Cholesterol homeostasis in the retina, a sensory organ in the back of the eye, has been studied in mice but not hamsters, despite the latter being more similar to humans than mice with respect to their whole-body cholesterol maintenance. The goal of this study was to begin to assess hamster retina and conduct initial interspecies comparisons. First, young (3-month old) and mature (6-month old) Syrian (golden) hamsters were compared with 3- and 6-month old mice for ocular biometrics and retinal appearance on optical coherence tomography and fluorescein angiography. Of the 30 evaluated hamsters, seven had retinal structural abnormalities and all had increased permeability of retinal blood vessels. However, hamsters did not carry the mutations causing retinal degenerations 1 and 8, had normal blood glucose levels, and only slightly elevated hemoglobin A1c content. Cholesterol and six other sterols were quantified in hamster retina and compared with sterol profiles in mouse and human retina. These comparisons suggested that cholesterol turnover is much higher in younger than mature hamster retina, and that mature hamster and human retinas share similarities in the ratios of cholesterol metabolites to cholesterol. This study supports further investigations of cholesterol maintenance in hamster retina.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period. The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch's membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch's membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ. This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

PURPOSE To describe an adaptation of an existing graph-theoretic method (initially developed for human optical coherence tomography [OCT] images) for the three-dimensional (3D) automated segmentation of 10 intraretinal surfaces in mice scans, and assess the accuracy of the method and the reproducibility of thickness measurements. METHODS Ten intraretinal surfaces were segmented in repeat spectral domain (SD)-OCT volumetric images acquired from normal (n = 8) and diabetic (n = 10) mice. The accuracy of the method was assessed by computing the border position errors of the automated segmentation with respect to manual tracings obtained from two experts. The reproducibility was statistically assessed for four retinal layers within eight predefined regions using the mean and SD of the differences in retinal thickness measured in the repeat scans, the coefficient of variation (CV) and the intraclass correlation coefficients (ICC; with 95% confidence intervals [CIs]). RESULTS The overall mean unsigned border position error for the 10 surfaces computed over 97 B-scans (10 scans, 10 normal mice) was 3.16 ± 0.91 μm. The overall mean differences in retinal thicknesses computed from the normal and diabetic mice were 1.86 ± 0.95 and 2.15 ± 0.86 μm, respectively. The CV of the retinal thicknesses for all the measured layers ranged from 1.04% to 5%. The ICCs for the total retinal thickness in the normal and diabetic mice were 0.78 [0.10, 0.92] and 0.83 [0.31, 0.96], respectively. CONCLUSION The presented method (publicly available as part of the Iowa Reference Algorithms) has acceptable accuracy and reproducibility and is expected to be useful in the quantitative study of intraretinal layers in mice. TRANSLATIONAL RELEVANCE The presented method, initially developed for human OCT, has been adapted for mice, with the potential to be adapted for other animals as well. Quantitative in vivo assessment of the retina in mice allows changes to be measured longitudinally, decreasing the need for them.

PURPOSEThese experiments assessed the ability of spectral-domain optical coherence tomography (SD-OCT) to accurately represent the structural organization of the adult zebrafish retina and reveal the dynamic morphologic changes during either light-induced damage and regeneration of photoreceptors or ouabain-induced inner retinal damage.METHODSRetinas of control dark-adapted adult albino zebrafish were compared with retinas subjected to 24 hours of constant intense light and recovered for up to 8 weeks or ouabain-damaged retinas that recovered for up to 3 weeks. Images were captured and the measurements of retinal morphology were made by SD-OCT, and then compared with those obtained by histology of the same eyes.RESULTSMeasurements between SD-OCT and histology were very similar for the undamaged, damaged, and regenerating retinas. Axial measurements of SD-OCT also revealed vitreal morphology that was not readily visualized by histology.CONCLUSIONSSD-OCT accurately represented retinal lamination and photoreceptor loss and recovery during light-induced damage and subsequent regeneration. SD-OCT was less accurate at detecting the inner nuclear layer in ouabain-damaged retinas, but accurately detected the undamaged outer nuclear layer. Thus, SD-OCT provides a noninvasive and quantitative method to assess the morphology and the extent of damage and repair in the zebrafish retina.

Kinetic measurements on a fluorescent peptide analog of the p17/p24 cleavage site of the Gag polyprotein demonstrate the conformational selectivity of human immunodeficiency virus, type 1 protease for the trans conformation of the Tyr-Pro bond, A mean cis/trans ratio of 0.3, and a cis --> trans isomerization rate constant of 0.022 s(-1) are determined at T = 22 degrees C. This rate is in excellent agreement with that predicted by F-19 NMR studies of structurally analogous peptides containing a fluorine/hydroxyl substitution on the tyrosyl residue, Addition of recombinant human cyclophilin resulted in a significant enhancement of this rate, and it is proposed that this enzyme, which has been shown to be associated with the Gag protein, functions as an auxiliary enzyme for the protease during cleavage in the virion.

Fluorine nuclear magnetic resonance studies of the cleavage of peptides containing a 4-fluorophenylalanine (FPhe)-Pro bond have been performed in order to determine the conformational specificity of FPhe-Pro bond cleavage by pepsin. The peptides selected were substrates of HIV protease or of avian sarcoma virus protease, both of which have been reported to be cleaved specifically at X-Pro by pepsin as well as by the corresponding viral protease enzyme. By working at 0 degrees C, it was possible to separate kinetically cleavage and cis/trans isomerization. For the case of the protease substrate, Ser-Gln-Asn-FPhe-Pro-Ile-Val-Gln, cleavage was shown to be specific for the trans conformation. A value for the rate constant for hydrolysis of the trans peptide divided by the Michaelis constant, ktH/KMtrans = 0.3 min-1 mM-1 was obtained with this substrate, and the Michaelis constant appears to be considerably higher than the substrate concentration, 3.7 mM, used in the study. On a slower time scale, additional cleavages can readily be detected. For the avian leukemia virus protease substrate, Thr-Phe-Gln-Ala-FPhe-Pro-Leu-Arg-Glu-Ala, the cleavage was both slower and less specific. In addition to the primary cleavage at the FPhe-Pro site, cleavage also occurs at the Ala-FPhe bond on a somewhat slower time scale. In addition to the conformational specificity of the cleavage reaction, these results indicate that pepsin is a better model for HIV protease than for avian leukemia virus protease.

19F NMR relaxation studies have been carried out on a fluorotryptophan-labeled E. coli periplasmic glucose/galactose receptor (GGR). The protein was derived from E. coli grown on a medium containing a 50:50 mixture of 5-fluorotryptophan and [2,4,6,7-2H4]-5-fluorotryptophan. As a result of the large λ-isotope shift, the two labels give rise to separate resonances, allowing relaxation contributions of the substituted indole protons to be selectively monitored. Spin-lattice relaxation rates were determined at field strengths of 11.75 T and 8.5 T, and the results were analyzed using a model-free formalism. In order to evaluate the contributions of chemical shift anisotropy to the observed relaxation parameters, solid-state NMR studies were performed on [2,4,6,7-2H4]-5-fluorotryptophan. Analysis of the observed 19F powder pattern lineshape resulted in anisotropy and asymmetry parameters of Δσ=−93.5 ppm and π=0.24. Theoretical analyses of the relaxation parameters are consistent with internal motion of the fluorotryptophan residues characterized by order parameters S2 of ∼1, and by correlation times for internal motion ∼10-11 s. Simultaneous least squares fitting of the spin-lattice relaxation and line-width data with τi set at 10 ps yielded a molecular correlation time of 20 ns for the glucose-complexed GGR, and a mean order parameter S2=0.89 for fluorotryptophan residues 183, 127, 133, and 195. By contrast, the calculated order parameter for FTrp284, located on the surface of the protein, was 0.77. Significant differences among the spin-lattice relaxation rates of the five fluorotryptophan residues of glucose-complexed GGR were also observed, with the order of relaxation rates given by: R inf1F sup183 >R inf1F sup127 ∼R inf1F sup133 ∼R inf1F sup195 >R inf1F sup284 . Although such differences may reflect motional variations among these residues, the effects are largely predicted by differences in the distribution of nearby hydrogen nuclei, derived from crystal structure data. In the absence of glucose, spin-lattice relaxation rates for fluorotryptophan residues 183, 127, 133, and 195 were found to decrease by a mean of 13%, while the value for residue 284 exhibits an increase of similar magnitude relative to the liganded molecule. These changes are interpreted in terms of a slower overall correlation time for molecular motion, as well as a change in the internal mobility of FTrp284, located in the hinge region of the receptor.

The broadening of the line spread function (LSF) in the drift direction with increasing drift distance in the kinestatic charge detector is substantially reduced when small amounts (less than 1%) of trimethylamine [(CH3)3 N] are added to the x‐ray detection medium (krypton or xenon). The LSF of a mixture of Kr and 0.01% trimethylamine (TMA) was measured as a function of distance at 15, 25, and 35 atm absolute pressure. The full width at half‐maximum (FWHM) of the LSF was reduced from about 1.0 mm to less than 0.5 mm at a drift distance of 4.0 mm for the three pressures. The LSF's of mixtures of xenon and TMA at concentrations ranging from 0.0004% to 0.4% in one run and 0.06% to 4.0% in a second run were measured at a constant pressure of 20 atm. The FWHM of the LSF was reduced from 0.6 to 0.4 mm at 4.0 mm for the xenon measurements. The optimum concentration of TMA in Xe was found to be in the neighborhood of 0.1%. The use of TMA reduced the drift distance‐dependent LSF broadening to the level expected from ionic diffusion, space charge repulsion, and electric field nonuniformity, and it may be possible to reduce the 0.4‐mm FWHM plateau through the use of an improved Frisch grid design. Observation of negative charge carriers showed that electron attachment increases with increasing TMA concentration, although this could be caused by impurities in the TMA. The implications of these results are discussed in terms of extending the maximum drift distance attainable in a kinestatic charge detector.

A prototype scanning imaging system which employs a kinestatic charge detector (KCD) and is under the control of a VAXstation II/GPX computer is described. The operating principles and advantages of the KCD method are reviewed. The detector is a 256-channel ionization drift chamber which creates a two-dimensional x-ray projection image by scanning the detector past the object of interest. The details of the drift chamber design, the signal collection electrodes (channels), and the Frisch grid geometry are given. Also described are the scanning gantry design, computer-controlled drive motor circuit, and safety features. The data acquisition system for the capture of a 1 M byte digital image is presented. This includes amplification, filtration, analog-to-digital conversion, data buffering, and transfer to the VAXstation II computer. The image processing and display techniques specific to the KCD are outlined and the first two-dimensional image taken with this system is presented.

Previous studies [1-3] of the capabilities of a digital radiographic imager employing a scanning strip (slot) beam and a Kinestatic Charge Detector (KCD) have focussed on spatial and contrast resolution. This paper compares the temporal resolution of such a KCD system with that of a system employing a wide area beam and a rare-earth phosphor. The results are largely based on theoretical and computer simulations, including use of a Monte Carlo x-ray transport program, and indicate that for equal image SNR, the KCD yields significantly improved temporal resolution and lower doses than the rare-earth screen system. However, the KCD suffers from tube loading problems and long scan times for patient thicknesses approaching the equivalent of 30 cm of water.

Recent breakthroughs in electronic detector technology have allowed digital radiographic images to become competitive with, or superior to, those produced with classical film-screen techniques. A summary of these technologies is given in ref. 1. Our group is involved in the research and development of a recently proposed imaging technology (2,3) based on the kinestatic charge detector (KCD). The modulation transfer function (MTF) of the KCD technique has been discussed in refs. 1 and 4. The low frequency detective quantum efficiency (DO(0)) of several KCD designs has been modeled and a value of approximately 0.75 is expected for future detectors (1,5). In this paper, the noise power spectrum (NPS) and the frequency-dependent DOE (MEM) are discussed. Noise contributions from x-ray quanta (random and structured) and data acquisition electronics are considered and preliminary experimental results are given for a recently installed imaging detector. A brief comparison is made of our theoretical and experimental results with published results for film-screen radiography.

Strip (or slot) beam digital radiography has been proposed as an ideal compromise between the excellent scatter rejection of pencil-beam or single-line scanned projection radiography systems and the excellent x-ray utilization of wide area beam systems. Moreover, the Kinestatic Charge Detector (KCD) has been proposed as a strip beam detector candidate with a potential for achieving a spatial resolution of over 5 cy/mm, a quantum detection efficiency (QDE) near unity ( > 90% and a local exposure time at a given contrast resolution which is less than other detection techniques (i.e., reduced motion blurring). Several laboratory KCDs containing various numbers of channels have now been constructed and tested which allow a better understanding of the practical performance which can be expected from a strip beam digital radiography system using a KCD.

The past decade has seen a marked increase in research on digital electronic detectors for x-radiographic imaging systems. This paper summarizes recent work on scanning strip detectors with emphasis on a new technique known as kinestatic charge detection (KCD). Principles of operation, theory and early experimental results are presented.

The goal of developing an on-line electronic digital radiographic (EDR) system to replace conventional film-screen radiography (FSR) is important for at least two reasons. First, theoretical arguments show that EDR can have improved diagnostic quality, reduced patient dose and faster image accessibility than FSR. Secondly, the availability of EDR systems will remove the final impediment to the realization of the PACS concept inasmuch as FSR is the only major nonelectronic imaging modality left in the modern radiology department. The Kinestatic Charge Detector (KCD) has properties which make it a candidate for an on-line EDR systems-10. The KCD is a strip detector with high spatial resolution in two dimensions. However, mechanically and electronically, it operates like a one-dimensional detector. Thus, it can effectively scan on the order of 64 to 128 parallel x-ray lines simultaneously but with a 64 to 128-fold reduction in the number of actual detector cells and electronic channels. Moreover, this can be done at quantum detection efficiencies approaching unity. In this paper, theoretical calculations and experimental measurements of the performance parameters of a KCD are presented. Some of the particular parameters discussed include spatial, contrast, and temporal resolution.