Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

43.Kirkpatrick JN, Spencer T, Manivannan A, Sharp PF, Forrester JV. Quantitative image analysis of macular drusen from fundus photographs and scanning laser ophthalmoscope images. Eye 1995;9:48–55.

44.Elsner AE, Burns SA, Weiter JJ, Delori FC. Infrared imaging of sub-retinal structures in the human ocular fundus. Vision Res 1996;36:191–205.

45.Beausencourt E, Remky A, Elsner AE, Hartnett ME, Trempe CL. Infrared scanning laser tomography of macular cysts. Ophthalmology 2000;107:375–385.

46.Kunze C, Elsner AE, Beausencourt E, Moraes L, Hartnett ME, Trempe CL. Spatial extent of pigment epithelial detachments in age-related macular degeneration. Ophthalmology 1999;106:1830–1840.

47.Miura M, Elsner AE, Beausencourt E, et al. Grading of infrared confocal scanning laser tomography and video displays of digitized color slides in exudative age-related macular degeneration. Retina 2002;22:300–308.

48.Zhang X, Hargitai J, Tammur J. Macular pigment and visual acuity in Stargardt macular dystrophy. Graefes Arch Clin Exp Ophthalmol 2002;240:802–809.

49.Goldbaum MH, Kouznetsova V, Cote BL, Hart WE, Nelson M. Automated registration of digital ocular fundus images for comparison of lesions. Proc SPIE 1993;187:94–99.

50.Hart WE, Goldbaum MH. Registering retinal images using automatically selected control point pairs. Image Processing, Proceedings. ICIP-94., IEEE International Conference. Nov. 1994;3:576–580.

51.Brown LG. A survey of image registration techniques. ACM Computing Surveys 1992; 24:326–376.

52.Can A, Shen H, Turner JN, Tanenbaum HL, Roysam B. Rapid automated tracing and feature extraction from retinal fundus images using direct exploratory algorithms. IEEE Trans Inf Technol Biomed 1999;3:125–138.

53.Can A, Stewart CV, Roysam B. Robust hierarchical algorithm for constructing a mosaic from images of the curved human retina. IEEE Computer Society Conference on Computer Vision and Pattern Recognition. Vol. 2, 23–25 June, 1999.

54.Can A, Stewart CV, Roysam B, Tanenbaum HL. A feature-based technique for joint, linear estimation of high-order image-to-mosaic transformations: application to mosaicing the curved human retina. IEEE Conference on Computer Vision and Pattern Recognition. June 2000; vol. 2:585–591.

55.Rivero ME, Bartsch DU, Otto T, Freeman WR. Automated scanning laser ophthalmoscope image montages of retinal diseases. Ophthalmology 1999;106:2296–2300.

56.Bogoni L. Extending dynamic range of monochrome and color images through fusion. 15th International Conference on Pattern Recognition. Sept. 2000; vol. 3:7–12.

57.Bogoni L, Hansen M, Burt P. Image enhancement using pattern-selective color image fusion. International Conference on Image Analysis and Processing. 27–29 Sept. 1999; pp. 44–49.

58.Burt PJ, Kolczynski RJ. Enhanced image capture through fusion. Fourth International Conference on Computer Vision. 11–14 May 1993; pp. 173–182.

59.Berger JW. Quantitative, spatio-temporal image analysis of fundus features in age-related macular degeneration. Proc SPIE (Ophthalmic Technologies) 1998;3246:48–53.

60.Sunness JS, Bressler NM, Tian Y, Alexander J, Applegate CA. Measuring geographic atrophy in advanced age-related macular degeneration. Invest Ophthalmol Vis Sci 1999;40:1761–1769.

61.Shin DS, Javornik NB, Berger JW. Computer-assisted, interactive fundus image processing for macular drusen quantitation [see comments]. Ophthalmology 1999;106:1119–1125.

62.Sebag M, Peli E, Lahav M. Image analysis of changes in drusen area. Acta Ophthalmologica 1991;69:603–610.

63.Morgan WH, Cooper RL, Constable IJ, Eikelboom RH. Automated extraction and quantification of macular drusen from fundal photographs. Aust New Zealand J Ophthalmol 1994;22:7–12.

64.Peli E, Lahav M. Drusen measurement from fundus photographs using computer image analysis. Ophthalmology 1986;93:1575–1580.

65.Rapantzikos K, Zervakis M, Balas K. Detection and segmentation of drusen deposits on human retina: Potential in the diagnosis of age-related macular degeneration. Med Image Anal 2003;7:95–108.

66.Sbeh B, Cohen LD, Mimoun G, Coscas G. A new approach of geodesic reconstruction for drusen segmentation in eye fundus images. IEEE Trans Med Imaging 2001;20(12):1321–1333.

67.Goldbaum MH, Katz NP, Nelson MR, Haff LR. The discrimination of similarly colored objects in computer images of the ocular fundus. Invest Ophthalmol Vis Sci 1990; 31:617–623.

68.Sivagnanavel V, Smith RT, Chong NHV. Digital drusen quantification in high-risk patients with age related maculopathy. Invest Ophthalmol Vis Sci 2003;44:E-5002.

69.Smith RT, Chan JK, Nagasaki T, et al. Automated detection of macular drusen using geometric background leveling and threshold selection. Arch Ophthalmol 2005;123:200–207.

70.Lee MS, Shin DS, Berger JW. Grading, image analysis, and stereopsis of digitally compressed fundus images. Retina 2000;20:275–281.

71.Scholl HPN, Dandekar SS, Peto T, et al. What is lost by digitizing stereoscopic fundus color slides for macular grading in age-related maculopathy and degeneration? Ophthalmology 2004;111:125–132.

72.Sajda P, Spence C, Pearson J. Learning contextual relationships in mammograms using a hierarchical pyramid neural network. IEEE Trans Med Imaging 2002;21:239–250.

73.Sajda P, Du S, Parra L, Stoyanova R, Brown T. Recovery of constituent spectra in 3D chemical shift imaging using non-negative matrix factorization. Proc. 4th International Symposium on Independent Component Analysis and Blind Signal Separation. April, 2003, Nara, Japan, pp. 71–76.

74.Sajda P, Du S, Brown TR, et al. (2004). Non-negative matrix factorization for rapid recovery of constituent spectra in magnetic resonance chemical shift imaging of the brain. IEEE Trans Med Imaging 2004;23(12):1453–1465.

75.Smith RT, Nagasaki T, Sparrow JR, Barbazetto I, Klaver CCW, Chan JK. A method of drusen measurement based on the geometry of fundus reflectance. BioMed Eng Online 2003;2:10.

76.Smith RT, Nagasaki T, Sparrow JR, Barbazetto I, Koniarek JP, Bickmann LJ. Patterns of reflectance in macular images: representation by a mathematical model. J Biomed Optics 2004;9:162–172.

77.Smith RT, Koniarek JP, Chan JK, Nagasaki T, Sparow J, Langton K. Autofluorescence characteristics of normal foveas and reconstruction of foveal autofluorescence from limited data subsets. Invest Ophthalmol Vis Sci 2005;46(8):2940–2946.

78.Smith RT, Chan JK, Nagasaki T, Sparrow JR, Barbazetto I. A method of drusen measurement based on reconstruction of fundus reflectance. B J Opthalmol 2005;89:87–91.

79.Otsu N. A threshold selection method from gray-level histograms. IEEE Trans Syst Man Cybern 1979;9:62–66.

80.Schweitzer D, Hammer M, Schweitzer F, et al. In-vivo measurement of time-resolved autofluorescence at the human fundus. J Biomed Optics 2004;9:1214–1222.

81.Carano R, Lynch JA, Redei J, et al. Multispectral analysis of bone lesions in the hands of patients with rheumatoid arthritis. Magn Reson Imaging 2004;22:505–514.

82.Ward J, Magnotta V, Andreason NC, Ooteman W, Nopoulos P, Pierson R. Color enhancement of multispectral MR images: improving the visualization of subcortical structures. J Comput Assist Tomogr 2001;25:942–949.

83.Akita K, Kuga H. A computer method of understanding ocular fundus images. Pattern Recognit 1992;15:431–433.

84.Undrill P. Towards the automatic interpretation of retinal images. Br J Opthalmol 1996; 80:937–938.

Fig. 1. Retinal pigment epithelial (RPE) lipofuscin dctected as autofluorescence in human retina. A section of human retina viewed by phase contrast (A) and epifluorescence (B) imaging. The brightness at the level of RPE in B is caused by lipofuscin autofluorescence. In an isolated human RPE cell (C), RPE lipofuscin has a granule-like appearance, indicative of its location within the lysosomal compartment of the cell. Scale bar, 10 M.

lipofuscin (2,6,7). Interest in the structures and modes of formation of these fluorophores stems from mounting evidence that these lipofuscin fluorophores may play a role in RPE cell dysfunction and death in some retinal disorders.

PACKAGING OF LIPOFUSCIN IN THE RPE

The lipofuscin of RPE cells is housed within membrane bound organelles—secondary lysosomes or residual bodies—that, because of their ultrastructural appearance in thin sections or after isolation by cell fractionation, are also called lipofuscin granules (8–11) (Fig. 1). Most of the macromolecules phagocytosed by the RPE are hydrolyzed by lysosomal enzymes to small molecules that can diffuse out of the lysosome (9), but those components that are indigestible accumulate as lipofuscin. The membrane that bounds the lipofuscin-containing organelle has been visualized as a surface structure detectable by atomic force microscopy (11) with chloroform/methanol exposure producing irregularities in the surface owing to solubilization of phospholipids (11).

Throughout life, the numbers of lipofuscin granules per RPE cell steadily increases (12) as does the optical density and fluorescence intensity of individual granules (10). The latter observation indicates an age-related increase in the fluorophore content of these residual bodies. In young eyes, lipofuscin granules are confined to basilar portions of the RPE,

whereas melanin granules are located apically (13). In older eyes, however, lipofuscin granules are located throughout the entire RPE cell. Melanosomes and lipofuscin granules also undergo a poorly understood age-associated modification that involves the formation of melanin-lipofuscin complexes (melanolipofuscin granules) (10,12).

CHEMICAL COMPOSITION OF RPE LIPOFUSCIN

The first comprehensive study of the composition of RPE lipofusin was performed by Eldred and Katz in 1988 (14). From their chromatographical analysis of chloroform/methanol extracts of RPE, these investigators described 10 fractions. Two of these were green-emitting pigments that co-chromatographed with all-trans-retinol and all-trans-retinyl palmitate, well known retinoids of the visual cycle not suited to the definition of lipofuscin (7,15). Three of the fractions were orange-emitting fluorophores with excitations in the visible range of the spectrum, features of particular interest because photoreactions in the RPE can only be initiated by visible light (>400), shorter wavelengths being absorbed by the cornea and lens. The most prominent of these three fractions was later named A2E (C42H58NO, mol wt 592) (16) because it could be synthesized from vitamin A aldehyde (16,17) and ethanolamine (2:1 ratio) and was shown to be readily detectable in extracts of human RPE by reverse phase high-performance liquid chromatography (HPLC) (18) (Fig. 2). The spectral characteristics of the other two orange-emitting fractions of Eldred and Katz suggest that one of them may have been iso-A2E, a less polar photoisomer of A2E (18,19) (Fig. 2). Whereas all of the double bonds of A2E assume the trans (E) configuration, iso-A2E has one cis (Z) olefin at the C13–14 position and its absorbance spectra is slightly blue shifted (~12 nm) relative to A2E. Under the influence of light, both in the eye and on the laboratory bench, A2E and iso-A2E are interconvertible although the trans configuration is favored at a ratio of 4:1 (18). At least five other less abundant isomers of A2E are also detectable in hydrophobic extracts of human RPE (19). These Z-isomers were originally identified because of HPLC retention times and absorbance spectra that were similar to A2E and an m/z (mass to charge ratio) (592) that was identical. Four of these isomers are readily accounted for by cis double bonds at the C9/9′–10/10′ and C11/11′–12/12′ positions whereas the fifth could contain two cis double bonds.

A2E is a wedge-shaped molecule with a positively charged pyridine ring and two side arms—a long and a short—each of which is derived from a molecule of ATR (16) (Fig. 2). Under in vivo conditions, the counterion of this pyridinium salt is presumably chloride (18). Although A2E is often said to be N-retinylidene-N- retinylethanolamine, a structure initially assigned to it and then withdrawn (17,20), this nomenclature is incorrect because it does not describe the pyridinium structure of the head group of A2E (16).

Another constitutent of RPE lipofuscin that has been structurally characterized is ATR dimer (21,22), a condensation product of two ATRs that forms under conditions of release of ATR from opsin (Fig. 2). Via its aldehyde group, ATR dimer forms conjugates with various amines, one of which is phosphatidylethanolamine (PE). A protonated Schiff-base ATR dimer–PE conjugate has been detected in RPE. Besides having a novel

Fig. 2. Structures of some known RPE lipofuscin fluorophores, A2E; its photoisomer, isoA2E; and all-trans-retinal (ATR)-dimer-phosphatylethanolamine (PE) conjugate.

structure, ATR dimer–PE has a distinctive absorbance of approx 506 nm that is redshifted relative to A2E (~440 nm) (22).

Additional components of RPE lipofuscin are generated by the photo-oxidation of A2E. These oxidative derivatives of A2E include oxiranes (23) and were originally identified because of the diminution in A2E fluorescence that accompanies their formation. By mass spectroscopy analysis, the addition of oxygens at carbon–carbon double bonds after blue light irradiation is detected as a series of peaks that differ in m/z by 16 starting from the M+ 592 peak attributable to A2E (23,24). Intracellular A2E has been shown to undergo photo-oxidation following blue light exposure (24). Moreover, it has been reported that a species with a mass of 608, corresponding to a monoepoxide of A2E, has been detected in hydrophobic extracts of human RPE cells (25) and A2E monoepoxides and bisepoxides have been detected in RPE isolated from Abcr–/––/– mice (26). The addition of oxygens at olefins of A2E leads to blue shifts in A2E

absorbance and to diminished A2E fluorescence; thus, an age-related increase in the proportion of A2E that is photooxidized could contribute to age-related changes in the spectral properties of RPE lipofuscin. The gradual weakening of A2E fluorescence could also account for the observation that lipofuscin fluorescence reaches a plateau or declines during the later decades of life (27,28). Of course, another explanation for the latter occurrence is the loss of RPE cells. Differences in the extent to which A2E is photo-oxidized in individual lipofuscin granules may also contribute to blue shifts in some granules relative to others (11,29).

The autofluorescent granules that form in RPE cultured at confluence for long periods of time likely represent inclusions derived from an autophagic source (30). On the other hand, it is not known whether the fluorescent inclusions that accumulate in cultured RPE incubated with ultraviolet (UV)-irradiated or nonirradiated OS (31–33) are the same as native RPE lipofuscin. Photoreceptor-specific proteins, identified according to their expected molecular weight and immunoreactivity with specific antibodies, have also been detected within lipofuscin-enriched preparations (34,35), but according to Feeney-Burns and colleagues, this occurs when the preparations are contaminated by phagosomes (34). Other retinoid-derived fluorophores in addition to A2E, its photoisomers and photooxidative products and ATR dimer, may also contribute to RPE lipofuscin. For instance, ATR at high concentrations can form covalent bonds at lysine residues in rhodopsin, with the result that bis-retinoid adducts having emission spectra similar to A2E, are generated (36). The accumulation of these adducts on rhodopsin, in vivo, could have the adverse effect of reducing light sensitivity, may necessitate the constant replacement of OS membrane (19,37), and may indicate that peptides fragments bearing nondegradable fluorescent A2-moieties can be deposited in RPE cells following OS phagocytosis.

Because it was presumed for several years that RPE lipofuscin originates from peroxidized lipid, there has been considerable interest in the lipid content of RPE lipofuscin. Accordingly, a study of the lipid composition of human RPE lipofuscin granules showed that the fatty acid composition of lipofuscin granule is different than that in photoreceptor OS, the source of the precursors that form the lipofuscin38. On the other hand, it is reasonable to expect that the phospholipids that are extractable from lipofuscin granules and that increase with age in parallel with the increase in lipofuscin granules (11,38) originate from the phospholipids bilayer that bounds these lipofuscin-containing organelles. OSderived products of lipid peroxidation may be present in RPE lipfuscin but are unlikely to be the major constituent because these blue-green-emitting fluorophores cannot account for the golden-yellow fluorescence of RPE lipofuscin (6,7,39). Additionally, because lipofuscin granules (40–42) and A2E (43) have both been shown to mediate light-dependent lipid peroxidation, it would not be surprising if the phospholipid membrane, of lipofuscincontaining residual bodies, was to undergo lipid peroxidative damage.

A2E BIOSYNTHETIC PATHWAYS AND MODULATION OF ITS FORMATION

A2E is a pyridinium bisretinoid (16,44), the formation of which begins in photoreceptor outer segments with condensation reactions between PE and first one and then a second ATR (Fig. 3). The ATR that enters the biosynthetic pathway is generated on

photoisomerization of 11-cis-retinal. The initial Schiff-base conjugate (N-retinylidene-PE [NRPE]) that forms between a single ATR and PE (45–47) may serve as a substrate for ABCA4 (ABCR), the photoreceptor-specific ATP-binding cassette transporter (46,48–54) that is the protein product of the Stargardt disease gene (55) (discussed below). The A2E biosynthetic pathway likely continues through a multi-step process that generates the fluorescent phosphatidyl-pyridinium bisretinoid, A2-PE (18,19,45). This pigment is the precursor of A2E that accumulates in photoreceptor OS with hydrolytic cleavage of A2-PE generating A2E. The intermediate that forms prior to A2-PE is an unstable dihydropyridinium molecule that immediately undergoes autoxidation (18,19,45). Because any of the intermediates formed before the oxidation is likely capable of reversal, this autoxidation step may be the last stage at which it is possible to intervene in the synthesis of A2E (56).

Evidence from a number of experimental approaches has shown conclusively that A2-PE, the precursor fluorophore, forms in photoreceptor OS (45), not in RPE cell lysosomes as originally thought. Nevertheless, A2-PE is not normally accrued in photoreceptor OS to detectable levels (19,45,57) because of continuous replacement of OS discs (58). Indeed, prevention of the latter accumulation may contribute to the need for constant turnover of OS membrane. Conversely, in Royal College of Surgeons (RCS) rats, a strain characterized by an inability to phagocytosis shed OS membrane, A2-PE contributes to the orange-colored pigment and golden-yellow fluorescence that characterizes the degenerating photoreceptor outer segment debris (59,60) (Fig. 4). Extracts of RCS rat retina were shown to exhibit peaks with absorbances consistent with A2-PE of variable fatty acid composition, and signals detected by mass spectroscopy were attributable to A2-PE because they were of the appropriate mass and because the permanent positive charge of the quaternary nitrogen of A2-PE, by positive ionization, was definitive for A2-PE identification (45). A2-PE is probably also at least one of the lipofuscin-like compounds that, because of AF, has been detected in photoreceptor cells in Stargardt disease and in some forms of retinitis pigmentosa (RP) (61–63). It is unlikely that acid hydrolysis of A2-PE within RPE lysosomes can account for A2PE cleavage (19). Rather, the generation of A2E from A2-PE is probably enzyme mediated and phospholipase D has been implicated in this process (45).

Only ATR that leaves the visual cycle and eludes reduction to all-trans-retinol by all- trans-retinol dehydrogenase, is available to form the ATR-derived fluorophores of RPE lipofuscin. It thus follows that conditions that increase the availability of ATR, thereby allowing random and inadvertent reactions between ATR and amine-containing molecules, enhance the opportunity for these fluorophores to form. Accordingly, because the generation of ATR in photoreceptor OS is light-activated, it is not surprising that light is a determinant of the rate of A2E formation. This assertion is supported by in vivo experiments demonstrating that the A2E precursor, A2-PE, in photoreceptor OS is augmented by exposing rats to bright light (19). Additionally, dark rearing of Abcr–/– mice impedes the deposition of A2E (57). Because A2E levels are not diminished if mice are raised in cyclic light and then transferred to darkness, it is also clear that once formed, A2E is not eliminated from the RPE (57). Another well known factor that modulates A2E formation is the activity of ABCA4 (ABCR), the photoreceptor-specific ATPbinding cassette transporter (49,50,51,53) that is thought to aid in the movement of ATR to the cytosolic side of the disc membrane (47,51,53,64,65) where it is accessible