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(Investigative Ophthalmology and Visual Science. 2002;43:2001-2006.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Retina-Specific Expression of PDGF-B Versus PDGF-A: Vascular Versus Nonvascular Proliferative Retinopathy

Keisuke Mori, Peter Gehlbach, Akira Ando, Gawain Dyer, Evan Lipinsky, Aneeka G. Chaudhry, Sean F. Hackett and Peter A. Campochiaro

From the Departments of Ophthalmology and Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Platelet-derived growth factor (PDGF) has been implicated in vascular proliferative retinopathies, such as diabetic retinopathy, and in nonvascular retinopathies, such as proliferative vitreoretinopathy. Traction retinal detachment is a central feature of both types of disease. Hemizygous rhodopsin promoter/PDGF-B (rho/PDGF-B) transgenic mice exhibit proliferation of vascular cells, glia, and retinal pigmented epithelial (RPE) cells, resulting in traction retinal detachment. Hemizygous rho/PDGF-A transgenic mice show mild proliferation of glial cells and no traction retinal detachments. This study was undertaken to determine whether higher levels of endogenously produced PDGF-A in the retinas of mice result in retinal detachment.

METHODS. To achieve high-level expression of PDGF-A in the retina, homozygous rho/PDGF-A (rho/PDGF-AA) mice were generated. The phenotype of these mice was compared with that of homozygous rho/PDGF-B (rho/PDGF-BB) mice and double hemizygous rho/PDGF-B-rho/PDGF-A (rho/PDGF-AB) mice.

RESULTS. Rho/PDGF-BB and rho/PDGF-AB mice showed a phenotype similar to that previously described in rho/PDGF-B mice. There was extensive proliferation of glial and vascular cells, resulting in fibrovascular membranes that detached the retina. PDGF-AA mice showed extensive proliferation of glial cells and traction retinal detachment.

CONCLUSIONS. High retinal expression of PDGF-A results in extensive proliferation of glial cells and traction retinal detachment without vascular cell involvement, similar to proliferative vitreoretinopathy in humans. High retinal expression of PDGF-B results in traction retinal detachment from proliferation of both vascular and nonvascular cells, similar to diabetic retinopathy in humans.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Development of new treatments for retinal diseases depends on identification of the molecular signals involved, which then become targets for intervention. Vascular endothelial growth factor (VEGF) has been demonstrated to play a central role in the development of retinal and choroidal neovascularization (for review, see Ref. 1 ). Although it is likely that other growth factors also participate,2 blockade of VEGF signaling has a dramatic inhibitory effect on ocular neovascularization.3 4 5 Also, increased expression of VEGF in the retina results in neovascularization.6 Therefore, VEGF is both necessary and sufficient for retinal neovascularization. Clinical trials have investigated VEGF antagonists in neovascular retinopathies.7 8

Nonneovascular proliferative retinopathies constitute a spectrum of diseases that are distinct from neovascular retinopathies. At one end of the spectrum is idiopathic macular pucker, which is characterized by the occurrence of an epiretinal membrane, composed primarily of ectopic retinal glia and to a lesser extent retinal pigmented epithelial (RPE) cells, that distorts the macula and thereby degrades vision.9 Idiopathic macular puckers are frequently associated with posterior vitreous detachment, but by definition are not associated with other ocular conditions. Macular pucker also occurs in association with ocular inflammatory disease or after retinal reattachment surgery. At the most severe end of the spectrum is proliferative vitreoretinopathy (PVR), the occurrence after retinal reattachment surgery of epiretinal and vitreous membranes composed primarily of retinal glia and RPE cells that often lead to recurrent retinal detachment.10

Numerous growth factors have been suggested as participants in the pathogenesis of PVR,11 leading some investigators to postulate that antagonizing a single growth factor may be useless.12 However, a similar view has been common regarding ocular neovascularization, before recent findings with VEGF antagonists.13 Therefore, despite the probable participation of several growth factors in PVR, attempting to identify and target a central critical factor is a reasonable strategy.

Several lines of evidence suggest that platelet-derived growth factor (PDGF) may play a critical role in PVR and other proliferative retinopathies.11 Multiple gene products collaborate to form PDGF, which is a dimer. PDGF-A and -B were discovered approximately 20 years ago, and effects of isoforms containing them—PDGF-AA, -BB, and -AB—have been extensively studied.14 15 16 17 18 There are two PDGF receptors: the PDGF {alpha} receptor (PDGF{alpha}R) and the PDGF ß receptor (PDGFßR). The PDGF{alpha}R binds both PDGF-A and -B and therefore PDGF-AA, -BB, and -AB can all activate {alpha} receptors. PDGFßR binds PDGF-B but not -A, and therefore PDGF-BB, but not -AA, can activate ß receptors. The action of PDGF-AB is more complex, but it appears that at physiologically relevant concentrations, PDGF-AB can activate PDGFßR only in combination with PDGF{alpha}R.19 Recently, two other family members have been identified: PDGF-C and -D.20 21 22 23 Unlike PDGF-A and -B, PDGF-C and -D are secreted as inactive proteins that are activated by proteolytic cleavage, but similar to PDGF-A and -B, they signal through PDGF{alpha}R and/or PDGFßR.

We have generated rhodopsin promoter/PDGF-A (rho/PDGF-A) and rhodopsin promoter/PDGF-B (rho/PDGF-B) transgenic mice with retina-specific expression of PDGF-A or -B, respectively.24 25 In rho/PDGF-A mice, increased expression of PDGF-A in photoreceptors causes migration and proliferation of astrocytes, so that they are increased in the inner nuclear and nerve fiber layers of the retina. This results in some irregularity of the inner nuclear layer, particularly in the peripheral portion of the retina, but the phenotype is quite subtle. In rho/PDGF-B transgenic mice there is proliferation of several cell types in the retina, including astrocytes, pericytes, and endothelial cells. The cells proliferate on the surface of the retina, and cords of cells migrate into the inner nuclear layer and exert traction on the retina, resulting in outer retinal folds and focal areas of detachment that enlarge and lead to total retinal detachment. This dramatic phenotype is strikingly different from the subtle phenotype exhibited in transgenic mice with increased expression of PDGF-A in photoreceptors.

Retinal glial cells possess PDGF{alpha}R, but not -ßR, whereas pericytes and endothelial cells possess PDGFßR, but not -{alpha}R.26 27 28 29 RPE cells possess both PDGF{alpha}R and -ßR, but they are downregulated unless the RPE cells become separated from the retina or surrounding RPE cells.30 Taken together, these observations explain why expression of PDGF-A in the retina results in proliferation of only glial cells. In contrast, PDGF-B activates both PDGF{alpha}R and -ßR, explaining why rho/PDGF-B mice show proliferation of pericytes, endothelial cells, and glial cells. However, it is not clear why rho/PDGF-A mice do not have traction retinal detachment. Is it because glial cells by themselves do not generate enough traction to cause retinal detachment? Is the severity of disease, ranging from macular pucker to PVR, determined by the relative amounts of PDGF-B and -A, with more severe disease having a greater proportion of PDGF-B? If the predominant isoform were PDGF-AB, would a different phenotype result? This study was designed to address these questions.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Transgenic Mice
Mice were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The generation of rho/PDGF-A and rho/PDGF-B transgenic mice has been previously described.24 25 Hemizygous rho/PDGF-A line 2 mice in a C57BL/6 background24 were crossed. Homozygous rho/PDGF-A (rho/PDGF-AA) mice were identified by Southern blot analysis of tail DNA, with a full-length cDNA of human PDGF-A used as a probe.24 Presumed rho/PDGF-AA mice were backcrossed with wild-type C57BL/6 mice, and two litters were genotyped by PCR of tail DNA. Tail pieces were digested overnight at 55°C in 50 mM Tris (pH 7.5), 100 mM EDTA, 400 mM NaCl, 0.5% SDS containing 0.6 µg/µL proteinase K. PCR was performed at 58°C, with primers that amplify 580 bp of transgene-specific sequence, P1 (5'-GTCCAGCCGGAGCCCCGTG-3') and P2 (5'-TGGCACTTGACACTGCTCGTGTTG-3'). Transgenic parents were considered confirmed rho/PDGF-AA mice if all offspring in both litters were transgene positive.

Hemizygous rho/PDGF-B line 1 mice in a C57BL/6 background25 were crossed with each other. Homozygous rho/PDGF-B (rho/PDGF-BB) mice were identified by Southern blot analysis of tail DNA, with a full-length cDNA of human PDGF-B used as a probe.25 Presumed rho/PDGF-BB mice were backcrossed with wild-type C57BL/6 mice, and two litters were genotyped by PCR of tail DNA. PCR was performed at an annealing temperature of 58°C, with primers that amplify 589 bp of transgene-specific sequence P1 (5'-GTCCAGCCGGAGCCCCGTG-3') and P2 (5'-CCGCACAATCTCGATCTTTCTCACC-3'). Transgenic parents were considered confirmed rho/PDGF-BB mice if all offspring in both litters were transgene positive. Rho/PDGF-AA and -BB mice were mated to generate double hemizygous rho/PDGF-AB mice.

Histochemical Staining with Griffonia simplicifolia Lectin
Rho/PDGF-BB and rho/PDGF-AB mice (n = 5 for each type at each time point) were killed at postnatal day (P)7, P10, P11, and P12. Rho/PDGF-AA mice (n = 3 for each time point) were killed at P12, P75, and P150. Eyes were rapidly removed and frozen in optimal cutting temperature (OCT) embedding medium (Miles Diagnostics, Elkhart, IN). Ten-micrometer frozen sections were fixed with 4% paraformaldehyde for 30 minutes and washed with 0.05 M Tris-buffered saline (TBS; pH 7.6). Slides were incubated in methanol-H2O2 for 10 minutes at 4°C, washed with 0.05 M TBS, and incubated for 30 minutes in 10% normal porcine serum. Slides were incubated for 2 hours at room temperature with biotinylated G. simplicifolia agglutinin (GSA; Vector Laboratories, Burlingame, CA), which selective labels vascular cells, and after rinsing with 0.05 M TBS, they were incubated with avidin coupled to peroxidase (Vector Laboratories) for 45 minutes at room temperature. After a 10-minute wash in 0.05 M TBS, slides were incubated with diaminobenzidine (Research Genetics, Huntsville, AL), to induce a brown reaction product, and counterstained with hematoxylin and eosin.

Immunohistochemical Staining for Glial Fibrillary Acidic Protein
Immunohistochemical staining of retinas for GFAP labels astrocytes and activated Müller cells. Ten-micrometer frozen sections were fixed with 4% paraformaldehyde for 30 minutes, washed with 0.05 M TBS, incubated in methanol-H2O2 for 10 minutes at 4°C, and washed with 0.05 M TBS. Specimens were blocked with 10% normal goat serum (NGS) in 0.05 M TBS for 30 minutes at room temperature and then incubated with 1:500 rabbit anti-bovine GFAP in 1% NGS and 0.05 M TBS and incubated in biotinylated goat anti-rabbit antibody for 30 minutes. After a wash, the slides were incubated in streptavidin-phosphatase and developed with HistoMark Red (Kirkegaard and Perry, Gaithersburg, MD) according to the manufacturer’s instructions. Sections were dehydrated and mounted with acrylic resin mounting medium (Cytoseal; Stephens Scientific, Cornwall, NJ).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In preliminary studies, rho/PDGF-AA, -BB, and -AB mice were examined by indirect ophthalmoscopy. Eyes in the majority of rho/PDGF-BB and -AB mice showed development of retinal detachment within 2 weeks of age, whereas those in rho/PDGF-AA mice took much longer to show any signs of retinal detachment. Therefore, early time points were selected for histopathologic evaluation of rho/PDGF-BB and -AB eyes, whereas rho/PDGF-AA eyes were examined at later times. At P7, line 1 eyes of rho/PDGF-B mice showed slightly more GSA staining on the surface of the retina than was shown in wild-type eyes.25 This was also the case in rho/PDGF-AB and -BB eyes (Figs. 1A 1E) . Similar to rho/PDGF-B eyes, there was increased GFAP staining on the retinal surface in rho/PDGF-AB and -BB eyes (Figs. 2A 2E) . By P10, rho/PDGF-AB and -BB eyes each showed a multilayered sheet of GSA-positive cells on the retinal surface and finger-like projections of cells extending into the inner nuclear layer (Figs. 1B 1F , arrows). Multiple layers of GFAP-positive cells were present on the surface of the retina, but did not extend into the retina as did the GSA-positive cells (Figs. 2B 2F) . At P11, there was a thick carpet of intermixed GSA- and GFAP-positive cells predominantly on the surface of the retina (Figs. 1C 1G 2C 2G) . There was evidence of traction with outer retinal folds and focal areas of detachment (asterisks). By P12, there were extensive detachments with thick epiretinal membranes composed of GSA- and GFAP-positive cells adherent to the back of the lens (Figs. 1D 1H 2D 2H) . Figure 3 shows high magnification views of the extensive epiretinal membranes resulting in traction retinal detachments. Compared with the evolution of the phenotype in line 1 rho/PDGF-B mice,25 the sequence of events was very similar, but substantially accelerated, in rho/PDGF-AB and -BB mice.



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  		Figure 1. Histochemical staining for GSA lectin in double-transgenic rho/PDGF-A–rho/PDGF-B (rho/PDGF-AB) mice, homozygous rho/PDGF-B (rho/PDGF-BB) mice, and homozygous rho/PDGF-A (rho/PDGF-AA) mice. At various ages, mice were killed, eyes were snap frozen, and 10-µm frozen sections were cut. Sections were stained with GSA, which selectively stains vascular cells, and were counterstained with hematoxylin and eosin. At least three mice for each time point were examined and showed similar findings. Eyes are shown at the various time points from (A–D) rho/PDGF-AB mice: (A) P7, (B) P10, (C) P11, and (D) P12; (E–H) rho/PDGF-BB mice: (E) P7, (F) P10, (G) P11, and (H) P12; and (I–L) rho/PDGF-AA mice: (I) P12, (J) P75, (K) P150, and (L) P75. Both rho/PDGF-AB and -BB mice showed rapid and extensive proliferation of vascular cells on the surface of the retina and invading the retina (B, F, arrows), resulting in outer retinal folds and focal detachments by P11 (C, G, *) and total retinal detachment by P12 (D, H). rho/PDGF-AA mice showed no evidence of abnormal proliferation of vascular cells. Retinal detachments developed slowly (J, P75) and eventually became funnel shaped with subretinal membranes similar to those in human PVR (K, arrows). (L) High-power view of detached retina from a P75 mouse showed normal retinal vessels and no ectopic vascular cells. Bar: (A–K) 500 µm; (L) 62.5 µm.

 


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  		Figure 2. Immunohistochemicalstaining for GFAP in double-transgenic rho/PDGF-A–rho/PDGF-B (rho/PDGF-AB) mice, homozygous rho/PDGF-B (rho/PDGF-BB) mice, and homozygous rho/PDGF-A (rho/PDGF-AA) mice. At various ages, mice were killed, eyes were snap frozen, and 10-µm frozen sections were cut. Sections were stained for GFAP, a marker for glial cells. At least three mice were examined at each time point and showed similar findings. Sections adjacent to those shown in Figure 1 are shown at various time points from eyes of (A–D) rho/PDGF-AB mice: (A) P7, (B) P10, (C) P11, and (D) P12; (E–H) rho/PDGF-BB mice: (E) P7, (F) P10, (G) P11, and (H) P12; and (I–L) rho/PDGF-AA mice: (I) P12, (J) P75, (K) P150, and (L) P75. Both rho/PDGF-AB and -BB mice showed rapid and extensive proliferation of glial cells on the surface of the retina (A–H), resulting in a thick epiretinal membrane and total retinal detachment by P12 (D, H). In rho/PDGF-AA mice, there was proliferation of glial cells on the surface of the retina resulting in a prominent epiretinal membrane by P12 (I). By P75, there was retinal detachment with a glial epiretinal membrane and prominent GFAP staining of Müller cells (J, L, arrows). At P150, there was a glial epiretinal membrane on the surface of the retina within a funnel detachment (K). Bar: (A–K) 500 µm; (L) 62.5 µm.

 


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  		Figure 3. High-magnification views of retinas from P12 PDGF-AB and -BB mice with traction retinal detachment. (A) GSA-stained retina of the P12 PDGF-AB double transgenic mouse in Figure 1D . (B) GFAP-stained retina of the P12 PDGF-AB double transgenic mouse in Figure 2D . (C) GSA-stained retina of the P12 PDGF-BB double transgenic mouse in Figure 1H . (D) GFAP-stained retina of the P12 PDGF-BB double transgenic mouse in Figure 2H .

 
At P12, rho/PDGF-AA eyes showed normal GSA staining in the retina (Fig. 1I) , but there was a multilayered sheet of GFAP-positive cells on the surface of the retina (Fig. 2I) . Similar to rho/PDGF-A eyes,24 rho/PDGF-AA eyes showed normal retinal vascular development, but unlike rho/PDGF-A eyes, rho/PDGF-AA eyes showed development of retinal detachments between 2 and 3 months of age (Figs.1J 2J) . A prominent epiretinal membrane appeared, made up of GFAP-positive cells. Staining for GSA was limited to underlying retinal blood vessels, which appeared normal, with no identifiable GSA-positive cells in epiretinal membranes (Figs. 1J 1L) . Once retinal detachment occurred, there was prominent GFAP staining of Müller cells within the retina and of astrocytes on the surface (Figs. 2J , arrows in 2L ). At the 2- to 3-month time point and beyond, the eyes of rho/PDGF-AA mice were larger than those of wild-type or rho/PDGF-A mice of the same age. Over the course of the next several months, the detachments became funnel-shaped, similar to those in human PVR (Fig. 1K) . Sheets of subretinal membranes appeared, resembling those in human PVR (Fig. 1K , arrows).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Several lines of evidence suggest that PDGF is an important stimulatory factor in proliferative retinopathies. It is a potent mitogen and chemoattractant for RPE cells, retinal glia, and pericytes, and expression of PDGF in the eye is upregulated after retinal detachment.30 31 32 Elevated levels of PDGF-AB have been demonstrated in the vitreous of patients with proliferative diabetic retinopathy (PDR)33 and PDGF-A and -B and PDGF{alpha}R and -ßR have been localized to epiretinal membranes obtained during surgery in patients with PDR or PVR.34 35 When injected into the vitreous cavity of rabbits, cells that do not respond to PDGF have greatly reduced ability to cause PVR compared with PDGF-responsive versions of the same cells.36

Recently, we have demonstrated that increased expression of PDGF-B in the retina causes formation of epiretinal membranes and traction retinal detachments, important features of proliferative retinopathies.25 In contrast, several lines of transgenic mice with increased expression of PDGF-A in the retina showed mild glial cell proliferation and no traction retinal detachments.24 Possible interpretations of these findings are that PDGF-B, but not -A, plays an important role in proliferative retinopathies and that proliferation of glial cells by themselves is not sufficient to cause retinal detachment. In this study, we have provided evidence that argues against these interpretations, because homozygous rho/PDGF-AA eyes had more extensive selective proliferation of glial cells than those in hemizygous rho/PDGF-A mice, and it resulted in the gradual onset of traction retinal detachment.

In view of these new data, it is reasonable to postulate that both PDGF-A and -B participate in the pathogenesis of proliferative retinopathies. Furthermore, compared with the phenotype of rho/PDGF-B, -BB, and -AB mice, the phenotype of rho/PDGF-AA mice is more like that seen in patients with macular pucker or PVR. In those disease processes, epiretinal membranes are made up predominantly of retinal glial and RPE cells with few or no vascular cells. The cells do not generally invade the retina, but rather grow over weeks or months on the surfaces of the retina, gradually increasing traction. In contrast, the phenotype of rho/PDGF-B, -BB, and -AB mice is more similar to that of severe PDR, in which vascular cells proliferate within and on the surface of the retina, and glial cells intermingle with vascular cells on the surface. The mouse models not only showed proliferation of the same cell types as in PDR, but also showed attachment of surface membranes to proliferating cells and vasculature within the retina. This feature of PDR membranes makes them more tenacious than PVR membranes and generally requires cutting connections with the retina for removal rather than simply peeling the membranes from the surface of the retina, as is usually possible with PVR membranes. Therefore, it is reasonable to hypothesize that PDGF-AA is an important stimulus in nonvascular proliferative retinopathies, whereas all the PDGF isoforms may participate in vascular proliferative retinopathies.

Findings in past studies are consistent with this hypothesis. Positive immunohistochemical staining for PDGF-A is much more prominent in PVR membranes than positive staining for PDGF-B.35 Cells transduced with expression constructs for dominant-negative PDGF{alpha}R mutants have perturbation of PDGF{alpha}R signaling and reduced ability to cause PVR when injected into the vitreous cavity of rabbits.36 Taken together, these observations, along with the findings in rho/PDGF-AA mice, suggest that PDGF-AA, acting through PDGF{alpha}R, is an important contributor to macular pucker and PVR. However, the demonstration of high levels of PDGF-AB in the vitreous cavity of patients with PDR33 is consistent with PDGF-B’s playing a major role in vascular proliferative retinopathies.

In some settings, PDGF-AB has effects that differ from those of PDGF-BB. For instance, PDGF-AB is a less potent mitogen for some types of cultured human vascular smooth muscle cells than PDGF-BB.16 There are significant differences in the ability of the various isoforms to increase intracellular calcium in smooth muscle cells. PDGF-BB causes a much larger increase than PDGF-AB, and PDGF-AA has no effect.18 These differences are probably because PDGF{alpha}R and -ßR have partially overlapping and partially distinct intracellular signaling pathways.37 Comparison of two lines of knockin mice in which the cytoplasmic domain of one PDGFR has been replaced by the cytoplasmic domain for the other, shows substantial rescue of normal development, but the cytoplasmic domain of PDGF{alpha}R fails to complement all aspects of PDGFßR, resulting in varying amounts of vascular disease.38 This demonstrates that PDGFßR signaling is necessary for optimal function of cells that coat the microvasculature, particularly in the brain and retina. This is consistent with the hypothesis that it is the recruitment and proliferation of pericytes by overexpression of PDGF-B that underlies the striking difference between rho/PDGF-B and -A mice. Rho/PDGF-AB mice have a phenotype that is indistinguishable from that of rho/PDGF-BB mice and is more severe than that of rho/PDGF-B mice. This indicates that although expression of PDGF-A alone does not result in a vascular proliferative retinopathy phenotype, coexpression of PDGF-A with -B does not perturb the effects of PDGF-B, but rather enhances them.

Based on the findings in this study and other recent studies in the literature, we propose the following working model. PDGF-AA is an essential stimulus for the development of macular pucker and PVR, with the level of expression being a critical distinguishing feature. The major source of PDGF-A in the eye is retinal ganglion cells and, to a lesser extent, the RPE.30 39 Perhaps vitreous traction on ganglion cells, as would be expected to occur during evolution of some, but not all, posterior vitreous detachments, increases production of PDGF-A by ganglion cells and stimulates proliferation of astrocytes, resulting in macular pucker. Withdrawal of inhibitory influences from hyalocytes may also contribute.40 After retinal detachment, production of PDGF-A by RPE cells increases, and the total amount of PDGF-AA in the eye may increase to levels sufficient to cause more severe epiretinal membranes and can cause traction retinal detachment similar to that in rho/PDGF-AA mice.

The expression of PDGF-B, similar to that of VEGF, is increased by hypoxia,41 42 and therefore it is likely that PDGF-B collaborates with VEGF in ischemic retinopathies. PDGF-A production may also be increased and hence the measurement of high levels of PDGF-AB in patients with diabetic retinopathy,33 but it is the participation of PDGF-B in either heterodimers or homodimers that fuels vascular proliferation. Our model predicts that blockage of signaling through both types of PDGF receptors constitutes an important target for treatment of proliferative retinopathies, and future studies will be designed to test this hypothesis.


   Footnotes
 
Supported by Grants EY-05951, EY-12609, K08 EY-13420 (PG), and P30-EY-1765 from the National Eye Institute; and by the Ruth and Milton Steinbach Foundation, the Juvenile Diabetes Research Foundation (PG), and a Lew R. Wasserman Merit Award (PAC) and unrestricted funds from Research to Prevent Blindness. PAC is the George S. and Dolores Dore Eccles Professor of Ophthalmology and Neuroscience.

Submitted for publication September 21, 2001; revised January 16, 2002; accepted January 24, 2002.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Peter A. Campochiaro, The Johns Hopkins University School of Medicine, Maumenee 719, 600 N. Wolfe Street, Baltimore, MD 21287-9277; pcampo{at}jhmi.edu.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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