Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999
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Figure 10.3. A1E is internalized by ARPE-19 cells. Detection by fluorescence confocal microscopy (1 mM optical section). Cell borders were labeled by immunostaining with antibody to ZO-1 (A,B) and nuclei were stained with propidium iodide (B). Internalized A1E presents as punctatefilling of the cells.
Figure 10.4. Comparison of effects of A1E and A2E on membrane integrity. A. Epifluorescence detection of A1E and A2E in ARPE-19 cells after 2 days of accumulation from 20 mM concentrations in media. Nuclei stained with DAPI. A1E visible in cytoplasmic region. A2E not accumulated to detectable levels. Note reduced nuclear density in A1E cultures due to cell loss. B. Percent permeabilized cells was determined by labeling with membrane impermeable dye. All nuclei labeled with DAPI. Percent of labeled cells in A2E-accumulating cultures is similar to that in control untreated cultures (data not shown). Mean ± SEM, 3-5 fields/well; 3 wells/condition. C. Loss of membrane integrity as evidenced by egress of LDH into culture medium, assayed colorimetrically after incubating with either A1E or A2E at various concentrations for 2 hours. D, DMSO; N, non-treated. Mean ± SEM.
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4. CONCLUSIONS
The properties of A2E that are likely to determine its behavior in a phospholipid bilayer are its amphiphilic structure, size, shape and cationic nature. Since A2E and A1E are both amphiphilic molecules, it is to be expected that both compounds would exhibit detergentlike properties. However the linear configuration of the non-biological compound A1E is more typical of a detergent and probably because of this stream-lined structure, A1E was able to penetrate the membrane more rapidly than A2E. In the current assays in which membrane permeabilization was assessed over a short period of time, A1E also induced a faster rate of loss in membrane integrity. The two widely displaced retinal-derived chains of A2E confer a bulky structure that likely displaces a relatively large area of membrane and may impede passage as it penetrates. Electrostatic attractions between amphiphiles with a cationic head-group and acidic phospholipids, such as phosphatidylserine can also influence the movement of the compound through the membrane and fluorescence anisotropy studies (De and Sakmar, 2002) suggest that this may be the case for A2E, despite the presence of its counterion.
As further evidence of the ability of A2E to perturb membrane integrity, we have shown here using the plasma membrane as a model phospholipid bilayer, that A2E can provoke membrane blebbing. These membrane blisters were observed in cells that had not yet undergone a change in permeability. While the mechanism by which A2E induces membrane blebbing has not been demonstrated, it is possible to speculate as to some of the events. For instance, A2E, because of its cationic head group may distribute preferentially in the inner leaflet of the membrane due to an attraction to negatively charged phosphatidylserine that is concentrated on the cytoplasmic side of the bilayer (Sheetz and Singer, 1974). Within the cytoplasmic leaflet of the membrane, wedge-shaped A2E would become oriented with its hydrophilic pyridinium portion interacting with the polar heads of the phospholipids and its broadly-spaced hydrophobic side-arms intermingling with the phospholipid hydrocarbon tails (Sparrow et al., 1999). Accordingly, the bulky side arms of A2E could force a large separation of the lipid acyl chains and expand the inner leaflet relative to the outer, the negative curvature of the inner leaflet producing a surface protrusion or bleb. Since A2E becomes sequestered within the lysosomal compartment of the cell, at sufficient concentration, A2E may exert similar effects on the lysosomal membrane.
5. ACKNOWLEDGEMENTS
The work was supported by National Institutes of Health Grants EY12951 and GM 34509, Macula Vision Research Foundation and American Health Assistance Foundation. JRS is the recipient of an Alcon Research Institute Award.
6. REFERENCES
De S, Sakmar TP. 2002, Interaction of A2E with model membranes. Implications to the pathogenesis of age-related macular degeneration. J Gen Physiol 120(2):147-157.
Eldred GE, Lasky MR. 1993, Retinal age pigments generated by self-assembling lysosomotropic detergents. Nature 361(6414):724-726.
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Jockusch S, Ren RX, Jang YP, Itagaki Y, Vollmer-Snarr HR, Sparrow JR, Nakanishi K, Turro NJ. 2004, Photochemistry of A1E, a retinoid with a conjugated pyridinium moiety: competition between pericyclic photooxygenation and pericyclization. J Am Chem Soc 126(14):4646-4652.
Lai C, Gouras P, Doi K, Lu F, Kjeldbye H, Goff SP, Pawliuk R, Leboulch P, Tsang SH. 1999, Tracking RPE transplants labeled by retroviral gene transfer with green fluorescent protein. Invest Ophthalmol Vis Sci 40(9):2141-2146.
Parish CA, Hashimoto M, Nakanishi K, Dillon J, Sparrow JR. 1998, Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium. Proc Natl Acad Sci U S A 95(25):1460914613.
Sakai N, Decatur J, Nakanishi K, Eldred GE. 1996, Ocular age pigment “A2E”: An unprecedented pyridinium bisretinoid. J Am Chem Soc 118:1559-1560.
Sheetz MP, Singer SJ. 1974, Biological membranes as bilayer couples. A molecular mechanism of drugerythrocyte interactions. Proc Natl Acad Sci U S A 71(11):4457-4461.
Sparrow JR, Nakanishi K, Parish CA. 2000, The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells. Invest Ophthalmol Vis Sci 41(7):1981-1989.
Sparrow JR, Parish CA, Hashimoto M, Nakanishi K. 1999, A2E, a lipofuscin fluorophore, in human retinal pigmented epithelial cells in culture. Invest Ophthalmol Vis Sci 40(12):2988-2995.
CHAPTER 11
AMINO-RETINOID COMPOUNDS IN THE HUMAN RETINAL PIGMENT EPITHELIUM
Heidi R. Vollmer-Snarr,* McKenzie R. Pew, Mary L. Alvarez,
D. Joshua Cameron, Zhibing Chen, Glenn L. Walker, Josh L. Price, and Jeffrey L. Swallow
1. INTRODUCTION
For many years, blue light damage associated with retinal pigment epithelial (RPE) cell lipofuscin (LF) has been implicated in the cause of age-related macular degeneration (AMD) (Young, 1988; Winkler et al., 1999; Bressler et al., 2000) and other retinal degenerative diseases, such as Stargardt’s disease, (Lopez et al., 1990; Birnbach et al., 1994), Best’s macular dystrophy (Weingeist et al., 1982) and cone-rod dystrophy (Rabb et al., 1986). A2E and isomers, pyridinium bis-retinoid compounds and only major blue-light absorbing fluorophores isolated from human LF, (Eldred and Katz, 1988; Eldred and Lasky, 1993; Sakai et al., 1996; Parish et al., 1998;) account for a small fraction of the composition of LF. Proteins account for 30-70% (Schutt et al., 2002; Haralampus-Grynaviski et al., 2003). The percentage of A2E oxidation products, retinol and retinyl palmitate has not been quantified, yet these compounds, too, make up only a fraction of the composition of LF. Evidence for A2E and especially A2E photo-oxidation products’ involvement in AMD is strong (Sparrow et al., 1999; Sparrow et al., 2000; Sparrow and Cai, 2001; Ben-Shabat et al., 2002a; Finnemann et al., 2002; Sparrow et al., 2002; Sparrow et al., 2003); however, their involvement does not rule out the possibility of AMD’s etiology being multifactorial. There are many compounds in LF that have not been characterized, some of which may also play a role in the pathogenesis of AMD and other retinal degenerative diseases.
The biosynthetic pathway of A2E begins with the formation of A2PE in the rod outer segments (ROS) of the eye (Parish et al., 1998; Liu et al., 2000; Mata et al., 2000; Ben-Shabat et al., 2002b). In the formation of A2PE, one molecule of phosphatidyl ethanolamine reacts with two molecules of all-trans-retinal. A2PE’s phosphatidyl group is then cleaved to form A2E. A2E is deposited into RPE LF during phagocytosis of ROS. In
* Department of Chemistry & Biochemistry, Brigham Young University, Provo, UT 84602.
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common with phosphatidyl ethanolamine, other biogenic amines found in the retina (Makino-Tasaka et al., 1985; Drujan et al., 1989; Djamgoz and Wagner, 1992; Gulcan et al., 1993; Taibi and Schiavo, 1993; Witkovsky et al., 1993) and specifically in ROS (Crain et al., 1978; Macaione and Calatroni, 1978; Aveldano and Bazan, 1983; Lentile et al., 1986; Taibi et al., 1995) also have a free amine functionality necessary for reaction with all-trans- retinal. Serotonin, tryptamine, norepinephrine, putrescine, spermidine, tyramine, spermine, and dopamine represent a small selection of biogenic amines with known abundances in the retina. All of these amines react with all-trans–retinal to form amino-retinoid compounds. These compounds may account for some of the uncharacterized compounds within RPE LF.
2. SYNTHETIC AMINO-RETINOID COMPOUNDS
Serotonin, tryptamine, norepinephrine, putrescine, spermidine, tyramine, spermine, and dopamine were reacted with all-trans-retinal in order to form amino-retinoid standard compounds for use in their detection in the RPE (Scheme 11.1). Fast atom bombardment (FAB+) mass spectrometry (MS), high performance liquid chromatography (HPLC) and nuclear magnetic resonance spectroscopy (NMR) data were used to characterize the structures of the products formed in these reactions (figs. 11.1 and 11.2). A mixture of pyridinium, “A2Elike,” products and single retinoid side-arm products resulting from Pictet-Spengler and modified Pictet-Spengler type mechanisms were formed in these reactions. Monoand bisretinoid compounds were observed in reactions of serotonin, tryptamine and norepinephrine with all-trans-retinal; bisand tetra-retinoid compounds were observed in reactions with putrescine, spermidine and spermine with all-trans-retinal; and only bis-retinoid products were observed in the reaction of tyramine with all-trans-retinal (fig. 11.1). In the reaction between dopamine and all-trans-retinal, the same Pictet-Spengler or mono-retinoid products were observed as reported by Pezzella and Prota (2002); bis-retinoid compounds were also observed (fig. 11.2).
Because the tetra-retinoid compounds observed in the reactions reported above have two bis-retinoid pyridinium ring moieties similar to the single bis-retinoid pyridinium structure of A2E, the bioactivity of these compounds may be similar to A2E. However, because A2E is most damaging to cells through its oxidation products, (Ben-Shabat et al., 2002a; Sparrow et al., 2002; Sparrow et al., 2003) products resulting from reactions with putrescine, spermidine and spermine (tetra-retinoid compounds) may form double the number of oxidation products of A2E. Therefore, their oxidation products may prove to be even more damaging to RPE cells than photo-oxidized A2E. The observed Pictet-Spengler or mono-retinoid reaction products may also exhibit different cytotoxic mechanisms to those observed by A2E. Furthermore, the new bis-retinoid compounds formed may also exhibit alternative mechanisms of RPE cytotoxicity to that of A2E. It is therefore important to determine whether any of these compounds exist in RPE LF.
Scheme 11.1. General reaction between all-trans-retinal – 1 and any given biogenic amine found in the retina.
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Figure 11.1. Reaction products observed for reactions of serotonin (A2S & A1S), tryptamine (A2- & A1-Tryp), norepinephrine (A2N & A1N), putrescine (A4P & A2P), spermidine (A2- & A4-Spd), tyramine (A2-Tyr) and spermine (A4and A2-Spm) with all-trans-retinal.
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Figure 11.2. Reaction products observed in reaction of dopamine with all-trans-retinal (A1D & A2D).
3. EVIDENCE OF AMINO-RETINOID COMPOUNDS IN THE HUMAN RPE
It has been suggested that extraction of RPE cells provide the same fluorophores as extraction of RPE LF (Eldred and Katz, 1988). Therefore, organic extractions were performed directly on RPE cells. Modifications using dichloromethane were made to extraction procedures described by Eldred and Katz (1988) and Parish et al. (1998) which have resulted in the observation of additional peaks on HPLC chromatograms that appear to be amino-retinoid compounds.
During HPLC analysis of the extracted material, a peak was observed with a retention time of eight minutes, which has a very similar UV spectrum to that of A2E (fig. 11.3). This
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Figure 11.3. A: UV spectrum of an unknown amino-retinoid compound; B: UV spectrum of A2E; C: HPLC chromatogram from which the UV spectra were observed. The peak at 8 minutes represents the unknown amino-retinoid and the peak at 13 minutes is A2E.
data suggests that the compound with the eight minute retention time may be an amino- bis-retinoid. A co-injection of A2E with the extracts confirmed that the new peak was not A2E. A2E was observed with a retention time of 13 minutes. The structure of the unknown amino-retinoid compound is in the process of being elucidated; it is not yet certain if the compound is one of the standard amino-retinoids described above, although the UV matches nicely with several of these compounds.
In addition to the peak observed at eight minutes, retention times and UV spectra associated with A2N and isomers are remarkably similar to those of A2E and isomers, which suggests that bis-retinoid compounds, such as A2N and isomers that are similar to A2E in structure may be masked on HPLC chromatograms by A2E. Co-injections of A2N and A2E have been made and confirm that the 2 sets of peaks are on top of each other. Method sets are being worked out to separate these isomers, and to determine if other bis-retinoid peaks in RPE extracts may be hidden underneath the A2E peaks.
The goals of the research efforts described above were to: 1) chemically synthesize amino-retinoid standard compounds for use in their detection in RPE LF; and 2) perform organic extractions on human RPE cells to determine whether standard compounds or other amino-retinoid compounds may be present. Standard amino-retinoid compounds have been made, which have proved useful in the search for these and related compounds in the RPE. New peaks on HPLC chromatograms of injected human RPE extracts have also been observed, which appear to be yet uncharacterized amino-retinoid compounds. The results of this research are promising, and further research will continue in the synthesis of additional amino-retinoid standard compounds and in the characterization of novel aminoretinoids from the human RPE. A long term goal is to extensively characterize human RPE LF, which has been implicated in the cause of AMD, but has never been completely characterized. As new RPE LF compounds are isolated, a more complete understanding of the role of LF in the RPE will begin to be established. It is hoped that this knowledge will
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enable new preventative and therapeutic approaches to the retinal degenerative diseases which afflict so many.
4. ACKNOWLEDGEMENTS
The authors graciously thank Drs. Paul Bernstein and Prakash Bhosale for providing human retinal pigment epithelial cells for described experiments and Brigham Young University’s Department of Chemistry and Biochemistry for funding.
5. REFERENCES
Aveldano, M. I., and Bazan, N. G., 1983, Molecular species of phosphatidylcholine, -ethanolamine, -serine, and -inositol in microsomal and photoreceptor membranes of bovine retina, J. Lipid Res. 24:620.
Ben-Shabat, S., Itagaki, Y., Jockusch, S., Sparrow, J. R., Turro, N. J., and Nakanishi, K., 2002a, Formation of a nona-oxirane from A2E, a lipofuscin fluorophore related to macular degeneration, and evidence of singlet oxygen involvement. Angew. Chem. Int. Ed. 41:814.
Ben-Shabat, S., Parish, C. A., Vollmer, H. R., Itagaki, Y., Fishkin, N., Nakanishi, K., Sparrow, J. R., 2002b, Biosynthetic studies of A2E, a major fluorophore of RPE lipofuscin. J. Biol. Chem. 277:7183.
Birnbach, C. D., Jarvelainen, M., Possin, D. E., and Milam, A. H., 1994, Histopathology and immunocytochemistry of the neurosensory retina in fundus flavimaculatus, Ophthalmol. 101:1211.
Bressler, S. B., Bressler, N. M., and Gragoudas, E. S., 2000, Age-related macular degeneration: drusen and geographic atrophy, in: Principles and Practice of Ophthalmology, Albert, D. M., Jakobiec, F. A., Azar, D. T., and Gragoudas, E. S., eds., vol 3. W. B. Saunders Co, Philadelphia, pp. 1982-1992.
Crain, R. C., Marinetti, G. V., and O’Brien, D. F., 1978, Topology of amino phospholipids in bovine retinal rod outer segment disk membranes, Biochem. 17:4186.
Djamgoz, M. B. A. and Wagner, H. J., 1992, Localization and function of dopamine in the adult vertebrate retina,
Neurochem. Int. 20:139.
Drujan, B. D., Jaffe, E. H., Urbina, M., Ayala, C., and Drujan, Y., 1989, Interaction of DA and other biogenic amines in the retina, Neurol. Neurobiol. 49:257.
Eldred, G. E. and Katz, M. L., 1988, Fluorophores of the human retinal pigment epithelium: separation and spectral characterization, Exp. Eye Res. 47:71.
Eldred, G. E. and Lasky, M. R., 1993, Retinal age pigments generated by self-assembling lysosomotropic detergents, Nature 361:724.
Finnemann, S. C., Leung, L. W., and Rodriguez-Boulan, E., 2002, The lipofuscin component A2E selectively inhibits phagolysosomal degradation of photoreceptor phospholipids by the retinal pigment epithelium, Proc. Natl. Acad. Sci. USA 99:3842.
Gulcan, H. G., Alvarez, R. A., Maude, M. B., and Anderson, R. E., 1993, Lipids of human retina, retinal pigment epithelium, and Bruch’s membrane/choroid: comparison of macular and peripheral regions, Invest. Ophthalmol. Visual Sci. 34:3187.
Haralampus-Grynaviski, N. M., Lamb, L. E., Clancy, C. M. R., Skumatz, C., Burke, J. M., Sarna, T., and Simon, J. D., 2003, Spectroscopic and morphological studies of human retinal lipofuscin granules, Proc. Natl. Acad. Sci. 100:3179.
Lentile, R., Russo, P., and Macaione, S., 1986, Polyamine localization and biosynthesis in chemically fractionated rat retina, J. Neurochem. 47:1356.
Liu, J., Itagaki, Y., Ben-Shabat, S., Nakanishi, K., and Sparrow, J. R., 2000, The biosynthesis of A2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane, J. Biol. Chem. 275:29354.
Lopez, P. F., Maumenee, I. H., de la Cruz, Z., and Green, W. R., 1990, Autosomal-dominant fundus favimaculatus. Clinicopathologic correlation, Ophthalmol. 97:798.
Macaione, S., and Calatroni, A., 1978, Polyamines and ornithine decarboxylase activity in the developing rat retina,
Life Sciences, 23:683.
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Makino-Tasaka, M., Suzuki, T., Nagai, K., and Miyata, S., 1985, Spatial distribution of visual pigment and dopamine in the bullfrog retina, Exp. Eye Res. 40:767.
Mata, N. L., Weng, J., and Travis, G. H., 2000, Biosynthesis of a major lipofuscin fluorophore in mice and humans with ABCR-mediated retinal and macular degeneration, Proc. Natl. Acad. Sci. USA, 97:7154.
Parish, C. A., Hashimoto, M., Nakanishi, K., Dillon, J., and Sparrow, J. R., 1998, Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium, Proc. Natl. Acad. Sci. USA 95:14609.
Pezzella, A., and Prota, G., 2002, Formation of novel tetrahydroisoquinoline retinoids by Pictet-Spengler reaction of dopamine and all-trans-retinal under conditions of relevance to biological environments, Tett. Lett., 43:6719.
Rabb, M. F., Tso, M. O., and Fishman, G. A., 1986, Cone-rod dystrophy. A clinical and histopathologic report,
Ophthalmol. 93:1443.
Sakai, N., Decatur, J., Nakanishi, K., and Eldred, G. E., 1996, Ocular age pigment “A2E”: An unprecedented pyridinium bisretinoid, J. Am. Chem. Soc. 118:1559.
Schutt, F., Ueberle, B., Schnolzer, M., Holz, F. G., and Kopitz, J., 2002, Proteome analysis of lipofuscin in human retinal pigment epithelial cells, FEBS Lett. 528:217.
Sparrow, J. R., and Cai, B., 2001, Blue light-induced apoptosis of A2E-containing RPE: involvement of caspase- 3 and protection by Bcl-2, Invest. Ophthalmol. Vis. Sci. 42:1356.
Sparrow, J. R., Nakanishi, K., and Parish, C. A., 2000, The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells, Invest. Ophthalmol. Vis. Sci. 41:1981.
Sparrow, J. R., Parish, C. A., Hashimoto, M., and Nakanishi, K., 1999, A2E, a lipofuscin fluorophore, in human retinal pigmented epithelial cells in culture, Invest. Ophthalmol. Vis. Sci. 40:2988.
Sparrow, J. R., Vollmer-Snarr, H. R., Zhou, J., Jang, Y. B., Jockusch, S., Itagaki, Y., and Nakanishi, K., 2003, A2Eepoxides Damage DNA in Retinal Pigment Epithelial cells. Vitamin E and other Antioxidants Inhibit A2E-epoxide formation, J. Biol. Chem. 278:18207.
Sparrow, J. R., Zhou, J., Ben-Shabat, S., Vollmer, H., Itagaki, Y., and Nakanishi, K., 2002, Involvement of oxidative mechanisms in blue light induced damage to A2E-laden RPE, Invest. Ophthalmol. Vis. Sci. 43:1222.
Taibi, G., and Schiavo, M. R., 1993, Simple high-performance liquid chromatographic assay for polyamines and their monoacetyl derivatives, Journal of Chromatography, Biomedical Applications, 614:153.
Taibi, G., Schiavo, M. R., and Nicotra, C., 1995, Polyamines and ripening [maturation] of photoreceptor outer segments in chicken embryos, International Journal of Developmental Neuroscience, 13:759.
Weingeist, T. A., Kobrin, J. L., and Watzke, R. C., 1982, Histopathology of Best’s macular dystrophy, Arch. Ophthalmol. 100:1108.
Winkler, B. S., Boulton, M. E., Gottsch, J. D., and Sternberg, P., 1999, Oxidative damage and age-related macular degeneration, Mol. Vis. 5:32.
Witkovsky, P., Nicholson, C., Rice, M. E., Bohmaker, K., and Meller, E., 1993, Extracellular dopamine concentration in the retina of the clawed frog, Xenopus laevis, Proc. Natl. Acad. Sci. 90:5667.
Young, R. W., 1988, Solar radiation and age-related macular degeneration. Surv Ophthalmol. 32:252.
CHAPTER 12
ANNEXINS IN BRUCH’S MEMBRANE AND DRUSEN
Mary E. Rayborn*, Hirokazu Sakaguchi, Karen G. Shadrach, John W. Crabb, and Joe G. Hollyfield
1. INTRODUCTION
Annexins (also known as lipocortins) are a family of calcium and phospholipid-binding proteins. At least 20 members of this family are known, and they have a wide range of potential functions, such as vesicular transport and trafficking, endocytosis, exocytosis and cellcell adhesion. Annexins have molecular weights ranging between 30 and 40 kDA (the exception is annexin VI which is 66 kDA) and possess striking structural features. To qualify as an annexin, a protein must have 1) the presence of a conserved 70 amino acid domain repeated either 4 or 8 times in the overall structure (annexin VI has an 8 repeating amino acid domain; whereas the rest have 4), 2) the ability to bind phosopholipids in the presence of calcium. Annexins are exported from the cytosol to the exterior of cells across the plasma membrane by an unknown mehanism. When located extracellular, some annexins have been shown to function as receptors for other extracellular proteins: annexin II binds to tenascin and tissue plasminogen activator, while annexin V binds to collagen (Kojima, 1997).
Several annexins were identified in a recent proteomic study of drusen (Crabb et al, 2002). Specifically, peptides from annexins I, II, IV, and VI were found by LC MS/MS Q- Tof analysis of trypsin digested drusen proteins. To define the precise the distribution of these annexins in drusen and Bruch’s membrane/choroid interface, we conducted immunocytochemical studies using a series of commercially available annexin antibodies.
2. METHODS
Bruch’s membrane/choroid complexes from 70–90 year old donor eyes were isolated as described previously (Crabb et al, 2002). After fixation in 4% paraformaldehyde, the tissues were embedded in paraffin. Three mm thick sections were cut, placed on microscope slides and deparaffinized. After washing in PBS for 15 min, sections were incubated in 0.3%
* Mary E. Rayborn, et al., Department of Ophthalmic Research, Cole Eye Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio 44195.
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