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1.  Ultrasound Characterization of Middle Ear Effusion 
Purpose
To further enhance and assess the ability to characterize middle ear effusion (MEE) using non-invasive ultrasound technology.
Materials and Methods
This is a prospective unblinded comparison study. Fifty-six children between the ages of 6 months and 17 years scheduled to undergo bilateral myringotomy with pressure equalization tube placement were enrolled. With the child anesthetized, the probe was placed into the external ear canal after sterile water was inserted. Ultrasound recordings of middle ear contents were analyzed by computer algorithm. Middle ear fluid was collected during myringotomy and analyzed for bacterial culture and viscosity.
Results
Ultrasound waveforms yielded a computer algorithm interpretation of middle ear contents in 66% of ears tested. When a result was obtained, the sensitivity and specificity for successfully characterizing middle ear fluid content as either void of fluid, thick fluid (mucoid), or thin fluid (serous or purulent) was at least 94%. Mucoid effusions had higher measured viscosity values (P=0.002). Viscosity measures were compared to culture result, and those with low viscosity (thin consistency) had a higher likelihood of having a positive culture (P=0.048).
Conclusion
The device sensitivity and specificity for fluid detection was 94% or greater among interpretable waveforms (66% of those tested). Although this technology provides important information of the middle ear effusion presence and characteristic, further technological improvements are needed.
doi:10.1016/j.amjoto.2012.08.005
PMCID: PMC3518560  PMID: 23084430
2.  Systems pharmacology identifies drug targets for Stargardt disease–associated retinal degeneration 
The Journal of Clinical Investigation  2013;123(12):5119-5134.
A systems pharmacological approach that capitalizes on the characterization of intracellular signaling networks can transform our understanding of human diseases and lead to therapy development. Here, we applied this strategy to identify pharmacological targets for the treatment of Stargardt disease, a severe juvenile form of macular degeneration. Diverse GPCRs have previously been implicated in neuronal cell survival, and crosstalk between GPCR signaling pathways represents an unexplored avenue for pharmacological intervention. We focused on this receptor family for potential therapeutic interventions in macular disease. Complete transcriptomes of mouse and human samples were analyzed to assess the expression of GPCRs in the retina. Focusing on adrenergic (AR) and serotonin (5-HT) receptors, we found that adrenoceptor α 2C (Adra2c) and serotonin receptor 2a (Htr2a) were the most highly expressed. Using a mouse model of Stargardt disease, we found that pharmacological interventions that targeted both GPCR signaling pathways and adenylate cyclases (ACs) improved photoreceptor cell survival, preserved photoreceptor function, and attenuated the accumulation of pathological fluorescent deposits in the retina. These findings demonstrate a strategy for the identification of new drug candidates and FDA-approved drugs for the treatment of monogenic and complex diseases.
doi:10.1172/JCI69076
PMCID: PMC3859412  PMID: 24231350
3.  In vivo two-photon imaging of the mouse retina 
Biomedical Optics Express  2013;4(8):1285-1293.
Though in vivo two-photon imaging has been demonstrated in non-human primates, improvements in the signal-to-noise ratio (SNR) would greatly improve its scientific utility. In this study, extrinsic fluorophores, expressed in otherwise transparent retinal ganglion cells, were imaged in the living mouse eye using a two-photon fluorescence adaptive optics scanning laser ophthalmoscope. We recorded two orders of magnitude greater signal levels from extrinsically labeled cells relative to previous work done in two-photon autofluorescence imaging of primates. Features as small as single dendrites in various layers of the retina could be resolved and predictions are made about the feasibility of measuring functional response from cells. In the future, two-photon imaging in the intact eye may allow us to monitor the function of retinal cell classes with infrared light that minimally excites the visual response.
doi:10.1364/BOE.4.001285
PMCID: PMC3756587  PMID: 24009992
(330.4460) Ophthalmic optics and devices; (180.4315) Nonlinear microscopy; (170.0110) Imaging systems
4.  Retinal cone and rod photoreceptor cells exhibit differential susceptibility to light–induced damage 
Journal of Neurochemistry  2012;121(1):146-156.
All-trans-retinal and its condensation-products can cause retinal degeneration in a light–dependent manner and contribute to the pathogenesis of human macular diseases such as Stargardt’s disease and age–related macular degeneration (AMD). Although these toxic retinoid by–products originate from rod and cone photoreceptor cells, the contribution of each cell type to light–induced retinal degeneration is unknown. Here the primary objective was to learn whether rods or cones are more susceptible to light–induced, all–trans–retinal–mediated damage. Previously, we reported that mice lacking enzymes that clear all–trans–retinal from the retina, ATP–binding cassette transporter 4 (ABCA4) and retinol dehydrogenase 8 (RDH8), manifested light-induced retinal dystrophy. We first examined early-stage-AMD patients and found retinal degenerative changes in rod-rich rather than cone-rich regions of the macula. We then evaluated transgenic mice with rod–only and cone–like–only retinas in addition to progenies of such mice inbred with Rdh8−/− Abca4−/− mice. Of all these strains, Rdh8−/− Abca4−/− mice with a mixed rod–cone population showed the most severe retinal degeneration under regular cyclic light conditions. Intense light exposure induced acute retinal damage in Rdh8−/− Abca4−/− and rod–only mice but not cone–like–only mice. These findings suggest that progression of retinal degeneration in Rdh8-/- Abca4-/- mice is affected by differential vulnerability of rods and cones to light.
doi:10.1111/j.1471-4159.2012.07647.x
PMCID: PMC3303932  PMID: 22220722
visual cycle; photoreceptor; retinoid; retina; Stargardt’s disease; age-related macular degeneration
5.  Imaging of protein crystals with two–photon microscopy† 
Biochemistry  2012;51(8):1625-1637.
Second–order non–linear optical imaging of chiral crystals (SONICC), that portrays second harmonic generation (SHG) by non–centrosymmetric crystals, is emerging as a powerful imaging technique for protein crystals in media opaque to visible light because of its high signal–to–noise ratio. Here we report the incorporation of both SONICC and two–photon excited fluorescence (TPEF) into one imaging system that allows visualization of crystals as small as ~10 μm in their longest dimension. Using this system, we then documented an inverse correlation between the level of symmetry in examined crystals and the intensity of their SHG. Moreover, because of blue-green TPEF exhibited by most tested protein crystals, we also could identify and image SHG–silent protein crystals. Our experimental data suggests that the TPEF in protein crystals is mainly caused by the oxidation of tryptophan residues. Additionally, we found that unspecific fluorescent dyes are able to bind to lysozyme crystals and enhance their detection by TPFE. We finally confirmed that the observed fluorescence was generated by a two-photon rather than a three-photon process. The capability for imaging small protein crystals in turbid or opaque media with non–damaging infrared light in a single system, makes the combination of SHG and intrinsic visible TPEF a powerful tool for non–destructive protein crystal identification and characterization during crystallization trials.
doi:10.1021/bi201682q
PMCID: PMC3293215  PMID: 22324807
6.  Primary amines protect against retinal degeneration in mouse models of retinopathies 
Nature chemical biology  2011;8(2):170-178.
Vertebrate vision is initiated by photoisomerization of the visual pigment chromophore, 11-cis-retinal, and is maintained by continuous regeneration of this retinoid through a series of reactions termed the retinoid cycle. However, toxic side reaction products, especially those involving reactive aldehyde groups of the photoisomered product, all-trans-retinal, can cause severe retinal pathology. Here we lowered peak concentrations of free all-trans-retinal with primary amine-containing FDA-approved drugs that did not inhibit chromophore regeneration in mouse models of retinal degeneration. Schiff base adducts between all-trans-retinal and these amines were identified by mass spectrometry. Adducts were observed in mouse eyes only when an experimental drug protected the retina from degeneration in both short-term and long-term treatment experiments. This study demonstrates a molecular basis of all-trans-retinal-induced retinal pathology and identifies an assemblage of FDA-approved compounds with protective effects against this pathology in a mouse model that displays features of Stargardt’s and age-related retinal degeneration.
doi:10.1038/nchembio.759
PMCID: PMC3518042  PMID: 22198730
Photoreceptor cells; A2E; RPE; retina; Stargardt’s disease; age-related macular degeneration; retinal degeneration; retinal condensation products
7.  Noninvasive multi–photon fluorescence microscopy resolves retinol and retinal–condensation products in mouse eyes 
Nature medicine  2010;16(12):1444-1449.
Multi–photon excitation fluorescence microscopy (MPM) can image certain molecular processes in vivo. In the eye, fluorescent retinyl esters in sub–cellular structures called retinosomes mediate regeneration of the visual chromophore, 11–cis–retinal, by the visual cycle. But harmful fluorescent condensation products were also identified previously. We report that in wild type mice, excitation with λ ~730 nm identified retinosomes in the retinal pigment epithelium, whereas excitation with λ ~910 nm revealed at least one additional retinal fluorophore. The latter fluorescence was absent in eyes of genetically modified mice lacking a functional visual cycle, but accentuated in eyes of older WT mice and mice with defective clearance of all–trans–retinal, an intermediate in the visual cycle. MPM, a noninvasive imaging modality that facilitates concurrent monitoring of retinosomes along with potentially harmful products in aging eyes, has the potential to detect early molecular changes due to age–related macular degeneration and other defects in retinoid metabolism.
doi:10.1038/nm.2260
PMCID: PMC3057900  PMID: 21076393
Photoreceptor cells; fluorescence microscopy; retinosomes; retinyl esters; A2E; RPE; retina; two–photon fluorescence excitation microscopy; three–photon fluorescence excitation microscopy
8.  Images of photoreceptors in living primate eyes using adaptive optics two-photon ophthalmoscopy 
Biomedical Optics Express  2010;2(1):139-148.
In vivo two-photon imaging through the pupil of the primate eye has the potential to become a useful tool for functional imaging of the retina. Two-photon excited fluorescence images of the macaque cone mosaic were obtained using a fluorescence adaptive optics scanning laser ophthalmoscope, overcoming the challenges of a low numerical aperture, imperfect optics of the eye, high required light levels, and eye motion. Although the specific fluorophores are as yet unknown, strong in vivo intrinsic fluorescence allowed images of the cone mosaic. Imaging intact ex vivo retina revealed that the strongest two-photon excited fluorescence signal comes from the cone inner segments. The fluorescence response increased following light stimulation, which could provide a functional measure of the effects of light on photoreceptors.
doi:10.1364/BOE.2.000139
PMCID: PMC3028489  PMID: 21326644
(010.1080) adaptive optics; (330.4460) Ophthalmic optics and devices; (330.5310) Vision – photoreceptors; (330.7327) Visual optics, ophthalmic instrumentation

Results 1-8 (8)