofluorescence is also proposed

Light Scattering from Intact Cells Reports Oxidative-Stress-Induced Mitochondrial Swelling

Angularly resolved light scattering measurements were performed on suspensions of EMT6 cells and on mitochondria isolated from rabbit liver. Mie theory analysis of the scattering from intact cells indicated that mitochondrial-sized organelles dominated scattering in the range 5–90°. This interpretation was supported by the analysis of scattering from isolated mitochondria. Intact cells were subjected to oxidative stress by photodynamic insult. After 3 h of incubation in the heme precursor aminolevulinic acid hexylester, EMT6 cells accumulated abundant protoporphyrin IX, an endogenous photosensitizer formed in mitochondria. Irradiation of aminolevulinic acid/protoporphyrin IX-sensitized cells with 10 J cm(−2) of 514 nm light led to pronounced changes in angularly resolved light scattering consistent with mitochondrial swelling. Electron microscopy of similarly treated EMT6 cell monolayers showed significant changes in mitochondrial morphology, which included distension of the outer unit membrane and bloating of the internal mitochondrial compartment. Informed by these electron microscopy results, we implemented a coated sphere model to interpret the scattering from intact cells subjected to oxidative stress. The coated sphere interpretation was compatible with the scattering measurements from these cells, whereas simpler Mie theory models based on homogenous swelling were dramatically unsuccessful. Thus, in this system, angularly resolved light scattering reports oxidative-stress-induced changes in mitochondrial morphology

Susceptibility of Candida Species to Photodynamic Effects of Photofrin

The in vitro susceptibility of pathogenic Candida species to the photodynamic effects of the clinically approved photosensitizing agent Photofrin was examined. Internalization of Photofrin by Candida was confirmed by confocal fluorescence microscopy, and the degree of uptake was dependent on incubation concentration. Uptake of Photofrin by Candida and subsequent sensitivity to irradiation was influenced by culture conditions. Photofrin uptake was poor in C. albicans blastoconidia grown in nutrient broth. However, conversion of blastoconidia to filamentous forms by incubation in defined tissue culture medium resulted in substantial Photofrin uptake. Under conditions where Photofrin was effectively taken up by Candida, irradiated organisms were damaged in a drug dose- and light-dependent manner. Uptake of Photofrin was not inhibited by azide, indicating that the mechanism of uptake was not dependent on energy provided via electron transport. Fungal damage induced by Photofrin-mediated photodynamic therapy (PDT) was determined by evaluation of metabolic activity after irradiation. A strain of C. glabrata took up Photofrin poorly and was resistant to killing after irradiation. In contrast, two different strains of C. albicans displayed comparable levels of sensitivity to PDT. Furthermore, a reference strain of C. krusei that is relatively resistant to fluconazole compared to C. albicans was equally sensitive to C. albicans at Photofrin concentrations of ≥3 μg/ml. The results indicate that photodynamic therapy may be a useful adjunct or alternative to current anti-Candida therapeutic modalities, particularly for superficial infections on surfaces amenable to illumination

Glaucoma - Next Generation Therapeutics: Impossible to Possible

The future of next generation therapeutics for glaucoma is strong. The recent approval of two novel intraocular pressure (IOP)-lowering drugs with distinct mechanisms of action is the first in over 20 years. However, these are still being administered as topical drops. Efforts are underway to increase patient compliance and greater therapeutic benefits with the development of sustained delivery technologies. Furthermore, innovations from biologics- and gene therapy-based therapeutics are being developed in the context of disease modification, which are expected to lead to more permanent therapies for patients. Neuroprotection, including the preservation of retinal ganglion cells (RGCs) and optic nerve is another area that is actively being explored for therapeutic options. With improvements in imaging technologies and determination of new surrogate clinical endpoints, the therapeutic potential for translation of neuroprotectants is coming close to clinical realization. This review summarizes the aforementioned topics and other related aspects

Nonclinical safety evaluation of scAAV8-RLBP1 (CPK850) for treatment of RLBP1 retinitis pigmentosa

Retinitis pigmentosa is a form of retinal degeneration usually caused by genetic mutations affecting key functional proteins. We have previously demonstrated efficacy in a mouse model of RLBP1 deficiency with a self-complementary AAV8 vector carrying the gene for human RLBP1 under control of a short RLBP1 promoter (CPK850)1. In this communication, we describe the nonclinical safety profile of this construct as well as updated efficacy data in the intended clinical formulation. In Rlbp1-/- mice dosed at a range of CPK850 levels, a minimum efficacious dose of 3x107 vg in a volume of 1ul was observed. For safety assessment in these and Rlbp1+/+ mice, optical coherence tomography (OCT) and histopathological analysis indicated retinal thinning that appeared to be dose-dependent for both Rlbp1 genotypes with no qualitative difference noted between Rlbp1+/+ and Rlbp1-/- mice. In a non-human primate study, RLBP1 mRNA expression was detected and dose dependent intraocular inflammation and retinal thinning were observed. Inflammation resolved slowly over time, and did not appear to be exacerbated in the presence of anti-AAV8 antibodies. Biodistribution was evaluated in rats as well as from satellite animals in the non-human primate study. The vector was largely detected in ocular tissues as well as at low levels in the optic nerve, superior colliculus and lateral geniculate nucleus with limited distribution outside of these tissues. These data suggest that an initial subretinal dose of ~3x107 vg/uL CPK850 could safely be used in clinical trials